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https://openalex.org/W2465106506	https://www.frontiersin.org/articles/10.3389/fnhum.2016.00348/pdf	English	null	Shutting Down Sensorimotor Interferences after Stroke: A Proof-of-Principle SMR Neurofeedback Study	Frontiers in human neuroscience	2,016	cc-by	11,195	Neurofeedback Study Johanna L. Reichert1,2, Silvia E. Kober1,2, Daniela Schweiger1, Peter Grieshofer3, Christa Neuper1 and Guilherme Wood1 Johanna L. Reichert1,2, Silvia E. Kober1,2, Daniela Schweiger1, Peter Grieshofer3, Christa Neuper1 and Guilherme Wood1 1 Institute of Psychology, University of Graz, Graz, Austria, 2 BioTechMed Graz, Graz, Austria, 3 Klinik Judendorf-Straßengel, Graz, Austria Introduction: Neurofeedback training aims at learning self-regulation of brain activity underlying cognitive, emotional or physiological functions. Despite of promising investigations on neurofeedback as a tool for cognitive rehabilitation in neurological diseases, such as after stroke, there is still a lack of research on feasibility and efﬁciency of neurofeedback in this ﬁeld. Methods: The present study aimed at investigating behavioral and electrophysiological effects of 10 sessions of sensorimotor rhythm (SMR) neurofeedback in a 74-years- old stroke patient (UG20). Based on previous results in healthy young participants, we hypothesized that SMR neurofeedback leads to a decrease in sensorimotor interferences and improved stimulus processing, reﬂected by changes in event- related potentials (ERPs) and electrophysiological coherence. To assess whether UG20 beneﬁted from the training as much as healthy persons of a similar age, a healthy control group of N = 10 elderly persons was trained as well. Before and after neurofeedback training, participants took part in a multichannel electroencephalography measurement conducted during a non-verbal and a verbal learning task. Keywords: neurofeedback, sensorimotor rhythm, memory, aging, stroke Edited by: Srikantan S. Nagarajan, University of California, San Francisco, USA Reviewed by: Vadim Nikulin, Charite University Hospital, Germany Márk Molnár, Hungarian Academy of Sciences, Hungary Results: Both UG20 and the healthy controls were able to regulate their SMR activity during neurofeedback training. Moreover, in a non-verbal learning task, changes in ERPs and coherence were observed after training: UG20 showed a better performance in the non-verbal learning task and a higher P3 amplitude after training than before, and coherence between central and parietal electrodes decreased after training. The control group also showed a behavioral improvement in the non-verbal learning task and tendencies for higher P3 amplitudes and decreased central-parietal coherence after training. Single-case analyses indicated that the changes observed in UG20 were not smaller than the changes in healthy controls. *Correspondence: Johanna L. Reichert Johanna.reichert@uni-graz.at Received: 14 March 2016 Accepted: 24 June 2016 Published: 15 July 2016 ORIGINAL RESEARCH published: 15 July 2016 doi: 10.3389/fnhum.2016.00348 INTRODUCTION Electroencephalography (EEG)-based neurofeedback is a promising tool for cognitive improvement and rehabilitation. While most traditional cognitive trainings consist of speciﬁc tasks that aim at improving cognitive functions, neurofeedback is aimed at directly regulating the brain activity underlying cognitive functioning. An important possible future area of application for neurofeedback is cognitive rehabilitation of neurological diseases, such as stroke (Doppelmayr et al., 2007; Hofer et al., 2014), as neurofeedback might be used to directly up-regulate certain aspects of brain activity while suppressing dysfunctional activations. During neurofeedback training, brain signals are recorded, processed and fed back to participants on a computer screen. In most cases, the feedback is presented auditorily or visually, for example as moving bars that have to be steered in a certain direction. This feedback enables participants to voluntarily control their own electrical brain activity. y During sensorimotor rhythm (SMR)-based neurofeedback, the power of SMR, a frequency band ranging from 12 to 15 Hz, is extracted from the EEG signal. SMR is an oscillatory rhythm recorded over central scalp regions, that is supposed to be originating from the thalamic nuclei, speciﬁcally from ventroposterior lateral and reticular nuclei (Sterman, 1996, 2000). SMR is observed when one is motionless but mentally focused and attentive and is suppressed when motor tasks or motor imagery are carried out (Pfurtscheller, 1981; Sterman, 1996). Since the beginnings of neurofeedback research, SMR has been utilized as a feedback frequency band. Several studies have shown that SMR neurofeedback training can lead to cognitive improvements, mainly in memory functions and attention (Vernon et al., 2003; Egner and Gruzelier, 2004; Hoedlmoser et al., 2008; Doppelmayr and Weber, 2011; Kober et al., 2015b). Underscoring its presumable eﬀect of reducing brain excitability, SMR neurofeedback has been successfully applied in diseases such as epilepsy and ADHD (Lubar et al., 1995; Tinius and Tinius, 2000; Sterman and Egner, 2006). It was suggested that SMR might facilitate thalamic inhibitory mechanisms (Sterman, 1996, 2000; Egner and Gruzelier, 2004), and block motor activity that interferes with information processing (Pfurtscheller, 1992; Sterman, 1996). In accordance with this assumption, we found in a previous study that SMR- based neurofeedback training of healthy young adults leads to cognitive improvements related to changes in task-related electrophysiological parameters. Components of event-related potentials (ERPs), the N1 and P3, were increased after SMR training as compared to pre-training, indicating more intensive stimulus processing (Kober et al., 2015b). Citation: Reichert JL, Kober SE, Schweiger D, Grieshofer P, Neuper C and Wood G (2016) Shutting Down Sensorimotor Interferences after Stroke: A Proof-of-Principle SMR Neurofeedback Study. Front. Hum. Neurosci. 10:348. doi: 10.3389/fnhum.2016.00348 Conclusion: Neurofeedback can be successfully applied in a stroke patient and in healthy elderly persons. We suggest that SMR neurofeedback leads to a shutting-down of sensorimotor interferences which beneﬁts semantic encoding and retrieval. July 2016 \| Volume 10 \| Article 348 1 Frontiers in Human Neuroscience \| www.frontiersin.org Shutting Down Sensorimotor Interferences Reichert et al. the functionality of SMR neurofeedback in elderly persons and in persons suﬀering from neurological diseases, such as stroke (Doppelmayr et al., 2007; Hofer et al., 2014), are still scarce. Therefore, we aimed to investigate the eﬀects of SMR neurofeedback in a single-case of stroke. Around 32% of stroke survivors still demonstrate cognitive sequelae 3 years after the incident (Patel et al., 2003). Particularly long-term deﬁcits in attention, memory and executive functions are common in stroke patients. Importantly, evidence suggests that speciﬁc neurofeedback protocols have largest eﬀects on speciﬁc cognitive functions. For instance, Theta/Beta neurofeedback has mainly proven successful in improving attention and executive functions (Monastra et al., 2002; Fox et al., 2005; Duric et al., 2012), while SMR neurofeedback has been consistently found to improve memory functions (Vernon et al., 2003; Hoedlmoser et al., 2008; Hofer et al., 2014; Schabus et al., 2014; Kober et al., 2015b). Thus, neurofeedback training protocols can be chosen according to the patient’s cognitive deﬁcits. In the present study, we chose to train a 74-years-old stroke patient with memory impairments with SMR neurofeedback to investigate training feasibility and the eﬀects of the training on cognition and electrophysiological parameters. We chose to train the selected patients due to his very speciﬁc cognitive deﬁcits that comprised memory impairments but no deﬁcits of attention or executive functions. In a previous study, we found that patients with heterogeneous lesion locations could reach control over their brain activity during SMR-based and Upper Alpha-based neurofeedback (Kober et al., 2015a). Therefore, in the present study we chose to base our patient selection on the cognitive deﬁcits observed rather than on lesion locations. Frontiers in Human Neuroscience \| www.frontiersin.org Stroke Patient UG20 UG20 (age 74, male) had suﬀered a stroke (ICD-10 diagnosis I63.1) due to basilar artery thrombosis (ICD-10 diagnosis I65.1) with lesions in right cerebellum, bilateral hippocampus, right mesencephalon, left occipital lobe, bilateral splenium 4.5 months before start of SMR training. At the time of stroke onset, UG20 was retired after working for several years as department head of an inventory department. He had completed 12 years of education. After the stroke, UG20 stayed in neurorehabilitation for 3 months, then he was released back home. Since his stroke, UG20 has suﬀered from memory deﬁcits (amnestic syndrome), while his score of 29 in the mini-mental state examination (MMSE, Folstein et al., 1975) did not indicate general cognitive deﬁcits. UG20 had normal corrected vision in the whole visual ﬁeld (no anopsia), intact hearing abilities and showed signs of slight dysarthria. He did not suﬀer from hemiparesis but showed a subtle motor restriction of the right foot and a slight unsteadiness in walking. UG20 was administered a detailed neuropsychological assessment before start of the training (pre- assessment) to ensure the selection of an adequate neurofeedback training for him. This neuropsychological test battery was repeated after the last neurofeedback training session (post- assessment). The results of the pre-assessment (see Figure 4) indicated that UG20 mainly suﬀered from short- and long- term memory deﬁcits, while other cognitive functions (alertness, cognitive ﬂexibility, divided attention, inhibition) remained unaﬀected. Thus, we decided to apply SMR neurofeedback training in UG20, as this training has proven to be speciﬁcally eﬀective in improving memory performance. Neurofeedback was conducted for 10 sessions in the course of 3 weeks at UG20s home. During neurofeedback training, UG20 did not receive medications that aﬀected vigilance or attention and did not take part in other forms of cognitive training. UG20 received an expense allowance of 7 Euro/hour. UG20 provided SMR-Based Neurofeedback Training Electroencephalography signals for SMR training were recorded at channel Cz, digitized at 256 Hz and ﬁltered with a 0.5 Hz high- pass and a 60 Hz low-pass ﬁlter. A 10-channel system (NeXus-10 MKII, Mind Media BV) and a g.USBamp 16-channel standard ampliﬁer (g.tec, Graz, Austria) were used for data recording. The ground electrode was located at the right mastoid, the reference at the left mastoid. Vertical electrooculogram (EOG) was recorded by two electrodes on top and below the left eye (bipolar montage). Each participant performed 10 training sessions (approximately 50 min each) on diﬀerent days. Elderly Control Sample As a reference for the evaluation of training-related changes in cognitive performance and EEG signal in UG20, we trained a control sample of N = 10 healthy elderly participants (mean age: 70.6, SE = 2.36, range: 60–84; see Table 1 for demographic data of UG20 and the control group). Healthy participants were recruited from the general population, by public bulletins and newspaper announcements and gave their written informed consent before participation. Exclusion criteria were any current or previous psychiatric or neurological disorders and history of severe head injuries. Controls received an expense allowance of 7 Euro/hour. The ethics committee of the University of Graz, Austria, approved all aspects of the present study (reference numbers GZ. 39/21/63 ex 2011/12 and GZ. 39/22/63 ex 2011/12) and the study was in line with the code of ethics of the World Medical Association, Declaration of Helsinki. Participants provided written informed consent before participation. INTRODUCTION UG20 Control Group Age 74 Mean 70.6 (SE = 2.36) Gender Male Five females, ﬁve males Handedness Right Nine right, one both Years of education 12 Mean 10.2 (SE = 0.63) TABLE 1 \| Demographic data of stroke patient UG20 and the healthy control group. remained unexplored. Thus, in the present study we set out to investigate electrophysiological parameters in detail in a stroke patients and a healthy elderly control group before and after neurofeedback training. Of note, we selected elderly participants as controls, as there is evidence that electrophysiological brain activity changes across the life span (Polich, 1997; Klimesch, 1999; Babiloni et al., 2006; Cummins and Finnigan, 2007; Rossini et al., 2007), which might also aﬀect the functionality of neurofeedback paradigms. As neurofeedback studies in older persons are still scarce, an additional aim of the present study was the assessment of the eﬃciency of SMR neurofeedback training in the elderly sample. It has been demonstrated that across the lifespan, cognitive decline is accompanied by a broad array of changes in brain activation and structure (Hedden and Gabrieli, 2004; Rabbitt et al., 2007; Finnigan and Robertson, 2011). Based on such observations, one may assume that self-regulation of brain activity might be an eﬃcient method to counteract age-related cognitive declines in older persons. written informed consent before participation and consented to publication of his data. INTRODUCTION Importantly, functional brain connectivity between motor areas and visual processing areas was reduced after SMR training, while performance in a verbal memory task was improved. Thus, these results support the idea that SMR up-training leads to an enhanced blocking of sensorimotor interference, which might be responsible for the observed improvements in stimulus processing and memory function (Kober et al., 2015b). However, these results were obtained in a healthy, young sample and studies assessing Hitherto, there is still a lack of studies assessing the eﬃciency and feasibility of neurofeedback protocols in stroke patients. In two studies reported by Doppelmayr et al. (2007), inconsistent results were observed regarding the eﬃciency of alpha and theta-based neurofeedback in stroke patients. While alpha neurofeedback proved superior to a control treatment in the ﬁrst study, this was not replicated in a second study investigating alpha and theta neurofeedback. In this second study, neither alpha neurofeedback nor theta was more eﬃcient than a control treatment. On the other hand, in a range of case studies, positive eﬀects of neurofeedback on cognitive performance in stroke patients were reported (Rozelle and Budzynski, 1995; Bearden et al., 2003; Cannon et al., 2010). Still, these studies lack healthy control groups to assess whether stroke patients can beneﬁt as much from the training as healthy persons. Therefore, in the present study we included an elderly control sample for comparison. Single-case control approaches allow the comparison of the patient’s improvement with the improvement observed in the healthy elderly (Crawford and Garthwaite, 2002, 2004). In a recent systematic study on stroke patients, Kober et al. (2015a) observed improvements in memory functions after SMR-based and Upper Alpha-based neurofeedback training. Eﬀects were stronger than the eﬀects of traditional cognitive training in a control group of stroke patients. While this study provided promising evidence that neurofeedback might be used as an eﬀective tool for cognitive rehabilitation in stroke patients, the neuronal basis of the observed behavioral improvements Frontiers in Human Neuroscience \| www.frontiersin.org July 2016 \| Volume 10 \| Article 348 Frontiers in Human Neuroscience \| www.frontiersin.org 2 Shutting Down Sensorimotor Interferences Reichert et al. TABLE 1 \| Demographic data of stroke patient UG20 and the healthy control group. Frontiers in Human Neuroscience \| www.frontiersin.org Stroke Patient UG20 Sessions were conducted 2–5 times a week and consisted of 10 min of preparation, a 3- min baseline trial in which the participants saw moving bars while instructed to relax themselves without trying to control the bars voluntarily, and six 3-min feedback runs. In these runs, participants were instructed to increase SMR power while reducing electromyography (EMG) and EOG artifacts. Three bars were presented on a computer screen: the height of the bar in the middle reﬂected real-time SMR power (12–15 Hz, band power), while the height of the left bar reﬂected eye- blink artifacts (4–7 Hz, EEG band power) and the height of the right bar EMG artifact power (EEG band power between 21 and 35 Hz which indicates movements and other high-frequency disturbances). The data recorded in the baseline trial was used to calculate individual thresholds for the three bars. For the artifact SMR-Based Neurofeedback Training SMR-Based Neurofeedback Training Electroencephalography signals for SMR training were recorded at channel Cz, digitized at 256 Hz and ﬁltered with a 0.5 Hz high- pass and a 60 Hz low-pass ﬁlter. A 10-channel system (NeXus-10 MKII, Mind Media BV) and a g.USBamp 16-channel standard ampliﬁer (g.tec, Graz, Austria) were used for data recording. The ground electrode was located at the right mastoid, the reference at the left mastoid. Vertical electrooculogram (EOG) was recorded by two electrodes on top and below the left eye (bipolar montage). July 2016 \| Volume 10 \| Article 348 3 Shutting Down Sensorimotor Interferences Reichert et al. EEG measurements were carried out while participants were presented with a non-verbal (NVLT) and a verbal (VLT) memory task adapted from Sturm and Willmes (1999). Both tasks were divided into eight blocks consisting of 20 stimuli each. Eight of these 20 stimuli appeared in each of the blocks (repetition items), while the other 12 stimuli were shown only once each. Stimuli were presented for 3 s each, then participants had 2 s to react by button press. Between stimuli a ﬁxation cross was displayed for 500 ms, followed by a black screen shown for a duration of 1.5–2.5 s (jittered; see Figure 1 for a depiction of task procedure). In the NVLT, participants saw geometric or irregular shapes consisting of black lines on a white rectangle presented on a black background screen. After seeing each shape, participants had to indicate by button press whether they had seen this ﬁgure before or not. The VLT followed the same procedure, but instead of ﬁgures, pseudo-words (neologisms) were used as stimuli. Neologisms were presented in black letters (2 cm font size) on a white rectangle over a black background screen. bars, thresholds were kept constant over all neurofeedback runs (baseline mean + 1 SD), while the threshold of SMR power was adapted automatically after each run on the basis of the previous run (mean). When the SMR band power exceeded the predeﬁned threshold, the color of the bar changed from red to green. When participants were able to move the SMR bar above its threshold while keeping the artifact bars below their thresholds for more than 250 ms, they were rewarded by getting points shown at the bottom of the screen. SMR-Based Neurofeedback Training Participants were not given any instruction of how to control the feedback bars but were only told to stay mentally focused and physically relaxed. For neurofeedback data analysis, SMR (12–15 Hz) band power was extracted by means of a procedure provided by the Vision Analyzer software (complex demodulation procedure, Brain Products GmbH, Munich, Germany). The resulting power values were averaged over all artifact free segments of each training run. To assess training eﬀects, SMR power was averaged per run of training across all diﬀerent sessions. SMR power values were z-transformed to ensure comparability across sessions and subjects. To analyze more closely the time course of SMR power over the training runs averaged over all sessions, we conducted linear regression analyses (predictor variable = run; dependent variable = SMR power). As during successful neurofeedback, a within-session increase of the feedback frequency power is expected, the slope of the regression line was used as an indicator of neurofeedback performance (see Zoefel et al., 2011; Escolano et al., 2012; Witte et al., 2013). UG20 additionally performed a comprehensive neuropsychological test battery before and after neurofeedback training to evaluate the cognitive proﬁle of the stroke patient in more detail. This test battery included standardized neuropsychological tests to assess attention [Subtest Alertness of the Test of Attentional Performance (TAP) test battery, Zimmermann and Fimm, 2009], divided attention (Subtest Divided Attention of the TAP test battery, Zimmermann and Fimm, 2009), inhibition (Subtest Go/NoGo of the TAP test battery, Zimmermann and Fimm, 2009), cognitive ﬂexibility (Subtest Flexibility of the TAP test battery, Zimmermann and Fimm, 2009), verbal long-term memory (California Verbal Learning Test CVLT, Niemann et al., 2008; Visual and Verbal Behavioral Assessment The same protocol was followed for UG20 and the healthy control sample: before and after neurofeedback training, 60-channel FIGURE 1 \| Task procedure of the (A) non-verbal learning task (NVLT) and (B) verbal learning task (VLT). After 3 s of stimulus presentation, participants were shown the stimulus and the question “Ja/Nein?” [German for “Yes/No”] for 2 s, asking them to indicate by button press whether they had seen the stimulus before. Then, a ﬁxation cross was shown for 0.5 s. Before the next stimulus presentation, a black screen was shown for a jittered duration of 1.5–2.5 s to avoid expectation effects of the participants. FIGURE 1 \| Task procedure of the (A) non-verbal learning task (NVLT) and (B) verbal learning task (VLT). After 3 s of stimulus presentation, participants were shown the stimulus and the question “Ja/Nein?” [German for “Yes/No”] for 2 s, asking them to indicate by button press whether they had seen the stimulus before. Then, a ﬁxation cross was shown for 0.5 s. Before the next stimulus presentation, a black screen was shown for a jittered duration of 1.5–2.5 s to avoid expectation effects of the participants. July 2016 \| Volume 10 \| Article 348 Frontiers in Human Neuroscience \| www.frontiersin.org 4 Shutting Down Sensorimotor Interferences Reichert et al. Memory Test VVM2 subscale “construction 2,” Schelling and Schächtele, 2001), non-verbal long-term memory (VVM2 subscale “city map 2,” Schelling and Schächtele, 2001), verbal short-term memory (VVM2 subscale “construction 1,” Schelling and Schächtele, 2001; CVLT List A Trial 1 and List B, Niemann et al., 2008; Wechsler Memory Scale WMS-R Digit Span test forward task, Härting and Wechsler, 2000), non-verbal short- term memory (VVM2 subscale “city map 1,” Schelling and Schächtele, 2001); Corsi Block Tapping Test (CBTT forward task, Schellig, 2011) and working memory (CBTT backward task, Schellig, 2011); Digit Span test backward task of the (WMS-R, Härting and Wechsler, 2000). Parallel forms of the memory tests were used to avoid learning eﬀects. Additionally, coherence analyses were applied to assess connectivity between motor areas and parietal brain areas during the learning tasks. EEG coherence is a measure of the degree of synchronous electrical activity in diﬀerent brain areas over time. Coherence was assessed during the baseline intervals from 500 ms prior to stimulus onset. Statistical Analysis of Pre–post Differences y p For the healthy participants, diﬀerences in scores of the learning tasks, ERP amplitudes and coherence were assessed by means of paired-sample t-test (evaluated at the two-sided signiﬁcance level of α = 5%). To ascertain the degree of homogeneity in the control group and to investigate the robustness of training results in the sample, non-parametric bootstrapping methods were employed. Pre–post comparisons were bootstrapped 10000 times (n = 10 with replacement). The median p-value generated by bootstrapping will be reported complementarily. To assess the response of UG20 to training, the diﬀerences in his test scores were compared to the pre–post diﬀerence of healthy controls applying single-case analysis based on methods by Crawford and Garthwaite, 2002). These methods enable the assessment of the probability that test scores and test score discrepancies of a patient and a modest-sized control sample belong to the same distribution (Crawford and Garthwaite, 2002). Data was preprocessed and analyzed with Brain Vision Analyzer software (Brain Products GmbH, Munich, Germany). The signal was ﬁltered oﬄine with a 0.1 Hz high-pass and 100 Hz low-pass. EOG artifact correction was carried out using independent component analysis (ICA) ocular correction. Other EEG artifacts (e.g., muscle artifacts) were automatically rejected when one of the following criteria was fulﬁlled: >50 µV voltage step per sampling point, absolute voltage value exceeding ±120 µV. Moreover, to analyze clinical relevance of pre–post changes of the neuropsychological test battery in UG20, critical diﬀerence analysis (Huber, 1973) were performed. To identify signiﬁcant improvement or decline, the critical diﬀerence of the relevant test parameter was compared with the test score diﬀerence obtained during the post-assessment minus the pre-assessment. The diﬀerence between pre- and post-assessment shown by UG20 was considered signiﬁcant when it was larger than the critical diﬀerence, which can be detected by each test and only occurs in the population with a probability lower than α < 10%. For the verbal and non-verbal learning task, we analyzed ERPs in the EEG after presentation of target stimuli participants responded correctly to. Based on previous results of our group (Kober et al., 2015b), we analyzed the ERPs P3 and N1. In a ﬁrst step, the period from 200 ms prior to 1000 ms following stimuli onset, relative to a 200 ms pre-stimulus baseline was extracted. For N1, signal over electrode FCz was analyzed, while for P3 we extracted the signal from Pz. Behavioral Assessment In a ﬁrst step, for each segment, Fast Fourier Transformation (FFT, maximum resolution of ∼0.98 Hz, 10% Hanning window including variance correction) was applied to calculate EEG power spectra. Coherence r in the SMR band 12–15 Hz was then calculated between the channels Cz and CPz, Cz and Pz, and Cz and POz (for a more detailed description of the procedure see Kober et al., 2015b). The resulting r-value is a generalization of the Pearson product correlation coeﬃcient to frequency domain variables and ranges from 0 (no correlation in frequency) to 1 (ideal constant correlation). Coherence values were Fisher’s z-transformed to normalize the distributions. For reporting, means were inverse transformed. Statistical Analysis of Pre–post Differences Then, the mean area ERP amplitude for the healthy group was obtained by averaging the amplitude of the signal for latency windows chosen depending on the ERP characteristics in each task: VLT: N1 = 110–170, P3 = 500–750 ms; NVLT: N1 = 110–170, P3 = 450–650 ms after stimulus onset. As UG20 showed a slightly later N1, for him a latency window of 180–250 was used in both tasks. We calculated diﬀerence values by subtracting the mean area ERP amplitude values during the post-measurement from the mean area ERP amplitude values during the pre-measurement. For N1, negative values indicate larger N1 amplitudes during post- compared to pre-measurement. For P3, positive values indicate larger P3 amplitudes during post- compared to pre-measurement. For both N1 and P3, a minimum number of 15 trials was used for averaging. Electrophysiological Parameters During the pre- and post-assessments, multichannel EEG was recorded from 60 electrode sites according to the International 10–20 EEG placement system with a sampling frequency of 500 Hz and band-pass ﬁlter (0.01–100 Hz). Multichannel EEG ampliﬁers (BrainAmp DC by Brain Products, Munich, Germany and g.USBamp by g.tec, Graz, Austria) were used (reference: linked-ear). Furthermore, horizontal and vertical EOG was recorded using an electrode placed at the nasion and two electrodes placed at the left and right temples, as in previous studies (Schlögl et al., 2007). Impedances were kept below 5 kOhms for EEG and below 10 kOhms for EOG recordings. Frontiers in Human Neuroscience \| www.frontiersin.org Neurofeedback Performance Figure 2, left side, illustrates the time course of SMR power over the training runs for the stroke patient UG20. A clear increase in SMR power across training runs can be observed in UG20, which was also reﬂected in the regression slope resulting from a linear regression analysis. As shown in Figure 2, right side, healthy participants were also able to increase SMR power over the training runs, as their July 2016 \| Volume 10 \| Article 348 Frontiers in Human Neuroscience \| www.frontiersin.org 5 Shutting Down Sensorimotor Interferences Reichert et al. FIGURE 2 \| Time course of sensorimotor rhythm (SMR) power over the neurofeedback training runs, averaged over all 10 NF training sessions, in stroke patient UG20 and in the healthy elderly group. FIGURE 2 \| Time course of sensorimotor rhythm (SMR) power over the neurofeedback training runs, averaged over all 10 NF training sessions, in stroke patient UG20 and in the healthy elderly group. a t-test [see Figure 3; pre–post: t(9) = −2.03, p < 0.05, d = −1.28, pbootstrapping = 0.035]. In the verbal learning task, no change in performance was observed in the controls [pre– post: t(9) = −0.74, n.s., d = −0.47, pbootstrapping = 0.30]. To further assess the improvement observed in UG20, the pre– post diﬀerence in his test scores was compared to the pre– post diﬀerence of healthy controls applying single-case analysis according to the approach of Crawford and Garthwaite (2002). The patient’s and the controls’ values did not diﬀer signiﬁcantly [t(9) = −0.127, p = 0.451, d = −0.08], indicating that UG20 improved at least as much as the controls in the NVLT after the training. linear regression slopes were signiﬁcantly larger than 0 in a two- sided t-test [t(9) = −2.04, p < 0.05, d = −1.29]. The results of this regression analysis indicated that participants showed consistent linear increase in SMR power over the training runs (averaged over all 10 training sessions). When analyzing the time course of SMR power over the training runs separately for each participant of the SMR group, 8 out of 10 participants (i.e., 80%) showed a positive gradient of the learning curve. Of the two remaining participants, one showed a ﬂat learning curve gradient and one a negative gradient. Neurofeedback Performance A single-case analysis (Crawford and Garthwaite, 2004) indicated that the patient’s regression slope did not diﬀer signiﬁcantly from the healthy participants’ slopes [t(9) = 1.93, p = 0.09, d = 1.22], indicating that his ability to alter SMR power was at least as good but not statistically diﬀerent from that of healthy controls. The increase in SMR power observed in UG20 across training runs indicated successful training of SMR in the stroke patient. In the neuropsychological test battery assessed before and after neurofeedback training, stroke patient UG20 showed signiﬁcant performance improvements in non-verbal short-term memory (VVM2 subscale “city map 1”) and working memory (CBTT backward task) tasks when comparing the pre- and post- assessment. The performance in several scales of the CVLT assessing verbal short- and long-term memory performance also improved (List B, Immediate Free Recall, Learning Eﬃciency). In the forward task of the CBTT and List A of the CVLT assessing short-term memory performance, UG20 showed a decreased performance after neurofeedback training compared to the pre- assessment. In sum, UG20 showed signiﬁcant improvements in Behavioral Results After neurofeedback training, UG20 improved in the non- verbal task, while performance in the verbal learning task remained unaltered after training (see left side of Figure 3). Healthy participants showed improvements in the non-verbal learning task after training as well, which were signiﬁcant in FIGURE 3 \| Means and standard errors of the performance in the verbal learning task (VLT) and the non-verbal learning task (NVLT) for UG20 and the control group before and after NF training. FIGURE 3 \| Means and standard errors of the performance in the verbal learning task (VLT) and the non-verbal learning task (NVLT) for UG20 and the control group before and after NF training. July 2016 \| Volume 10 \| Article 348 July 2016 \| Volume 10 \| Article 348 Frontiers in Human Neuroscience \| www.frontiersin.org 6 Reichert et al. Shutting Down Sensorimotor Interferences FIGURE 4 \| Results of the detailed cognitive assessment carried out in stroke patient UG20 before and after neurofeedback training. Shaded area ndicates norm scores. Test performance is expressed in T-scores with population mean M = 50 and standard deviation SD = 10. Neuropsychological test scores nd conﬁdence intervals for measurements of alertness, divided attention, cognitive ﬂexibility and inhibition scores were derived from tasks of the Testbatterie zur Aufmerksamkeitsprüfung (Zimmermann and Fimm, 2009). STM, short-term memory; LTM, long-term memory; CBTT, Corsi Block Tapping Test (Schellig, 2011); VVM2, Visueller und Verbaler Merkfähigkeitstest 2 (Schelling and Schächtele, 2001); CVLT, California Verbal Learning test (Niemann et al., 2008). Signiﬁcant ifferences between pre- and post-test (critical difference analysis on the group level, Huber, 1973) are marked with asterisks (∗signiﬁcant). FIGURE 4 \| Results of the detailed cognitive assessment carried out in stroke patient UG20 before and after neurofeedback training. Shaded area indicates norm scores. Test performance is expressed in T-scores with population mean M = 50 and standard deviation SD = 10. Neuropsychological test scores and conﬁdence intervals for measurements of alertness, divided attention, cognitive ﬂexibility and inhibition scores were derived from tasks of the Testbatterie zur Aufmerksamkeitsprüfung (Zimmermann and Fimm, 2009). STM, short-term memory; LTM, long-term memory; CBTT, Corsi Block Tapping Test (Schellig, 2011); VVM2, Visueller und Verbaler Merkfähigkeitstest 2 (Schelling and Schächtele, 2001); CVLT, California Verbal Learning test (Niemann et al., 2008). Signiﬁcant differences between pre- and post-test (critical difference analysis on the group level, Huber, 1973) are marked with asterisks (∗signiﬁcant). Behavioral Results FIGURE 4 \| Results of the detailed cognitive assessment carried out in stroke patient UG20 before and after neurofeedback training. Shaded area indicates norm scores. Test performance is expressed in T-scores with population mean M = 50 and standard deviation SD = 10. Neuropsychological test scores and conﬁdence intervals for measurements of alertness, divided attention, cognitive ﬂexibility and inhibition scores were derived from tasks of the Testbatterie zur Aufmerksamkeitsprüfung (Zimmermann and Fimm, 2009). STM, short-term memory; LTM, long-term memory; CBTT, Corsi Block Tapping Test (Schellig, 2011); VVM2, Visueller und Verbaler Merkfähigkeitstest 2 (Schelling and Schächtele, 2001); CVLT, California Verbal Learning test (Niemann et al., 2008). Signiﬁcant differences between pre- and post-test (critical difference analysis on the group level, Huber, 1973) are marked with asterisks (∗signiﬁcant). memory tasks, whereas attentional and executive functions did not change when comparing the results of the pre- and post- assessment (Figure 4). pre- and post-measurement. For UG20, an alteration in P3 amplitude was observed in the non-verbal learning task (see Figure 6): P3 amplitude was enhanced post-training compared to pre-training. N1 amplitude in the non-verbal task was slightly increased in UG20 after training as well. In the verbal learning task, no clear pre–post diﬀerences in P3 or N1 amplitudes could be observed in UG20 (see Figure 6). Electrophysiological Results Event-Related Potentials In Figures 5 and 6, the grand average ERPs from Pz are illustrated for UG20 and the healthy elderly group during July 2016 \| Volume 10 \| Article 348 Frontiers in Human Neuroscience \| www.frontiersin.org 7 Shutting Down Sensorimotor Interferences Reichert et al. FIGURE 5 \| Grand average event-related potentials (FCz and Pz) during the encoding phase of the non-verbal learning task (NVLT) and the verbal learning task (VLT), for the pre- and post-measurement of the healthy elderly group. The analyzed latency windows are marked with rectangles. FIGURE 5 \| Grand average event-related potentials (FCz and Pz) during the encoding phase of the non-verbal learning task (NVLT) and the verbal learning task (VLT), for the pre- and post-measurement of the healthy elderly group. The analyzed latency windows are marked with rectangles. FIGURE 6 \| Means of ERP amplitudes (FCz and Pz) during the encoding phase of the non-verbal learning task (NVLT) and the verbal learning task (VLT), for the pre- and post-measurement of stroke patient UG20. The analyzed latency windows are marked with rectangles. The healthy group showed a tendency for a higher P3 amplitude in the post-compared to the pre-measurement during stimulus processing of the non-verbal task [t(9) = 1.91, p = 0.08, d = 1.21, pbootstrapping = 0.078; see Figure 5]. When comparing the N1 amplitude between pre- and post-test, no diﬀerences amplitudes [t(9) = 1.12, n.s., d = 0.71, pbootstrapping = 0.27] nor in N1 amplitudes [t(9) = 0.66, n.s., d = 0.42, pbootstrapping = 0.41]. A single-case-analysis (Crawford and Garthwaite, 2002) comparing the post-pre diﬀerence in P3 amplitudes during the FIGURE 5 \| Grand average event-related potentials (FCz and Pz) during the encoding phase of the non-verbal learning task (NVLT) and the verbal learning task (VLT), for the pre- and post-measurement of the healthy elderly group. The analyzed latency windows are marked with rectangles. FIGURE 6 \| Means of ERP amplitudes (FCz and Pz) during the encoding phase of the non-verbal learning task (NVLT) and the verbal learning task (VLT), for the pre- and post-measurement of stroke patient UG20. The analyzed latency windows are marked with rectangles. FIGURE 6 \| Means of ERP amplitudes (FCz and Pz) during the encoding phase of the non-verbal learning task (NVLT) and the verbal learning task (VLT), for the pre- and post-measurement of stroke patient UG20. The analyzed latency windows are marked with rectangles. Coherence Coherence analyses for the non-verbal task indicated a decrease in coherence between electrodes Cz and CPz during the non-verbal task for UG20. In the verbal learning task, coherence decreased as well (see Figure 7). For healthy controls, coherence during the non-verbal learning task between electrodes Cz and CPz showed a marginally signiﬁcant decrease after NF compared to pre-training values [t(9) = 1.92, p = 0.09, d = 1.21, pbootstrapping = 0.06]. For the other two electrode pairs, coherence did not diﬀer signiﬁcantly after training when compared to before training [Cz-Pz: t(9) = 1.75, p = 0.11, d = 1.11, Cz-Poz: t(9) = 0.45, p = 0.66, d = 0.28, pbootstrapping = 0.11]. For the verbal task, no diﬀerences in coherence were observed [Cz-CPz: t(9) = 0.06, p = 0.95, d = 0.04, pbootstrapping = 0.50, Cz-Pz: t(9) = 0.83, p = 0.43, d = 0.52, pbootstrapping = 0.35, Cz-Poz: t(9) = 1.48, p = 0.17, d = 0.94, pbootstrapping = 0.16]. Before and after neurofeedback training, UG20 was confronted with a verbal learning task and a non-verbal learning task. UG20 showed an improvement in the non-verbal learning task after neurofeedback training. The control group’s results mirrored those of UG20, as they showed a signiﬁcant improvement of performance in the non-verbal learning task as well. A single-case Crawford analysis indicated that UG20 improved his non-verbal memory performance as much after the training as the healthy elderly group. These results are well in line with studies demonstrating improved memory abilities after SMR neurofeedback training (Vernon et al., 2003; Hoedlmoser et al., 2008; Schabus et al., 2014; Kober et al., 2015b). Further, they support results of a previous study of our group showing improved memory performance after SMR neurofeedback in healthy young participants (Kober et al., 2015b). Based on these ﬁndings, we suggested that up-regulation of SMR leads to a reduction of sensorimotor interferences. This inhibition of sensorimotor interferences enhances stimulus processing and thereby leads to improved memory performance (Kober et al., 2015b). Thus, in our present study we found that these mechanisms could also be triggered in a stroke patient suﬀering from broad memory deﬁcits as well as in healthy elderly persons not aﬀected by memory deﬁcits. Coherence pp g Single-case analysis (Crawford and Garthwaite, 2002) for the non-verbal learning task showed that there was a signiﬁcant diﬀerence between the patient’s change in coherence between Cz and CPz and the healthy controls’ [t(9) = −1.871, p = 0.047, d = −1.18]: the decrease in UG20s coherence values was larger than the decrease in the healthy elderly. In the verbal learning task, according to single-case analysis, the decrease in coherence was also signiﬁcantly larger in UG20 than in healthy controls [t(9) = −2.080, p = 0.03, d = −1.32]. There were no signiﬁcant diﬀerences between pre–post coherence changes of UG20 and the controls in the other two electrode pairs and in the verbal learning task (all ps > 0.1, see Figure 7). Electrophysiological Results Event-Related Potentials Also, there was no signiﬁcant diﬀerence between the patient’s and the healthy participants’ change in N1 according to single-case analysis based on Crawford and Garthwaite (2002; t(9) = −0.016, p = 0.494, d = −0.016), indicating no signiﬁcant change in N1 in the patient. d = −0.003] between these values, thus indicating a tendency of amplitude increase of similar extent as observed in the controls. Also, there was no signiﬁcant diﬀerence between the patient’s and the healthy participants’ change in N1 according to single-case analysis based on Crawford and Garthwaite (2002; t(9) = −0.016, p = 0.494, d = −0.016), indicating no signiﬁcant change in N1 in the patient. Electrophysiological Results Event-Related Potentials amplitudes [t(9) = 1.12, n.s., d = 0.71, pbootstrapping = 0.27] nor in N1 amplitudes [t(9) = 0.66, n.s., d = 0.42, pbootstrapping = 0.41]. amplitudes [t(9) = 1.12, n.s., d = 0.71, pbootstrapping = 0.27] nor in N1 amplitudes [t(9) = 0.66, n.s., d = 0.42, pbootstrapping = 0.41]. The healthy group showed a tendency for a higher P3 amplitude in the post-compared to the pre-measurement during stimulus processing of the non-verbal task [t(9) = 1.91, p = 0.08, d = 1.21, pbootstrapping = 0.078; see Figure 5]. When comparing the N1 amplitude between pre- and post-test, no diﬀerences were found [t(9) = 0.82, n.s., d = 0.51, pbootstrapping = 0.36]. In the verbal task, there were neither pre–post diﬀerences in P3 pp g A single-case-analysis (Crawford and Garthwaite, 2002) comparing the post-pre diﬀerence in P3 amplitudes during the non-verbal task between the patient and healthy participants indicated no signiﬁcant diﬀerence [t(9) = −0.005, p = 0.498, Frontiers in Human Neuroscience \| www.frontiersin.org July 2016 \| Volume 10 \| Article 348 8 Shutting Down Sensorimotor Interferences Reichert et al. Regression analysis showed that during the neurofeedback training sessions, UG20s SMR activity increased linearly, indicating that he was able to voluntarily increase SMR activity. The control group also showed consistent increases in SMR power over the training runs averaged, indicating successful up-training of SMR activity. This ﬁnding is consistent with previous studies demonstrating the applicability of slow cortical potentials-, alpha- and theta-based neurofeedback paradigms in healthy older persons (Kotchoubey et al., 2000; Angelakis et al., 2007; Wang and Hsieh, 2013). A single-case Crawford analysis (Crawford and Garthwaite, 2002, 2004) comparing the regression slope of UG20 and the control group showed that there was no diﬀerence in learning ability between UG20 and the controls. These results support previous case studies showing that stroke patients are able to control their brain activity during neurofeedback training (Bearden et al., 2003; Cannon et al., 2010) and extend the evidence by showing that UG20 was equally able to up-regulate SMR activity as age-matched healthy controls. d = −0.003] between these values, thus indicating a tendency of amplitude increase of similar extent as observed in the controls. Frontiers in Human Neuroscience \| www.frontiersin.org DISCUSSION The present study investigated the applicability of SMR-based neurofeedback training in one stroke patient (UG20) and a healthy elderly control sample and assessed behavioral and electrophysiological eﬀects of the training. Results indicated that participants learned to increase SMR voluntarily during neurofeedback. After 10 sessions of SMR up-training, UG20 and the control group showed behavioral improvements in a non-verbal learning memory task, an increase in P3 amplitudes and a decrease in electrophysiological coherence during this task. Single-case Crawford analyses indicated that the changes observed in UG20 were as large as the changes in the age- matched controls, suggesting that UG20 beneﬁted as much from the training as the control group. These results will be discussed in more detail below. y y While UG20 as well as healthy elderly controls showed improvements in their non-verbal memory performance after SMR based neurofeedback training, no change in performance was observed in the verbal learning task. This ﬁnding contrasts with our previous study (Kober et al., 2015b), where we observed behavioral improvements in a verbal memory task after training. One reason for this discrepancy could lie in the nature of the applied tasks: while in our previous study, participants improved in a free-recall task of verbal material presented in a story-like format (subtest “construction” of the Visual and Verbal Memory Test; Visueller und verbaler Merkfähigkeitstest2 – Schelling and Schächtele, 2001), in the present study a recognition task with pseudo-words was used. As suggested in Kober et al. (2015b), we suppose that SMR up-training leads to an improved suppression of sensorimotor interferences, which facilitates the formation In a ﬁrst step, we assessed whether UG20 was able to increase SMR activity voluntarily through neurofeedback training. July 2016 \| Volume 10 \| Article 348 9 Reichert et al. Shutting Down Sensorimotor Interferences FIGURE 7 \| Coherence values (M and SE) in the SMR band (12–15 Hz) for the electrode pairs Cz-CPz, Cz-Pz and Cz-Poz for the healthy elderly group and stroke patient UG20. FIGURE 7 \| Coherence values (M and SE) in the SMR band (12–15 Hz) for the electrode pairs Cz-CPz, Cz-Pz and Cz-Poz for the healthy elderly group and stroke patient UG20. of deep semantic associations and therefore enhances later retrieval. The verbal task used in the present study, however, does not allow the formation of deep semantic associations as the items that should be memorized were pseudo-words. Frontiers in Human Neuroscience \| www.frontiersin.org DISCUSSION in line with previous ﬁndings in healthy people that indicated that SMR neurofeedback training leads to memory improvements (Vernon et al., 2003; Hoedlmoser et al., 2008; Gruzelier, 2014; Hofer et al., 2014; Schabus et al., 2014; Kober et al., 2015b). Of note, UG20 did not participate in other cognitive training and did not take medication while taking part in our study. Thus, we can exclude that the improvements we observed are due to these factors. However, we cannot entirely exclude the inﬂuence of other factors, for instance socialization on the changes in memory function. Some small performance declines could be observed in UG20 in two scales of the short-term memory assessment (CBTT forward task and CVLT List A). The reduced performance in these two tasks after neurofeedback training compared to the pre-assessment might be caused by reduced motivation or inattention, since an improvement in working memory performance (CBTT backward task) would not be possible if short-term memory performance in UG20 had declined seriously. For successful short-term memory performance, items have to be memorized and retrieved after a short delay. For successful working memory performance, items have to be memorized, retrieved and mentally transformed. Working memory refers to the structures and processes used for temporarily storing and manipulating information. Hence, an intact short-term memory can be regarded as a prerequisite for successful working memory performance. Therefore, we expected an enhancement of this component after training. The lack of a change in N1 in the control group could be due to large inter-individual variations in N1 amplitude observed in our study. Moreover, the comparatively small number of trials in our task might have contributed to this lack of an eﬀect on N1 amplitude. Furthermore, coherence analyses were carried out assessing electrophysiological coherence between the feedback region (electrode Cz) and parietal areas. In UG20, coherence between Cz and CPz decreased strongly after training compared to before and this change was apparent in both the verbal and the non- verbal learning task. This reduced coherence between motor and parietal areas indicates lower functional connectivity between these brain areas after training. Thus, activity in these regions was less synchronized after training, which could be a further indicator of a reduction of sensorimotor interferences achieved by SMR neurofeedback training. DISCUSSION In the control group, coherence in the non-verbal learning task was marginally decreased after training compared to pre-training, and there were no changes in coherence in the verbal learning task. Single-case analyses indicated that electrophysiological coherence during the verbal and non-verbal learning task decreased more strongly in UG20 than in the healthy participants, further corroborating that SMR training was eﬀective in UG20. Of note, compared to our previous results in healthy young participants (Kober et al., 2015b), the connectivity changes were less widespread in the stroke patient and the elderly persons as we could not observe changes in coherence between Cz and more posterior electrodes (POz, Pz). While completing the non-verbal and verbal learning tasks, our participants underwent 60-channel EEG measurements enabling us to assess ERPs during stimulus processing. In line with the behavioral results, in UG20 changes in EEG parameters after neurofeedback training were only apparent in the non- verbal learning task: in this task, he showed higher P3 amplitudes after training compared to the pre-measurements. N1 amplitude was also slightly higher in UG20 after training than before. In the healthy elderly group, a similar pattern was observed: analyses of ERPs showed a tendency for higher P3 amplitudes in the post-compared to the pre-measurements during presentation of correctly identiﬁed target stimuli. However, there was no change in N1 amplitude after training in the control group. Single-case analyses showed that the pre–post change in P3 amplitude in UG20 was not signiﬁcantly diﬀerent from the pre-post-change observed in healthy controls. Further, the change in N1 amplitude visible in UG20 was not larger than the change observed in the healthy elderly group. Thus, while neither UG20 nor the control group showed a reliable diﬀerence in N1 amplitude after training, they showed a tendential increase in P3 amplitude after training. The observed increase in P3 amplitude is consistent with previous results (Egner and Gruzelier, 2001; Kober et al., 2015b) and indicates more intensive stimulus processing after SMR up- training (Kok, 2001; Polich, 2007). In the context of memory tasks, increased P3 amplitudes are regarded as an indicator of more successful encoding, which facilitates later retrieval and recognition (Polich, 2007). Thus, this ﬁnding ﬁts well with the behavioral improvements in the task apparent in UG20 and the control group. DISCUSSION This might be a reason why this task was not suitable to capture changes induced by SMR neurofeedback training. Still, the question arises why participants then improved in the non-verbal learning task in our study. In contrast to the verbal learning task with pseudo-words, the non-verbal learning task contains partially associative ﬁgures (e.g., a symbol resembling a house) that could allow for the formation of semantic associations. Another possible explanation for our ﬁndings is that non-verbal memory might be aﬀected by aging earlier than verbal memory (Jenkins et al., 2000; Murre et al., 2013). This might lead to the non-verbal learning task being more sensitive to performance improvements induced by SMR neurofeedback. The results of the neuropsychological test battery, which was performed by UG20 before and after neurofeedback training, support this assumption. UG20 improved his performance in the non-verbal short-term memory task of the VVM2 (subscale “city map 1”), but not in the verbal subtest “construction” of the VVM2 when comparing pre- and post-assessment. However, the healthy elderly group did not show performance diﬀerences between the verbal and non-verbal learning task during the pre-assessment, which is in contrast to the hypothesis that non-verbal memory might be aﬀected by aging earlier than verbal memory. Further studies applying more varieties of memory tasks to healthy elderly controls are necessary to elucidate the speciﬁcity of SMR training eﬀects on verbal and non-verbal memory functions and on the formation of semantic associations. The results of the neuropsychological test battery suggest that SMR neurofeedback training had mainly eﬀects on memory functions in stroke patient UG20. Before the start of the neurofeedback training, UG20 showed severe memory deﬁcits (T-scores < 40). After SMR neurofeedback, UG20 showed a higher performance in diﬀerent memory functions compared to the pre-assessment. For instance, working memory performance as assessed with the CBTT backward task was below average during the pre-assessment. After SMR neurofeedback training, performance in the CBTT backward task was in a normal range. Short-term memory performance (VVM2 “city map 1” and CVLT List B) was also far below average before the start of the neurofeedback training. During the post-assessment, performance in these short-term memory tasks was only slightly lower than the normal range. Attentional and executive functions did not change due to SMR neurofeedback training. This result is July 2016 \| Volume 10 \| Article 348 10 Shutting Down Sensorimotor Interferences Reichert et al. Frontiers in Human Neuroscience \| www.frontiersin.org DISCUSSION N1 is has also been related to cognitive processing, especially in attention tasks involving expectancy eﬀects and in tasks assessing short-term memory (Golob and Starr, 2000; Herrmann and Knight, 2001; Correa et al., 2006; Fu et al., 2008). The behavioral and electrophysiological results of our study partially replicate a previous study of our group on SMR neurofeedback in healthy young participants (Kober et al., 2015b). In this study, healthy young participants showed improved verbal memory, higher P3 and N1 amplitudes and decreased coherence between motor areas and parietal-occipital brain areas after SMR up-training. Based on these ﬁndings, we suggested that SMR up-training might lead to an improved shutting-down of sensorimotor interferences after training, which in turn results in enhanced stimulus processing (Kober et al., 2015b). In a recent study, we could show that stroke patients can beneﬁt from SMR-based neurofeedback training, as they showed improved memory performance after training (Kober et al., 2015a). The present study set out to explore whether the observed memory improvements in stroke patients might also be related to the mechanism of shutting-down of sensorimotor interferences we proposed for younger participants (Kober et al., 2015b). As there is plenty of evidence showing that both resting-state EEG and task-related EEG activity change across the lifespan (Polich, 1997; Klimesch, 1999; Babiloni et al., 2006; Cummins and Finnigan, 2007; Rossini et al., 2007), in the present study we also set out to further investigate these processes in an elderly sample. Our results are in line with an assumed enhanced shutting-down of sensorimotor intereferences, as we observed a similar pattern of electrophysiological results as in Kober et al. (2015b): our ERP results provide evidence that up- regulation of SMR is related to enhanced stimulus processing of task relevant information. Furthermore, we observed a reduced July 2016 \| Volume 10 \| Article 348 11 Shutting Down Sensorimotor Interferences Reichert et al. coherence between motor areas (Cz) and more parietal areas which are more related to visual processing. As proposed by Sterman (1996), we assume that motor activity might disengage cortical visual processing areas, resulting in a compromising eﬀect on information processing. Thus, we hypothesize that up-regulation of SMR activity is associated to a reduction of motor interferences and reduces the somatosensory information ﬂow to motor areas. According to our interpretation, this “shutting down of sensorimotor interferences” in turn leads to an enhanced information processing (Kober et al., 2015b). FUNDING This work was supported by the European STREP Program – Collaborative Project no. FP7-287320 – CONTRAST. DISCUSSION Our results indicate that SMR up-training could enhance the suppression of sensorimotor interferences not only in healthy young, but also in elderly participants and in a stroke patient. These results are also in line with a previous study of our group, in which age was not a signiﬁcant predictor of the ability to regulate SMR in a sample with a wide age-range (Reichert et al., 2015) and extend this ﬁnding by showing that SMR training has similar behavioral and electrophysiological eﬀects in young and elderly participants and even in a stroke patient. have facilitated the implementation of neurofeedback training tremendously. In the future, even the independent realization of neurofeedback training at the homes of patients will be possible, as there are already suﬃciently small ampliﬁers and easy-to-use headsets that can be set up by caregivers or patients themselves. Thus, neurofeedback provides an enormous potential for home- based rehabilitation that could add to the currently utilized traditional cognitive training programs. ACKNOWLEDGMENTS Possible inaccuracies of information are under the responsibility of the project team. The text reﬂects solely the views of its authors. The European Commission is not liable for any use that may be made of the information contained therein. The authors are grateful to Katharina Farveleder and Margit Krenn for data acquisition. None of the authors have potential conﬂicts of interest to be disclosed. AUTHOR CONTRIBUTIONS JR: participated in the design of the study and data acquisition, analyzed, and interpreted data; and prepared the manuscript. SK: participated in the study design, analyzed data, and critically revised the manuscript. DS: acquired data, contributed to study design, carried out patient recruitment and the patient’s clinical assessment. PG: contributed to study coordination, patient recruitment, data collection, and medical care of the patient. GW: participated in the study design and coordination, performed statistical analysis and critically revised the manuscript. CN: participated in conception and supervision of the study, interpreted data and contributed to manuscript revision. All authors read and approved the ﬁnal manuscript. CONCLUSION In the present case-study, we could show that a stroke patient and healthy elderly controls were able to self-regulate their SMR activity during 10 sessions of SMR neurofeedback training. Moreover, observed behavioral improvements after training in a non-verbal learning task were accompanied by increased P3 amplitudes and a decreased coherence between motor and parietal areas during this task. These results oﬀer support for the hypothesis that SMR neurofeedback enhances the inhibition of sensorimotor interferences, thereby facilitating stimulus processing and the formation of deep semantic associations. Furthermore, the improvements we observed in the UG20 were not smaller than those in the healthy controls, suggesting that neurofeedback might be a suitable tool for stroke patients and should be investigated further in future studies. Research on neurofeedback for stroke rehabilitation is of special importance as positive eﬀects of traditional cognitive trainings in this domain remain disputed (das Nair and Lincoln, 2007; Hoﬀmann et al., 2010). Also, the technological developments in the last years REFERENCES Correa, Á., Lupiáñez, J., Madrid, E., and Tudela, P. (2006). Temporal attention enhances early visual processing: a review and new evidence from event- related potentials. Brain Res. 1076, 116–128. doi: 10.1016/j.brainres.2005. 11.074 Angelakis, E., Stathopoulou, S., Frymiare, J. L., Green, D. L., Lubar, J. F., and Kounios, J. (2007). EEG neurofeedback: a brief overview and an example of peak alpha frequency training for cognitive enhancement in the elderly. Clin. 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https://openalex.org/W3042527134	https://zenodo.org/records/3982331/files/IJRR0015.pdf	English	null	Malnutrition Related Diabetes Mellitus in Indian Population	Zenodo (CERN European Organization for Nuclear Research)	2,020	cc-by	4,029	Malnutrition Related Diabetes Mellitus in Indian Population Suku Sneha Fortis Hospital, Sector 62, Noida, Uttar Pradesh - 201301 Suku Sneha Fortis Hospital, Sector 62, Noida, Uttar Pradesh - 201301 young population cloaking the prospective effects of genetic, environmental(acquired) and behavioural factors(urban migration, lifestyle changes and rising living standards). Although obesity has been a recognised risk factor for diabetes, malnutrition has shown potential effects in development of insulin dependent diabetes mellitus. WHO recognised malnutrition as distinct entity and had incorporated Malnutrition Related Diabetes Mellitus as a category in classification of diabetes in 1985. [3,8] The present review aims to highlight the impact of malnutrition on diabetes mellitus in Indian population and its relationship with the diabetes mellitus. The literature search strategy involved a retrieval of relevant articles from Google and PubMed database using appropriate search terms. International Journal of Research and Review Vol.7; Issue: 7; July 2020 Website: www.ijrrjournal.com E-ISSN: 2349-9788; P-ISSN: 2454-2237 International Journal of Research and Review Vol.7; Issue: 7; July 2020 Website: www.ijrrjournal.com E-ISSN: 2349-9788; P-ISSN: 2454-2237 International Journal of Research and Review Vol.7; Issue: 7; July 2020 Website: www.ijrrjournal.com E-ISSN: 2349-9788; P-ISSN: 2454-2237 Review Article ABSTRACT Malnutrition related diabetes mellitus (MRDM) is rare type of diabetes associated with long term malnutrition and is not uncommon in India. Diabetes mellitus is the most common endocrine disorder of multifactorial aetiology and has been linked to both chronic under nutrition and obesity. Malnutrition is often related to deficient micro and macro nutrients, unhealthy behaviours and low socioeconomic status. Various clinical studies have underlined the relationship between malnutrition and diabetes mellitus. In the present review, we mainly explored malnutrition related diabetes mellitus in Indian population. Maternal under nutrition during pregnancy causes a decrease in the fetal intrauterine development rate known as intrauterine growth retardation (IUGR). Malnutrition majorly causes persistent insulin deficiency, glucose intolerance and insulin resistance and there by increases the risk of diabetes in Indian population. In this study we have also discussed the pathogenesis of MRDM and their management like self- dietary management and evidence based individualized nutritional therapy. CLASSIFICATION OF MRDM: Malnutrition is a nutritional condition which results from the imbalance of energy, protein and nutrients, and poses effects in the form of composition and function of body tissues. [9] MRDM is characterized by insulinopenia, insulin resistance, [10] hyperglycemia and partial failure of the beta-cells in the pancreas. [11] The American Diabetes Association reclassified MRDM into 2 subgroups for research purposes. Keywords: Malnutrition related diabetes mellitus (MRDM), under nutrition, obesity, Diabetic risk, intrauterine growth retardation (IUGR), insulin resistance and glucose tolerance. The American Diabetes Association reclassified MRDM into 2 subgroups for research purposes. INTRODUCTION There has been an exponential increase in prevalence of Diabetes globally and if the current scenario prevails then the cases are expected to double by 2050 with estimated 79.4 million affected individuals in India alone. [2] This could be attributed to the current Indian demographic constituting 2-subgroups of MRDM include: 1. Fibrocalcific or fibrocalculous pancreatic diabetes (FCPD): [6,11] It is characterized socioeconomic background of poverty and malnutrition International Journal of Research and Review (ijrrjournal.com) Vol.7; Issue: 7; July 2020 136 Suku Sneha et.al. Malnutrition related diabetes mellitus in Indian population radical and peroxide removal and environmental agents such as organic nitrides or cytochrome p450 causes pancreatic injury in the form of endocrine B cell damage and acinar and ductal damage manifesting clinically as diabetes, calculi and pain abdomen) plays key role and was demonstrated by restriction fragment length polymorphism (RFLP). [6] Although heavy alcohol consumption is also a major role in the pancreatic damage in older individuals. [5] FCPD develops mainly due to chronic calculous pancreatopathy and hyperparathyroidism. Pancreatic calculi are usually large multiple and intraductal. Marked ductal dilation and fibrosis usually occurs due to inflammatory changes. [12] FCPD is associated with increased risk of pancreatic carcinoma. [12] and it mostly observed in young adults mainly below the age of 30 years. It is recognized by the clinical evidence of malnutrition, insulin-requirement for control, ketosis-resistance, and radiological evidence of pancreatic calcification and the presence of exocrine pancreatic dysfunction. and it mostly observed in young adults mainly below the age of 30 years. It is recognized by the clinical evidence of malnutrition, insulin-requirement for control, ketosis-resistance, and radiological evidence of pancreatic calcification and the presence of exocrine pancreatic dysfunction. p y 2. Protein-deficient pancreatic diabetes (PDPD)/ protein deficient diabetes mellitus (PDDM): PDPD mostly observed in young malnourished individuals in developed countries similar to FCPD but differs from FCPD in absence of clinical and radiological evidence of pancreatic dysfunction and relative to insulin resistance. [12] Having a clinical feature of low BMI, malnutrition and often growth retardation. [12] 2. Protein-deficient pancreatic diabetes (PDPD)/ protein deficient diabetes mellitus (PDDM): PDPD mostly observed in young malnourished individuals in developed countries similar to FCPD but differs from FCPD in absence of clinical and radiological evidence of pancreatic dysfunction and relative to insulin resistance. [12] Having a clinical feature of low BMI, malnutrition and often growth retardation. CONSIDERATIONS OF MRDM: CONSIDERATIONS OF MRDM: Globally, many factors are involved in the etio-pathology of malnutrition related diabetes mellitus are malnutrition, infection, food habits like increased consumption of cassava, immunological destruction of pancreatic beta cells. But the exact etio- pathogenesis of MRDM remains controversial. [13] MRDM is mostly develops in poor populations. The individuals are mostly less than 40 years of age, are underweight or cachectic and exhibit hair and skin changes and parotid gland enlargement typical of severe malnutrition. [5] Fibrocalcific or fibrocalculous pancreatic diabetes (FCPD): The radiological evidence of pancreatic calcification or ultrasound signs of ductal dilatation favour pancreatic disease as the cause of the diabetes. [14] Shrunken pancreas shows extensive fibrosis involving the acinar and islet tissues. Exocrine pancreatic and B cell dysfunction is involved in the pathogenesis of FCPD. [5] Genetic susceptibility (undernutrition, deficiency of micronutrients and sulfur containing amino acids, decreased free INTRODUCTION [12] Protein-deficient pancreatic diabetes (PDPD)/ protein deficient diabetes mellitus (PDDM): The aetiology of PDPD has remained a subject of debate. Several months and years of survival without treatment after the onset of diabetes, indicates resistance to ketoacidosis. Ketosis resistance(liver fails to reduce glucose production in response to insulin) due to subnormal B cell function further leads to insulin interruption. [5] It shows shrunken fibrotic pancreas without calculi and polymorpho-nuclear and mononuclear inflammatory cell infiltration in addition to extensive fibrosis. [5] Malnutrition with deficiency of micronutrients initiates the functional impairment of the pancreatic B cell. [6] CHRONIC UNDERNUTRITION AND DIABETES IN INDIAN POPULATION: Diabetes mellitus was strongly associated with the poverty and malnutrition particularly under nutrition has remained a major health challenge in children in low- income developing countries. [15,16] Previous retrospective study demonstrated the association between chronic under nutrition and diabetes; reported that protein-energy malnutrition (PEM) majorly causes 137 International Journal of Research and Review (ijrrjournal.com) Vol.7; Issue: 7; July 2020 Suku Sneha et.al. Malnutrition related diabetes mellitus in Indian population persistent insulin deficiency, glucose intolerance and insulin resistance. [4] The condition is characterized by the presence of chronic pancreatitis and was first defined in Indonesia in 1959. [4] Clinical studies have demonstrated the mechanisms underlying the feature of malnutrition is impaired insulin secretion, as low circulating insulin levels and inadequate insulinotropic responses to glucose, glucagon, as well as amino acids. [7] Indians typically develop diabetes at younger ages than Europeans and are predicted to reach a harmful threshold of metabolic load earlier, increase in BMI in Indian populations elevate diabetes risk to a greater extent than European populations. [17] Previous determined that Indians have a higher proportion of fat mass in their body weight relative to Europeans. Therefore, we can highlight several ways in which Europeans and Indians differ in their size, shape, and body composition which further increase the risk of diabetes. [18] Beyond this greater ratio of fat mass to lean mass, Indians also have greater metabolic sensitivity to adipose tissue than other ethnic groups. [19] Reduced maternal malnutrition during pregnancy increases the risk of diabetes in offspring. Specifically in Indians, shorter maternal stature and lower BMI are associated with lower birth weight. [20] Previous study noticed that long term influence of maternal micronutrient deficiencies on diabetes risk and is demonstrated by an association between low maternal B12 during pregnancy and insulin resistance in the offspring at 6 years, exacerbated by high folate levels. [21] A recent study in rural areas from Pune has highlighted low intake vitamin B12 and functional folate deficiency as maternal dietary determinants of low birth weight, mediated through their impact on maternal homocysteine levels. [22] Although the risk of diabetes in south Asians may be explained in terms of a reduced metabolic capacity that renders them particularly susceptible to the i d b li l d i d i h h lifestyles that track urbanization, or migration to higher-income settings. [23] or Prenatal factors: Malnutrition and low protein diet: Various studies described the link between maternal malnutrition, low protein diet, and T2DM. Reductions in total food intake during pregnancy or early postnatal life lead to decreased glucose tolerance and diabetes in the offspring. [15] A study conducted by Orozco-Sol´ıs et al analysed that low protein diet during pregnancy and lactating may cause permanent altered hypothalamic expression of genes in rat offspring; these genes involved in insulin signalling and lipid and glucose metabolism, which may outline the metabolic diseases. [24] Maternal under nutrition during pregnancy causes a decrease in the fetal intrauterine development rate known as intrauterine growth retardation (IUGR). [25] In IUGR, the developing fetus engages different mechanisms include increased sensitivity to insulin for more efficient glucose utilization, increased gluconeogenesis rates in liver, decreased muscle sensitivity to insulin, and abnormal development of the pancreas. [25] Under nutrition during pregnancy affects the methylation and alters the activity of many genes that control hepatic and pancreatic function. [15] Low protein diet during pregnancy may lead to increased oxidative stress, fibrosis, decreased HNF4a expression, defected mitochondriogenesis, and mitochondria dysfunction, and increased cell differentiation instead of proliferation was found in β-cell of adult animal offspring. These may cause β-cell dysfunction and consequently increase the incidence of T2DM in postnatal life. [26] Over nutrition: Over nutrition: A study done by Maurer and Reimer in brown fat tissue of Wistar rats in 35-day- old offspring has noticed that high protein diet during pregnancy and lactating may cause increased resistin and IL-6 mRNA levels, which are involved in pathogenesis International Journal of Research and Review (ijrrjournal.com) Vol.7; Issue: 7; July 2020 138 Suku Sneha et.al. Malnutrition related diabetes mellitus in Indian population of insulin resistance. [27] Most of human and animal studies have founded that high fat diet may cause obesity and insulin resistance. [28] A study conducted by Masuyama and Hiramatsu et al noticed that mice offspring exposed to high fat diet during pregnancy developed insulin resistance and hyperlipidemia at 24wks of age, which was associated with altered levels of leptin in adipose tissue. [29] BMI and early age of onset might raise suspicion of type 1 diabetes, however, it can be differentiated by decreased levels of islet specific antibodies, lower risk of ketoacidosis on insulin withdrawal and male preponderance in MRDM. Key distinctive feature of MRDM from classic type 2 diabetes is defect in insulin secretion as opposed to resistance to peripheral insulin. Postnatal factors: [33] Population-based studies from India have documented the association between overconsumption of refined grains that lack dietary fibers and vital micronutrients and saturated fats with insulin resistance and poor glycemic control, whereas intake of fiber-rich foods, fruits and vegetables has shown a negative correlation. [34] study conducted in Indian participants with type 2 diabetes on oral hypoglycemic medications demonstrated the success of a structured diet plan comprising a modified traditional Indian to lower GI and glycemic load (GL) diet. [35] Multivitamin supplementation may be Postnatal factors: Malnutrition during postnatal life may lead to decreased glucose tolerance and increases the risk of diabetes in the offspring. [15] Socioeconomic status has significant effect on diabetic risk. Some studies have demonstrated the link between low childhood socioeconomic status and type 2 diabetes. [30] Poor nutrition, unhealthy behaviours, and limited access to material goods and limited socioeconomic opportunities may contribute to altered body composition in later life, which might explain the relationship between childhood socioeconomic position and metabolic disorders in adult. A recent study suggested that Under nutrition during childhood has been found to be associated with an increased type 2 diabetes risk in adulthood. [31] According to American Diabetes Association, self-dietary management plays key role in the prevention of diabetes. [1] A study conducted in Indian men with impaired glucose tolerance (IGT) has suggested that healthy lifestyle adjustments comprising a calorie-restricted diet were associated with a decreased incidence of diabetes. [32] Balanced diet and regular physical activity improves insulin sensitivity and beta cell preservation. [32] The major goals of dietary strategies was to restrict fat to > 30% of calorie intake and reduce intake of high-GI carbohydrates such as sugar, flavored beverages and high-calorie snacks. [33] A 12-month prospective study from India reported the success of a dietician-led evidence-based individualized nutritional therapy for macronutrient intake and restricted intake of sugars. Participants with T2DM, randomized to nutritional therapy, achieved significant improvements in HbA1c in all lipid parameters, especially triglyceride levels. [33] Population-based studies from India have documented the association between overconsumption of refined grains that lack dietary fibers and vital micronutrients and saturated fats with insulin resistance and poor glycemic control, whereas intake of fiber-rich foods, fruits and vegetables has shown a negative correlation. [34] study conducted in Indian participants with type 2 diabetes on oral hypoglycemic medications demonstrated the success of a structured diet plan comprising a modified traditional Indian to lower GI and glycemic load (GL) diet. [35] Multivitamin supplementation may be A 12-month prospective study from India reported the success of a dietician-led evidence-based individualized nutritional therapy for macronutrient intake and restricted intake of sugars. Participants with T2DM, randomized to nutritional therapy, achieved significant improvements in HbA1c in all lipid parameters, especially triglyceride levels. DISCUSSION WHO introduced widely accepted classification of Diabetes mellitus in 1980 which included type 1 and type 2 diabetes. It was later modified in 1985 post study group report and renamed type 1 and type 2 diabetes as Insulin Dependent Diabetes Mellitus and Non-Insulin Dependent Diabetes Mellitus and introduced Malnutrition related diabetes mellitus as a new class. In 1999, International Workshop reviewed characteristic of diabetes in malnourished population. It established the influence of malnourishment exposure leading to diabetes however the evidence provided was insufficient and not convincing, hence MRDM class was deleted from WHO classification. Later in 2006 WHO report, subtype of MRDM which is Fibro calculous Pancreatic Diabetes was classified as disease of exocrine pancreas which may lead to diabetes and it remains till date as per 2019 WHO classification of Diabetes. 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There is a need for prospective multicenter study in India to elucidate the pathogenetic mechanism of malnutrition related diabetes with consideration to predisposing factors such as insufficient nutrition in intrauterine or early postnatal environment causing genetic predisposition to fragile beta cell mass. 5. Abdulkadir J. Malnutrition-related Diabetes Mellitus in Africa. INT J DIAB DEV COUNTRIES. 1993;13:22-28. 6. Taksande A, Taksande B, Kumar A, Vilhekar K. Malnutrition-related diabetes mellitus. Journal of Mahatma Gandhi Institute of Medical Sciences. 2008; 13(ii):19-24. 7. Al-Amin A, Ahrén B. Relation Between Malnutrition and Development of Diabetes Mellitus. International Journal of Pancreatology,. 2019;26(3):125-130. DIAGNOSIS Malnutrition related diabetes mellitus was noted to be similar to other clinical syndromes described worldwide such as Tropical Diabetes, J type Diabetes, Z type Diabetes, Type 3 or M Type Diabetes and Phasic insulin dependent diabetes. [37,38] Criteria for identification of malnutrition related diabetes mellitus included the disease onset 10-30 years of age, poor socioeconomic status or history of childhood malnutrition, BMI <18Kg/m2, blood glucose >200mg/dl or 11.1mmol/L, absence of ketosis on insulin withdrawal and insulin requirement of >1.5U/kg/day. Clinical features of MRDM appear to overlap with features of type 1 and type 2 diabetes mellitus on several occasions. Low International Journal of Research and Review (ijrrjournal.com) Vol.7; Issue: 7; July 2020 139 Suku Sneha et.al. Malnutrition related diabetes mellitus in Indian population recommended in deficiencies in vitaminA, C and E, thiamine, pyridoxine, B12 and biotin, which are common patients with diabetes. Particularly treatment with metformin requires B12 and folic acid supplements, as prolonged use of metformin decreases the absorption of these vitamins. [36] postnatal life results in decreased glucose tolerance and increases the risk of diabetes in the offspring. Poor nutrition, unhealthy behaviours, and limited socioeconomic opportunities may contribute to altered body composition, which might describe the relationship between childhood socioeconomic position and metabolic disorders in adult. In this review, we concluded the association between malnutrition and diabetes mellitus. Malnutrition majorly causes persistent insulin deficiency, glucose intolerance and insulin resistance and there by increases of risk of diabetes in Indian population. CONCLUSION The review of various studies established the relationship between malnutrition and diabetes mellitus. Malnutrition during pregnancy or early 8. a a as ir S. . Diabetes and malnutrition / by Jasbir S. Bajaj. World 8. a a as ir S. . Diabetes and malnutrition / by Jasbir S. Bajaj. 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The Elevated Susceptibility to Diabetes in India: An Evolutionary Perspective. Frontiers in Public Health. 2016;4. 17. Bhargava SK, Sachdev HS, Fall CHD, Osmond C, Lakshmy R, Barker DJP, et al. Relation of serial changes in childhood body-mass index to impaired glucose tolerance in young adulthood. N Engl J Med (2004) 350(9):865–75. doi:10.1056/NEJMoa035698 26. Jiang X, Ma H, Wang Y, Liu Y. Early Life Factors and Type 2 Diabetes Mellitus. Journal of Diabetes Research. 2013;2013:1- 11. 27. A.D.MaurerandR. A. Reimer “Maternal consumption of highprebiotic fibre or - protein diets during pregnancy and lactation differentially influences satiety hormones and expression of genes involved in glucose and lipid metabolism in offspring in rats ”The ritish ournal of Nutrition, vol. 105, no. 3,pp. 329–338, 2011. doi:10.1056/NEJMoa035698 18. Deurenberg P, Deurenberg-Yap M, Guricci S. Asians are different from Caucasians and from each other in their body mass index/body fat per cent relationship. 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Insights from the Diabetes in India Nutrition Guidelines Study: adopting innovations using a knowledge transfer model. Top Clin Nutr. 2017;32:69–86. ****** 142 International Journal of Research and Review (ijrrjournal.com) Vol.7; Issue: 7; July 2020 International Journal of Research and Review (ijrrjournal.com) Vol.7; Issue: 7; July 2020
https://openalex.org/W2767168938	http://journals.rudn.ru/polylinguality/article/download/17051/15044	Russian	null	LINGUISTIC SITUATION IN ISLAMIC COMMUNITY OF RUSSIA	Vestnik Rossijskogo universiteta družby narodov. Seriâ Voprosy obrazovaniâ: âzyki i specialʹnostʹ	2,017	cc-by	2,816	2017 Vol. 14 No. 3 369—374 http://journals.rudn.ru/education-languages DOI 10.22363/2312-8011-2017-14-3-369-374 УДК 81 DOI 10.22363/2312-8011-2017-14-3-369-374 УДК 81 ЯЗЫКОВАЯ СИТУАЦИЯ В РОССИЙСКОМ МУСУЛЬМАНСКОМ СООБЩЕСТВЕ О.И. Александрова Российский университет дружбы народов ул. Миклухо-Маклая, д. 10, корп. 2А, Москва, Россия, 117198 В статье рассматривается языковая ситуация в российском мусульманском сообществе. Объектом исследования является мусульманский дискурс как один из религиозных субдис- курсов, характеризующихся наличием диглоссии, под которой понимается одновременное сосуществование двух языков, применяемых в разных функциональных сферах. В российской мусульманской полиэтнической среде наблюдается ситуация полиглоссии, когда в коммуни- кации используются не только сакральный арабский язык и национальный язык верующих, но и русский язык, традиционно имеющий статус официального языка общения всех мусуль- ман Российской Федерации. Использование верующими одного из нескольких языков обу- словлено типом коммуникации в религиозном дискурсе. Так, общение с Богом — чтение мо- литв и священных текстов — осуществляется на арабском языке, как предписывает Коран. Личная и коллективная коммуникация может проходить как на национальном, так и на рус- ском языке в зависимости от уровня владения коммуникантами указанными языками. Мас- совая коммуникация, которая включает общение на институциональном уровне, осуществля- ется, как правило, на русском языке с помощью средств массовой информации. Русский язык, традиционно используемый духовными управлениями мусульман, на наш взгляд, способству- ет консолидации российского этнополитического и полиэтнического мусульманского сообще- ства, отличающегося терпимостью и стремлением к миру. Ключевые слова: исламский дискурс, диглоссия, полиглоссия, билингвизм, полиэтническая среда, коммуникативное пространство Российское мусульманское сообщество В связи с активизацией процессов исламизации и реисламизации, наблюда- ющихся не только в России, но и во всем мире, повлекших за собой изменения в общественно-политической ситуации, возникает острая необходимость изуче- ния особенностей ислама, в том числе его дискурсивных параметров. Специфи- ческими в нем по сравнению с другими религиями будут признаны не только ценности и ключевые концепты, но и коммуникативные характеристики, кото- рые, на наш взгляд, заслуживают особого внимания. Участниками исследуемой нами религиозной коммуникации являются члены мусульманской общины, так называемой уммы (от арабского — ﺃ ﱠﻣ ﺓнация, народ, — ﺃ ﱠﻣ ﺓ ﻤﺤ ﱠﻣﺪобщина Мухаммеда, мусульмане [1]), насчитывающей в Российской 369 LANGUAGES IN CONTACT: THEORY AND PRACTICE LANGUAGES IN CONTACT: THEORY AND PRACTICE Александрова О.И. Вестник РУДН. Серия: Вопросы образования: языки и специальность. 2017. Т. 14. № 3. С. 369—374 Федерации по разным оценкам от 16 до 20 миллионов человек. Мусульманская община очень разнородна по своему составу, что обусловлено ее многонацио- нальностью и принадлежностью ее представителей к различным направлениям ислама. Большая часть мусульман Российской Федерации причисляет себя к сун- нитам (сторонникам религиозно-правовой школы ислама, признающей досто- верность канонических сборников хадисов — жизнеописаний пророка Мухам- мада) различных толков. Мусульмане Поволжья, внутренней России и Сибири придерживаются суннитского мазхаба ханафитского толка, а мусульмане Север- ного Кавказа — шафиитского (направления суннизма различаются ритуальными, бытовыми и социальными правилами). Кроме того, в России есть сунниты хан- балитского и маликитского мазхаба, шииты, салафиты и ваххабиты. С точки зре- ния возрастного состава мусульманская община наполовину состоит из молоде- жи (десять лет назад мусульманская община почти наполовину (48,8%) состояла из молодежи (до 40 лет)) [2. С. 26], по данным социологической службы амери- канского института по исследованию общественного мнения Pew Forum on Religion and Public Life, к 2013 году около половины мусульман — люди моложе 30 лет. Этнический состав российской мусульманской общины разнообразен: ее пред- ставляют татары, башкиры, чеченцы, дагестанцы, кабардинцы, черкесы, балкар- цы, аварцы, кумыки, ингуши и другие народы и народности [2. С. 27]. Кроме «этнических» мусульман в умме начинают формироваться новые сообщества из тех этносов, которые ранее исповедовали иные религии: в ислам обращаются славяне, евреи, народы Сибири и Крайнего Севера и др., так называемые «ново- обращенные», «русские мусульмане» и т.п. [3]. Часть представителей указанных этнических групп относится к билингвам, владеющим в равной (или неравной) степени национальным и русским, нацио- нальным и арабским или русским и арабским языками. Российское мусульманское сообщество Довольно большая часть мусульман (главным образом городское население) является русскоязычной и плохо владеет национальным языком, интересуясь при этом исламом, историей исламской цивилизации и национальной историей [2. С. 27], и лишь малая часть, наиболее образованная, владеет несколькими языками — национальным, русским и арабским (как вариант — национальным, русским и турецким), в таком случае речь идет о многоязычии. Комбинация языков, которыми владеют мусульмане, может варьироваться. Например, в сельской местности мусульманин может вла- деть национальным и инонациональным, частично русским и арабским языками (например, аварским, кумыкским, русским, арабским). При этом очень важно разграничивать билингвизм/многоязычие за пределами религиозного общения и в его границах. Билингвизм российских мусульман вне религиозного общения может быть национально-русским, русско-национальным, национально-инонациональным [4. С. 30—31], при этом под билингвизмом понимается «совершенное владение двумя языками, умение в равной степени использовать их в необходимых усло- виях общения» [5. С. 120]. Здесь должна быть сделана оговорка: так как за русским языком закреплен статус государственного языка и языка межнационального общения народов России, функционально используемые билингвами языки не ЯЗЫКОВЫЕ КОНТАКТЫ: ТЕОРИЯ И ПРАКТИКА 370 Aleksandrova O.I. RUDN Journal of Language Education and Translingual Practices, 2017, 14 (3), 369—374 вполне равноправны. Несмотря на разнообразие социально-коммуникативных систем, сложившихся на разных территориях России, активизацию процессов повышения национального самосознания в национальных республиках, статус русского языка как государственного остается более высоким по сравнению с национальными. Следовательно, в данном случае целесообразно говорить о ди- глоссии, при которой говорящий осознанно выбирает язык коммуникации. Функ- циональная закрепленность языков значительно усиливается в пределах религи- озного дискурса. Типы коммуникации и языковая закрепленность В условиях религиозного общения использование разных языков, как прави- ло, соответствует определенному типу коммуникации — гиперкоммуникации, личной, коллективной или массовой коммуникации. Арабский язык в мусульманстве признается сакральным, так как представля- ет собой код, на котором в священном Коране запечатлены истины, переданные Богом пророку Мухаммаду. Перевод Корана на другие языки не желателен во избежание инотолкования, хотя неоднократно предпринимались попытки его перевода (точнее, перевода смыслов Корана, так называемые тафсиры) на разные языки, включая русский, татарский, кумыкский, чеченский, ингушский и др. В аспекте дискурсивного исследования арабский язык является основным сред- ством гиперкоммуникации [6. С. 20], т.е. общения со Всевышним, осуществляе- мого через молитву и чтение священных книг. В исламе знакомство с Кораном и обращение к Богу в соответствии с мусульманскими канонами требует от верую- щего знания арабского языка, отстоящего от национальных языков мусульман России и с точки зрения исторического родства, и с точки зрения типологии. Кроме того, сложность представляет графика арабского языка, затрудняющая самостоятельное чтение священных текстов. Следовательно, содержание «са- крального слова как воплощения Божественной сущности» [6. С. 20] может быть затемненным для мусульманина, не владеющего арабским, и тогда для него как участника дискурсивной ситуации более значимыми становятся экстралингви- стические факторы дискурсивного события и его тональность. В случае неспособности верующего подобрать арабоязычную «формулу», со- ответствующую коммуникативному событию, или в иных случаях (например, при внутренней молитве) в качестве средства коммуникации используется родной язык. Таким образом, большое значение приобретает религиозное образование верующего и уровень владения арабским языком. Национальный язык (в том числе русский) используется в личной и коллек- тивной религиозной коммуникации, под которой подразумевается диалогичное или групповое общение между верующими [6. С. 20], в частности, общение пред- ставителя религиозного института, владеющего теологическим знанием (муфтия, имама-мухтасиба, имама), и верующего (личная коммуникация) или верующих (групповая коммуникация), не владеющих или владеющих в меньшей степени LANGUAGES IN CONTACT: THEORY AND PRACTICE 371 Александрова О.И. Вестник РУДН. Серия: Вопросы образования: языки и специальность. 2017. Т. 14. № 3. С. 369—374 таким знанием. Значение роли мусульманского духовенства заметно возрастает в связи с проблемой невысокого уровня владения мусульманами арабским языком и, как следствие, неспособностью к самостоятельному чтению оригинальных священных текстов. В местных (особенно сельских) мусульманских организаци- ях — махаллях (от арабского — ﻣﺤڶ ֵۍместный [1]) имамы обсуждают с верующими вопросы веры и соблюдения шариата исключительно на национальном языке. таким знанием. Значение роли мусульманского духовенства заметно возрастает в связи с проблемой невысокого уровня владения мусульманами арабским языком и, как следствие, неспособностью к самостоятельному чтению оригинальных священных текстов. Типы коммуникации и языковая закрепленность В местных (особенно сельских) мусульманских организаци- ях — махаллях (от арабского — ﻣﺤڶ ֵۍместный [1]) имамы обсуждают с верующими вопросы веры и соблюдения шариата исключительно на национальном языке. В крупных российских городах все чаще наблюдается объединение мусульман по этническому или субконфессиональному принципу (анклавизация), особен- но это касается трудовых мигрантов, вследствие чего открываются узбекские, таджикские, азербайджанские и другие мечети [2. С. 26], так называемые мусал- ля (от арабского — ﻣﺻﱠﻠﯽместо для молитвы, молельня [1]), в которых групповое общение осуществляется только на национальном языке. С другой стороны, в мегаполисах и крупных городах действуют мечети, в которых религиозное обще- ние после чтения молитв на арабском языке проходит преимущественно на рус- ском языке, что обусловлено полиэтническим составом приходящих в них веру- ющих. Муфтий, принимающий решения по шариату, имам, читающий хутбу (пятничную проповедь) или имам-мухтасиб, отвечающий на вопросы верующе- го, могут быть разной с верующим национальности, и тогда языком коммуника- ции для них будет русский язык. Такая ситуация общения является очень важной, так как для этих коммуникантов язык общения является неродным и не являет- ся сакральным, но при этом обсуждаются вопросы веры и религии, по которым инотолкования, подмены и вольная интерпретация нежелательны. Русский язык в силу статуса государственного языка Российской Федерации становится основным средством массовой коммуникации в исламском дискурсе. К массовой коммуникации относятся обращения мусульманского духовенства к верующим через СМИ (специализированные телеканалы, радиостанции, мусуль- манские сайты и др.). Учитывая стремительно растущую численность мусульман в России и общественно-политические настроения, СМИ становятся для мусуль- манского духовенства одним из важнейших инструментов воздействия на веру- ющих. На русском языке осуществляется и общение мусульман России на институ- циональном уровне — на уровне духовных управлений мусульман, которых в Рос- сии насчитывается более 60. В задачи управлений входит объединение мусульман России и сохранение российской мусульманской традиции, а также администра- тивная деятельность, следовательно, и русский язык выполняет консолидирую- щую функцию. В исламе основным сводом религиозно-правовых норм является шариат, со- блюдение которого является обязательным. Шариат, основанный на извлечени- ях из Корана, охватывает почти все сферы деятельности человека и в некоторых мусульманских странах является единственной правовой системой. В Российской Федерации, где юридический кодекс является светским, следование шариату регулируется и контролируется духовенством. Толкования законов шариата, из- влеченные из арабских первоисточников, переводятся представителями духовен- ства и на национальный, и на русский язык, что может привести к различному восприятию и смысловому смещению. ЯЗЫКОВЫЕ КОНТАКТЫ: ТЕОРИЯ И ПРАКТИКА 372 Aleksandrova O.I. RUDN Journal of Language Education and Translingual Practices, 2017, 14 (3), 369—374 История статьи: р Поступила в редакцию: 20.04.2017 Поступила в редакцию: 20.04.2017 Принята к публикации: 27.06.2017 Модератор: С.В. Дмитрюк Модератор: С.В. Дмитрюк Конфликт интересов: отсутствует Конфликт интересов: отсутствует Конфликт интересов: отсутствует СПИСОК ЛИТЕРАТУРЫ 1. Баранов Х.К. Арабско-русский словарь: ок. 42000 слов. 6-е изд., стереотип. М.: Рус. яз., 1985. 944 с. 1. Баранов Х.К. Арабско-русский словарь: ок. 42000 слов. 6-е изд., стереотип. М.: Рус. яз., 1985. 944 с. 2. Кобищанов Ю. Мусульмане России, коренные российские мусульмане и русские мусуль- мане (окончание) // Россия и мусульманский мир. № 11. М.: Изд-во Института научной информации по общественным наукам РАН, 2003. С. 24—48. 3. Герей ханов Г.П. Мусульманская умма в фокусе социально-философского анализа. Ч. 2. Историко-культурные аспекты формирования мусульманской уммы в России. Электрон- ное научное издание Альманах Пространство и Время. Т. 2. Вып. 1. 2013. URL: http:// www.j-spacetime.com (дата обращения: 16.09.2016). 4. Магомедова Т.И. Типологическая картина двуязычия в Дагестане: история и современное состояние. Rhema. Рема № 12. М.: Изд-во МПГУ, 2011. С. 28—34. 5. Ахманова О.С. Словарь лингвистических терминов. М.: Советская энциклопедия, 1966. 606 с. 6. Прохватилова О.А. Экстралингвистические параметры и языковые характеристики рели- гиозного стиля // Вестник Волгоградского государственного университета. Серия 2. Язы- кознание. 2006. С. 19—26. Для цитирования: Александрова О.И. О языковой ситуации в российском мусульманском сообществе // Вестник Рос- сийского университета дружбы народов. Серия: Вопросы образования: языки и специальность. 2017. Т. 14 № 3. С. 369—374. DOI 10.22363/2312-8011-2017-14-3-369-374 Александрова О.И. О языковой ситуации в российском мусульманском сообществе // Вестник Рос- сийского университета дружбы народов. Серия: Вопросы образования: языки и специальность. 2017. Т. 14 № 3. С. 369—374. DOI 10.22363/2312-8011-2017-14-3-369-374 3. ЗАКЛЮЧЕНИЕ Российское мусульманское сообщество представляет собой многочисленную полиэтническую группу верующих, объединенных общностью религиозных взгля- дов и направляемых в вероисповедании исламским духовенством, использующих в своем коммуникативном пространстве одновременно несколько языков. За каждым из используемых языков закреплены определенные функции, обуслов- ленные типом коммуникации верующих, что указывает на диглоссию или по- лиглоссию. Арабский язык является средством передачи божественной истины, кодом гиперкоммуникации — общения со Всевышним, маркером исламского дискурса. Национальный язык может использоваться верующими во всех типах коммуникации от обращения к Богу через внутреннюю молитву до массовой ком- муникации, но в основном является средством группового общения на религи- озные и околорелигиозные темы. Русский язык как государственный использу- ется в массовой коммуникации и выполняет консолидирующую функцию в рос- сийском мусульманском сообществе. © Александрова О.И., 2017 © Александрова О.И., 2017 р Александрова О.И. О языковой ситуации в российском мусульманском сообществе // Вестник Рос- сийского университета дружбы народов. Серия: Вопросы образования: языки и специальность. 2017. Т. 14 № 3. С. 369—374. DOI 10.22363/2312-8011-2017-14-3-369-374 Сведения об авторе: Сведения об авторе: Александрова Оксана Ивановна — кандидат филологических наук, доцент кафедры общего и рус- ского языкознания, филологический факультет РУДН. E-mail: alexandrova_oi@pfur.ru 373 LANGUAGES IN CONTACT: THEORY AND PRACTICE Александрова О.И. Вестник РУДН. Серия: Вопросы образования: языки и специальность. 2017. Т. 14. № 3. С. 369—374 LINGUISTIC SITUATION IN ISLAMIC COMMUNITY OF RUSSIA O.I. Aleksandrova Peoples’ Friendship University of Russia Miklukho-Maklaya str., 10-2 A, Moscow, Russia, 117198 O.I. Aleksandrova Peoples’ Friendship University of Russia Miklukho-Maklaya str., 10-2 A, Moscow, Russia, 117198 The article deals with the problem of linguistic situation in RF Islamic community. The object of the research is Islamic discourse as a type of religious discourse, characterized by diglossia, that is understood as simultaneous use of two languages in different functional areas. In islamic polyethnic community in Russia it is possible to tell about polyglossia, since RF Moslems of different nationalities use not only sacral Arabic and their national languages but also Russian, that has a status of official language used by members of Islamic community. Using of a particular language depends on the type of communication in the religious discourse. Communication with God through namaz and reading sacred texts is possible only in Arabic. Personal and group communication is usually carried out in national language, including Russian. Mass communication on the institutional level is carried out in Russian through media. Russian language used by Spiritual Directorates of the Muslims helps to unite RF polyethnic Islamic community that tends to be tolerant and dovish. Key words: Islamic discourse, diglossia, polyglossia, bilingualism, polyethnic community, communicative space Article history: Article history: Received: 20.04.2017 Accepted: 27.06.2017 Moderator: S.V. Dmitryuk Conflict of interests: none For citation: Aleksandrova Oksana I. (2017). Linguistic Situation In Islamic Community of Russia. RUDN Journal of Language and Translingual Practices, 14 (3), 369—374. DOI 10.22363/2312-8011-2017-14-3-369-374 Bio Note: Alexandrova Oksana Ivanovna is a PhD in Russian Language, is Assistant-professor at General and Russian Linguistics Department of Peoples Friendship University of Russia (RUDN University), Moscow, Russia. E-mail: alexandrova_oi@pfur.ru Received: 20.04.2017 Accepted: 27.06.2017 Conflict of interests: none REFERENCES 1. Baranov H.K. Arabsko-russkij slovar’ [Arabic-Russian Dictionary]: Ok. 42000 slov. 6-e izd., stereotip. Moskva: Rus. jaz., 1985. 944 s. 2. Kobishhanov Ju. Musul’mane Rossii, korennye rossijskie musul’mane i russkie musul’mane [Muslims of Russia, Aboriginal Muslims of Russian and Russians Muslims.] (okonchanie). Rossija i musul’manskij mir. № 11. Moskva: Izd-vo Instituta nauchnoj informacii po obshhestvennym naukam RAN, 2003. S. 24—48. 3. Gerejhanov G.P. Musul’manskaja umma v fokuse social’no-filosofskogo analiza. Ch. 2. Istoriko- kul’turnye aspekty formirovanija musul’manskoj ummy v Rossii [Muslim Umma in a Focus of Social and Philosophical Analysis. Part 2. Historical and Cultural Aspects of Muslim Umma Forming in Russia]. Jelektronnoe nauchnoe izdanie Al’manah Prostranstvo i Vremja. T. 2. Vyp. 1. 2013. URL: http://www.j-spacetime.com (date 16.09.2016). 3. Gerejhanov G.P. Musul’manskaja umma v fokuse social’no-filosofskogo analiza. Ch. 2. Istoriko- kul’turnye aspekty formirovanija musul’manskoj ummy v Rossii [Muslim Umma in a Focus of Social and Philosophical Analysis. Part 2. Historical and Cultural Aspects of Muslim Umma Forming in Russia]. Jelektronnoe nauchnoe izdanie Al’manah Prostranstvo i Vremja. T. 2. Vyp. 1. 2013. URL: http://www.j-spacetime.com (date 16.09.2016). 4. Magomedova T.I. Tipologicheskaja kartina dvujazychija v Dagestane: istorija i sovremennoe sostojanie [Typology of Bilingualism in Dagestan: Past and Current Situation]. Rhema. Rema № 12. Moskva: Izd-vo MPGU, 2011. S. 28—34. 4. Magomedova T.I. Tipologicheskaja kartina dvujazychija v Dagestane: istorija i sovremennoe sostojanie [Typology of Bilingualism in Dagestan: Past and Current Situation]. Rhema. Rema № 12. Moskva: Izd-vo MPGU, 2011. S. 28—34. 5. Ahmanova O.S. Slovar’ lingvisticheskih terminov [Dictionary of Linguistic Terms]. M.: Sov. Jenciklopedija, 1966. 606 s. 5. Ahmanova O.S. Slovar’ lingvisticheskih terminov [Dictionary of Linguistic Terms]. M.: Sov. Jenciklopedija, 1966. 606 s. 6. Prohvatilova O.A. Jekstralingvisticheskie parametry i jazykovye harakteristiki religioznogo stilja [Non- linguistic Parameters and Language Features of Religious Style]. Vestnik Volgogradskogo gosudarstvennogo universiteta. Serija 2. Jazykoznanie. 2006. S. 19—26. 6. Prohvatilova O.A. Jekstralingvisticheskie parametry i jazykovye harakteristiki religioznogo stilja [Non- linguistic Parameters and Language Features of Religious Style]. Vestnik Volgogradskogo gosudarstvennogo universiteta. Serija 2. Jazykoznanie. 2006. S. 19—26. For citation: Aleksandrova Oksana I. (2017). Linguistic Situation In Islamic Community of Russia. RUDN Journal of Language and Translingual Practices, 14 (3), 369—374. DOI 10.22363/2312-8011-2017-14-3-369-374 Bio Note: Bio Note: Alexandrova Oksana Ivanovna is a PhD in Russian Language, is Assistant-professor at General and Russian Linguistics Department of Peoples Friendship University of Russia (RUDN University), Moscow, Russia. E-mail: alexandrova_oi@pfur.ru o Note: Alexandrova Oksana Ivanovna is a PhD in Russian Language, is Assistant-professor at General and Russian Linguistics Department of Peoples Friendship University of Russia (RUDN University), Moscow, Russia. E-mail: alexandrova_oi@pfur.ru 374 ЯЗЫКОВЫЕ КОНТАКТЫ: ТЕОРИЯ И ПРАКТИКА
https://openalex.org/W2071687499	https://sjtrem.biomedcentral.com/counter/pdf/10.1186/1757-7241-17-11	English	null	Undisclosed cocaine use and chest pain in emergency departments of Spain	Scandinavian journal of trauma, resuscitation and emergency medicine	2,009	cc-by	3,048	Published: 2 March 2009 Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine 2009, 17:11 doi:10.1186/1757-7241- 17-11 This article is available from: http://www.sjtrem.com/content/17/1/11 © 2009 Burillo-Putze 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 Aims: Illicit cocaine consumption in Spain is one of the highest in Europe. Our objective was to study the incidence of undisclosed cocaine consumption in patients attending in two Spanish Emergency Departments for chest pain. Methods: We analysed urine samples from consenting consecutive patients attending ED for chest pain to determine the presence of cocaine, and other drugs, by semiquantative tests with fluorescence polarization immunoassay (FPIA). Results: Of 140 cases, 15.7 presented positive test for drugs, and cocaine was present in 6.4%. All cocaine-positive patients were younger (p < 0.001); none was admitted to Hospital (p = 0.08). No significant differences in ED stay or need for hospitalization were found between cocaine-positive and negative patients. Conclusion: This finding in chest pain patients who consented to urine analysis suggests that the true incidence of cocaine use leading to such ED visits may be higher. The relationship between cocaine use and episodes of cor- onary ischemia or chest pain is clear, and cocaine is con- sidered a new risk factor for cardiovascular events in chronic users [3-5]. Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine BioMed Central Open Access p Original research Undisclosed cocaine use and chest pain in emergency departments of Spain Guillermo Burillo-Putze1, Beatriz López2, Juan María Borreguero León1, Miquel Sánchez Sánchez2, Martin García González3, Alberto Domínguez Rodriguez3, Eva Vallbona Afonso1, Alejandro Jiménez Sosa4 and Oscar Mirò2 Address: 1Emergency Department, Hospital Universitario de Canarias, La Laguna, Santa Cruz de Tenerife, Tenerife, Spain, 2Emergency Department, Hospital Clínic, Barcelona, Spain, 3Cardiac Intensive Care Unit, Hospital Universitario de Canarias, La Laguna, Santa Cruz de Tenerife, Tenerife, Spain and 4Research Unit, Hospital Universitario de Canarias, La Laguna, Santa Cruz de Tenerife, Tenerife, Spain mail: Guillermo Burillo-Putze - gburillo@telefonica.net; Beatriz López - blopez@clinic.ub.es; Juan María Email: Guillermo Burillo-Putze* - gburillo@telefonica.net; Beatriz López - blopez@clinic.ub.es; Juan María Borreguero León - gburillo@huc.canarias.org; Miquel Sánchez Sánchez - msanchez@cllnic.ub.es; Borreguero León - gburillo@huc.canarias.org; Miquel Sánchez Sánchez - msanchez@cllnic.ub.es; Martin García González - gburillo@huc.canarias.org; Alberto Domínguez Rodriguez - gburillo@huc.canarias.org; g g @ g q @ n García González - gburillo@huc.canarias.org; Alberto Domínguez Rodriguez - gburillo@huc.canarias.org; g g g g g g va Vallbona Afonso - evallbona6@yahoo.com; Alejandro Jiménez Sosa - ajimenez@huc.canarias.org; Oscar Mirò - o Corresponding author Eva Vallbona Afonso - evallbona6@yahoo.com; Alejandro Jiménez Sosa - ajimenez@huc.canarias.org; Oscar Mirò - omiro@clinic.ub.es * Corresponding author * Corresponding author Received: 10 December 2008 Accepted: 2 March 2009 Introduction Illi i i Illicit cocaine consumption in Spain is, together with the United Kingdom, the highest in Europe, mainly in young people [1,2]. Page 1 of 4 (page number not for citation purposes) Page 1 of 4 (page number not for citation purposes) Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine 2009, 17:11 http://www.sjtrem.com/content/17/1/11 Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine 2009, 17:11 http://www.sjtrem.com/content/17/1/11 Statistical analysis To our knowledge, few studies have been performed in Spain on the prevalence of cocaine consumption in patients seeking Emergency Department attention when this was not the direct reason for the visit [6,7]. Results for categorical variables are expressed as frequen- cies and percentages and 95% confidence intervals. Results for numerical and ordinal variables are expressed as means and standard deviations. Proportions were com- pared with Chi-square test or Fisher's exact test whenever required. Ranks between groups were compared with Mann-Whitney U test or Wilcoxon-Mann-Whitney test whenever required. A P value of less than 0.05 was consid- ered to indicate statistical significance. The objective of this work was to study the incidence of undisclosed cocaine consumption in patients attending the Emergency Department (ED) of two hospitals for chest pain. Patients and methods Statistical analysis was carried out with SPSS v. 14.0.1 (Chicago, ILL) and StatXact 5.0 (Cytel Co., Cambridge, MA). Between May and June 2006, we prospectively studied urine samples from consecutive patients over 18 years who were attended at two University Hospitals Emergency Departments (Tenerife, Canary Islands and Barcelona, Catalonia) for non-traumatic chest pain of probable car- diovascular origin, initially not-related with cocaine con- sumption. Results Of 190 recorded patients, 140 agreed to participate in the study and complete information was obtained. There was some drug consumption in 15.7% (95% confidence inter- val: 9.6%–21.7%) and 6.4% (95% confidence interval: 2.0%–10.4%) showed cocaine-positive test. Polycon- sumption was present in 4.3% of patients. Demographic features, ED management, and drug test results are shown in Table 1. There were differences between the two Hospi- tals in age, sex, hospital stay, cannabis consumption and polyconsumption. Barcelona Hospital Clinic is a large, inner city, university tertiary-care hospital, with a specialised Chest Pain Unit within its ED. All Barcelona patients included in this study were ED patients with chest pain attended by this unit. The University Hospital of the Canary Islands (HUC) is a large, suburban, university tertiary-care hospital, whose ED has a special circuit for the attention of chest pain patients, with similar features to the Barcelona chest pain unit. All Tenerife patients included in this study were ED patients with chest pain who were attended on this circuit. We found an inverse relation between cocaine consump- tion and Hospital admission. One in two cocaine users also used cannabis. No differences were observed between cocaine users and non users regarding the concomitant use of opiods and amphetamines (Table 2). We found an association between cocaine and polyconsumption (p < 0.001). Informed consent for urianalysis and participation in this study was obtained from all participants. Urine samples were stored at -80°C for subsequent analysis. Attending physicians had no information of drugs test results. The following variables were studied: age, sex, outcome (death, hospital admission, discharge from ED), duration of ED stay for non-admissions, days of hospital stay and positive drugs test. Not unexpectedly, all cocaine-positive patients were young men, ranging in age from 22 to 34 years. With respect to follow-up data, all cocaine-positive patients were discharged home from ED after attention We measured in urine samples the levels of cocaine (ben- zoylecgonine and methylecgonine ether), cannabis (delta-9-tetrahidrocannabinol), amphetamine/metaam- phetamine, opioids (morphine, and N-morphine). Drug detection was performed by semiquantative tests with flu- orescence polarization immunoassay (FPIA)(AxSYM Sys- tem, Abbott laboratories, Illinois, USA.). We considered the following values as positive: cocaine > 300 ng/ml, can- nabis > 50 ng/ml, opioids > 300 ng/ml and ampheta- mine/metaamphetamine > 1000 ng/ml. Polyconsumption was defined as the presence of two or d i h l l d Page 2 of 4 (page number not for citation purposes) Discussion Our findings of low incidence of occult users added to those of other series in declared or suspected cases may provide a more realistic picture of cocaine consumption in these ED patients in Spain. According to the literature, it seems probable that the real incidence of cocaine use in non-traumatic chest pain patients is around 30% [6,7,10]. The presence of cocaine in urine does not necessarily imply that this substance was the cause of the chest pain leading to their ED visit. Although urianalysis is usually positive in the first 48–72 hours alter consumption, chronic users can have positive urines for up to 2 weeks [15]. Our data on the prevalence of cocaine use in young people suggest that ED staff should be alert to possible consumption that is not disclosed by the patient. Junior doctors are less likely to routinely ask about cocaine use compared to other classical risk factors [5,16]. As in other studies, the great majority of our cocaine-con- suming patients with chest pain were young people, in their third decade of life, and in general presumably at low risk of adverse cardiovascular events [10,12,13]. Thus none of them required admission to hospital. However, caution must be exercised when evaluating these patients with chest pain since there are no reliable tests to predict adverse cardiovascular outcomes in cocaine-associated chest pain [14]. Despite the fact that we studied two demographically dis- parate groups of patients, we found no significant differ- ences in clinical characteristics such as ED stay, need for hospitalization or length of hospital stay. Nor did we find differences in cocaine consumption between the two groups, but this could very well be explained by the small sample size. The high mean age of the Barcelona Hospital group probably accounts for the low number of cocaine- positive patients. In addition, this group was attended at Table 2: Demographics, ED management and other drug consumption in cocaine-positive/negative patients. Discussion In USA, with similar cocaine consumption rates to Spain, the Drug Abuse Warning Network DAWN estimates that cocaine was involved in 10% of drug misuse/abuse ED visits [8,9]. In the study of Hollander et al prevalence of cocaine use in chest pain of possible ischemic origin was 17%, ranged from 20% in Urban Hospitals ED to 7.45% in Suburban Hospital EDs [10]. With respect to other Spanish studies, our finding of 6.4% cocaine-positive chest pain patients was lower than the 25% reported by Sanjurjo et al [6,7]. This could be due to features of our study population who were patients with undisclosed cocaine-related chest pain, when in other series the patient visit was related with declared consump- The project was approved by the local ethical research committee. Page 2 of 4 (page number not for citation purposes) Page 2 of 4 (page number not for citation purposes) Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine 2009, 17:11 http://www.sjtrem.com/content/17/1/11 Table 1: Demographic, ED Management and drug results by Hospital. Total n = 140 Tenerife Hospital N = 40 Barcelona Hospital n = 100 P value Age (years) 58.76 ± 19.3 49 ± 15.6 63 ± 19.3 < 0.001 Male sex – no (%) 90 (65) [57–73] 32 (80) [67.6–92.4] 58 (59) [49–68] 0.019 Emergency Dept. stay (hours) 4.43 ± 6.1 4.56 ± 7.67 4.3 ± 4.93 0.99 Hospital admission – no (%) 55 (40) [32–49] 21 (53) [37–68] 34 (35) [26–45] 0.08 Hospital stay (days) 7.1 ± 6.5 5.9 ± 7.2 8.5 ± 5.3 0.01 Cocaine (positive test) – no (%) 9 (6) [2–10] 5 (12.5) [2.3–22.7] 4 (4) [1–8] 0.12 Cannabinoids (positive test) – no (%) 9 (6) [2–10] 6 (15) [3.9–26.1] 3 (3) [0–6] 0.016 Opioids (positive test) – no (%) 9 (6) [2–10] 2 (5) [0–11.7] 7 (7) [2–12] 0.5 Amphetamines (positive test) – no (%) 1 (1) [0–2] 0 (0) [0-0] 1 (0) [0-0] 0.99 Any drug consumption – no (%) 22 (15.7) [1–22] 9 (22.5) [9.6–35.4] 13 (13) [6–20] 0.099 Polyconsumption – no (%) 6 (4.2) [0.1–8] 6 (15) [3.9–26.1] 0 (0) [0-0] < 0.001 Table 1: Demographic, ED Management and drug results by Hospital. tion or clinical toxic cocaine-related signs suggestive of consumption. As Perrone et al propose, drug screening for substance abuse in addition to clinical history is necessary for optimal identification of drug use in ED patients [11]. Acknowledgements g This study was supported by the National Plan on Drugs, Ministry of Health, Government of Spain, in 2004 http://www.pnsd.msc.es/. Authors' contributions GB, MS and OM were responsible for study design, ana- lyzing and interpretation data. BL, MG, EV and AD partic- ipated in collecting data. JB carried out the immunoassays. AJ performed the statistical analysis and interpretation data. Al the authors read and approved the final manuscript. Conclusion 14. Chase M, Brown AM, Robey JL, Zogby KE, Shofer FS, Chmielewski L, et al.: Application of the TIMI risk score in ED patients with cocaine-associated chest pain. Am J Emerg Med 2007, 25:1015-8. This study found undisclosed cocaine consumption in 6.4% (95% confidence interval: 2.0%–10.4%) of adult patients presenting at Emergency Department for chest pain. This finding in chest pain patients who consented to urine analysis suggests that the true incidence of cocaine use leading to such ED visits may be higher. p J g 15. Weiss RD, Gawin FH: Protracted elimination of cocaine metabolites in long-term high-dose cocaine abusers. Am J Med 1988, 85:879-80. 16. Wood DM, Hill D, Gunasekera A, Greene SL, Jones AL, Dargan PI: Is cocaine use recognised as a risk factor for acute coronary syndrome by doctors in the UK? Postgrad Med J 2007, 83:325-8. 17 S h M L B B l E G A l E J S O 16. Wood DM, Hill D, Gunasekera A, Greene SL, Jones AL, Dargan PI: Is cocaine use recognised as a risk factor for acute coronary syndrome by doctors in the UK? Postgrad Med J 2007, 83:325-8. 17. Sanchez M, Lopez B, Bragulat E, Gomez-Angelats E, Jimenez S, Ortega M, et al.: Triage flowchart to rule out acute coronary syn- drome. Am J Emerg Med 2007, 25:865-72. 17. Sanchez M, Lopez B, Bragulat E, Gomez-Angelats E, Jimenez S, Ortega M, et al.: Triage flowchart to rule out acute coronary syn- drome. Am J Emerg Med 2007, 25:865-72. Discussion Total n = 140 Cocaine-positive Patients n = 9 Cocaine-negative Patients n = 131 P value Age (years) 58.76 ± 19.3 27.89 ± 5.77 60.89 ± 18.04 0.046 Male sex – no (%) 90 (65) [55–74] 9 (100) [100-100] 81 (62) [54–70] 0.017 ED stay (hrs) 4.43 ± 6.1 7.2 ± 2.3 4.13 ± 8.16 0.046 Hospitalization – no (%) 55 (40) [31–50] 0 (0) [0-0] 55 (42) [34–50] 0.008 Hospital stay (days) 7.1 ± 6.5 0 ± 0 7.6 ± 6.4 0.046 Cannabinoids (positive test) – no (%) 9 (6) [2–11] 5 (55.6) [23.1–88.1] 4 (3) [0–6] < 0.001 Opioids (positive test) – no (%) 9 (6) [2–11] 0 (0) [0-0] 9 (7) [3–11] 0.54 Amphetamines (positive test) – no (%) 1 (0.7) [0–2] 1 (11.1) [0–31.6] 0 (0) [0-0] 0.99 Polyconsumption – no (%) 6 (4) [0–8] 6 (66.7) [35.1–96.9] 0 (0) [0-0] < 0.001 gement and other drug consumption in cocaine-positive/negative patients. e 2: Demographics, ED management and other drug consumption in cocaine-positive/negative patients. Table 2: Demographics, ED management and other drug consumption in cocaine-positive/neg Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine 2009, 17:11 http://www.sjtrem.com/content/17/1/11 Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine 2009, 17:11 http://www.sjtrem.com/content/17/1/11 the Chest Pain Unit, without any fast-track circuit patients (mostly young), as in previous studies by this group [6,7,17]. 11. Perrone J, De Roos F, Jayaraman S, Hollander J: Drug screening versus history in detection of substance abuse in ED psychi- atric patients. Am J Emerg Med 2001, 19:49-51. p J g 12. Hollander JE, Hoffman RS, Gennis P, Fairweather P, DiSano MJ, Schumb DA, et al.: Prospective multicenter evaluation of cocaine-associated chest pain. Cocaine Associated Chest Pain (COCHPA) Study Group. Acad Emerg Med 1994, 1:330-9. Further research with longer study periods and greater number of patients are required to confirm these findings. ( ) y p g 13. Baumann BM, Perrone J, Hornig SE, Shofer FS, Hollander JE: Cardiac and hemodynamic assessment of patients with cocaine-asso- ciated chest pain syndromes. J Toxicol Clin Toxicol 2000, 38:283-90. Competing interests The authors declare that they have no competing interests. References 1. The European Monitoring Centre for Drugs and Drug Addic- tion Annual report on the state of the drugs problem in Europe 2007 [http://www.emcdda.europa.eu/html.cfm/index419EN.html]. 1. The European Monitoring Centre for Drugs and Drug Addic- tion Annual report on the state of the drugs problem in Europe 2007 [http://www.emcdda.europa.eu/html.cfm/index419EN.html]. 2. Delegacion del Gobierno para el Plan Nacional sobre Dro- gas. Observatorio español sobre drogas. Informe 2004. Min- isterio de Sanidad y Consumo [http://www.pnsd.msc.es/ Categoria2/publica/pdf/oed-2004.pdf] 3. Hahn I, Hoffman RS: Cocaine use and acute myocardial infarc- tion. Emerg Med Clin North Am 2001, 19:493-510. g 4. Weber JE, Shofer FS, Larkin GL, Kalaria AS, Hollander JE: Validation of a brief observation period for patients with cocaine-asso- ciated chest pain. N Engl J Med 2003, 348:507-10. p g J , 5. Burillo-Putze G, Hoffman RS, Dueñas-Laita A: Cocaine as possible cardiovascular risk factor. Rev Esp Cardiol 2004, 57(6):595-596. 5. Burillo-Putze G, Hoffman RS, Dueñas-Laita A: Cocaine as possible cardiovascular risk factor. Rev Esp Cardiol 2004, 57(6):595-596. 6. Sanjurjo E, Montori E, Nogue S, Sánchez M, Munne P: Urgencias por cocaína: un problema emergente. Med Clin (Barc) 2006, 126:616-9. cardiovascular risk factor. Rev Esp Cardiol 2004, 57(6):595 596. 6. Sanjurjo E, Montori E, Nogue S, Sánchez M, Munne P: Urgencias por cocaína: un problema emergente. Med Clin (Barc) 2006, 126:616-9. 7. Sanjurjo E, Camara M, Nogue S, Negredo M, Garcia S, To-Figueras J, et al.: Urgencias por consumo de drogas de abuso: confront- ación entre los datos clínicos y los analíticos. Emergencias 2005, 17:26-31 [http://www.semes.org/revista/vol16_6/5.pdf]. References Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Page 4 of 4 (page number not for citation purposes) Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Publish with BioMed Central and every scientist can read your work free of charge 17:26-31 [http://www.semes.org/revista/vol16_6/5.pdf]. 8. United Nations: Office on Drugs and Crime. World Drug Report – Global Illicit Drug Trends 2007 [http://www.unodc.org/unodc/en/data- and-analysis/WDR-2007.html]. 9. Substance Abuse and Mental Health Services Administration, Office of Applied Studies: Drug Abuse Warning Network, 2005: National Estimates of Drug-Related Emergency Depart- ment Visits. DAWN Series D-29, DHHS Publication No. (SMA) 07- 4256, Rockville, MD 2007 [http://www.mayatech.com/cti/sbirtgsm07/ doc/Resources/DAWN-ED-2005-Web.pdf]. p 10. Hollander JE, Todd KH, Green G, Heilpem KL, Karras DJ, Singer AJ, et al.: Chest pain associated with cocaine: an assesment of prevalence in suburban and urban Emergency Department. Ann Emerg Med 1995, 26:671-6.
W4313404339.txt	https://www.mdpi.com/2076-3417/12/24/12938/pdf?version=1671184813	en		Analysis of Factors Influencing the Prices of Tourist Offers	Applied sciences	2,022	cc-by	14,152	applied sciences Article Analysis of Factors Influencing the Prices of Tourist Offers Agata Kołakowska * and Magdalena Godlewska Department of Intelligent Interactive Systems, Faculty of Electronics, Telecommunications and Informatics, Gdańsk University of Technology, 80-233 Gdańsk, Poland * Correspondence: agatakol@eti.pg.edu.pl Abstract: Tourism is a significant branch of many world economies. Many factors influence the volume of tourist traffic and the prices of trips. There are factors that clearly affect tourism, such as COVID-19. The paper describes the methods of machine learning and process mining that allow for assessing the impact of various factors (micro, mezzo and macro) on the prices of tourist offers. The methods were used on large sets of real data from two tour operators, and the results of these studies are discussed in this paper. The research presented is part of a larger project aiming at predicting trip prices. It answers the question of which factors have the greatest impact on the price and which can be omitted in further work. Nevertheless, the dynamic world situation suggests that the ranking of factors may change and the presented universal methods may provide different results in the coming years. Keywords: machine learning; process mining; factors; price predictor; tourism 1. Introduction Citation: Kołakowska, A.; Godlewska, M. Analysis of Factors Influencing the Prices of Tourist Offers. Appl. Sci. 2022, 12, 12938. https://doi.org/10.3390/ app122412938 Academic Editor: Giacomo Fiumara Received: 1 November 2022 Accepted: 10 December 2022 Published: 16 December 2022 Publisher’s Note: MDPI stays neutral The paper reports on the part of a research project under the ePredyktour project, which is an attempt to solve a problem of predicting trip prices on the basis of various factors. The presented research was carried out for data provided by two Polish tour operators in 2021. In Poland, expenditure on tourism and recreation in the consumption basket amounted to 8.2% (in 2018). Poles more often choose organized trips than trips on their own. They account for 1.6% of the consumption basket compared to 1% in the European Union. The COVID-19 pandemic had a heavy impact on the travel industry. In most countries, the contribution of tourism to GDP has decreased significantly; in Poland, this decrease amounted to about 2 percentage points in total GDP [1]. Therefore, in 2021, the COVID pandemic is the overarching factor influencing the tourism industry, and certainly has an impact on the conclusions drawn from the presented research. Nevertheless, the methods proposed in the paper are universal and can be repeated for data before and after the pandemic. The entire ePredyktour project consists of the following stages (presented in Figure 1): with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// Stage I: Theoretical analysis and definition of various factors that may affect the price of the trip, finding the sources of these factors and performing initial normalization. This stage was made by specialists in the tourism industry. At this stage, it turned out that it is not possible to access all the databases that were theoretically considered. For example, various opinions posted on social media could not be used in this project due to a lack of authorization. The identified factors have been divided into three groups: micro (factors directly related to a given trip, such as hotel properties, transport type, type of meals, etc.), mezzo (factors related to the vicinity of the holiday destination, such as weather, historic attraction, terrorist attacks risk, etc.), and macro (global factors such as fuel prices, exchange rates, economic indicators, etc.) creativecommons.org/licenses/by/ 4.0/). Appl. Sci. 2022, 12, 12938. https://doi.org/10.3390/app122412938 https://www.mdpi.com/journal/applsci Appl. Sci. 2022, 12, 12938 2 of 31 Stage II: The raw data from the various databases were downloaded, transformed into predefined structures and saved on the Hadoop Distributed File System (HDFS) [2]. These data are available in the form of Data Frames via the API of the Apache Spark [3]. The tourism, IT and big data specialists were responsible for this stage. Data were collected from the beginning of 2021 and initially collections were created for two tour operators. Each trip was recorded almost daily to a new record. In this way, millions of records were created. Stage III: This stage is the subject of the paper and the authors were responsible for its implementation. Its goal was the analysis of factors in order to determine their impact on both the entry price and the change in the price of trips. The latter task was of particular importance, as the whole project aims to design a system that predicts changes in trip prices. Initially, the data were transformed into structures corresponding to the input of the machine learning algorithms and several algorithms were used to evaluate the influence of various factors on the changes of trip prices. Theses methods and the results achieved are the subject of the following sections of this paper. Stage IV: The use of machine learning algorithms, which, based on the factors examined in Stage III, will predict the price of the trip currently offered by the tour operator. During the preparation of this paper, this stage was still in progress. Tou r op er Raw Data ato r da ta Hote l pro perti es Weather forecast Data Frames 1 Data Analysis 2 Prediction 3 ... rates ange Exch tor ic a d in ic om n o Ec Stage I Stage II Stage III Stage IV Figure 1. Stages of the ePredyktour project. As a result of Stage II, six datasets have been created: offers, trips, properties, nbp, opec, and tradingeconomics. When a client comes to a travel agency, he/she first chooses an offer, whose name in the case of stays is the same as the name of the hotel (eg. Hotel Sea), and, in the case of touring trips, it is related to the scope of the trip (e.g., the most beautiful towns of Provence). This information is included in the offers set. Then, the details of the trip are selected: day of departure and return, transport and food type, standard of the room, number of persons, etc. These details are included in the trips dataset. The properties set provides general information about hotels. The sets: nbp (dollar, euro, and pound exchange rates), opec (oil barrel price), and tradingeconomics (consumer spending, GDP per capita, inflation rate, personal savings, and unemployment rate) represent the macro factors. The price applies to each trip and it depend on numerous factors; it is set at the presentation time and may change over time. All factors considered in the project can be divided into constant and changing over time within a given trip. This division is important for the analysis, because the constant factors play a different role than the changing ones. The constant factors are the basis for setting initial prices of trips and grouping trips according to certain features. On the other hand, the changing factors have a hypothetical greater impact on the change of the price of a given trip over time; hence, they may be more useful for price prediction. All these factors, divided into constant and time varying are described in Section 3. Appl. Sci. 2022, 12, 12938 3 of 31 It is worth mentioning that not all of these factors change with the same frequency. The barrel price and exchange rates change daily, but economic indicators rarely change: once a month, once every six months, or even once a year. The current weather may also change every day, while the forecast weather on the day of departure does not start to change until two weeks before departure due to the reliability of weather forecasts. The aim of this research it to evaluate which of all these factors, both constant and timevarying ones, are worth being considered while analyzing the dynamics of trip prices. The factors identified as having significant influence on trip prices will be used to create a price prediction model in stage IV of the project. A tool created in this way might be implemented in a meta-search engine of trips and offered as Software, or as a Service, useful from the point of view of tour operators, travel agents, or hotel owners. The market demand for the project results has been confirmed by potential recipients of the system. The rest of the paper is organized as follows. Section 2 presents other studies on predicting some parameters related to tourism, e.g., tourism demand or tourists’ satisfaction. It also mentions methods applied in other areas but that are potentially adequate in this application. Section 3 of the paper presents a feature selection approach applied to evaluate the predictive power of individual trip attributes to the changes of prices. These methods belong to the category of feature engineering and are commonly implemented in machine learning systems. They not only enable one to evaluate the features independently, but also to identify subsets of these parameters optimal for a given task. Section 4 outlines a different approach to the problem at hand: creating and analyzing a model of the price change process. For this purpose, various tools and algorithms for process mining [4] were used. This approach allows one to look at the price change process in the context of changing other factors and to find other interesting relationships. Section 5 presents conclusions drawn on the basis of the obtained results and some ideas for future works. 2. Related Work To select all the factors that influence the price of a trip, enormous data from around the world would have to be analyzed. This is because tourism covers almost the entire world and is available to people of various affluence level, views, culture, style of recreation, preferred climate, etc. Of course, such calculations are practically impossible. In this project, a set of factors identified by a team of tourism experts and technically feasible were used (see Table 1). There are papers in the literature that also select certain factors in order to automatically analyze their impact on tourism. A variable often treated as a target is tourism demand. In [5], the first challenge in the research was also to determine the set of factors influencing tourism in order to forecast the tourism demand. Initially, the factors were divided into three categories: the first contained tourist origin, the economic level, and the personal situation of tourists, the second was related to the supply of tourist destinations, including the attraction, price, infrastructure, and travel organization, the third contained consumer price comparison, promotion, exchange rate, policy coordination, and socio-economic and cultural heterogeneity. As a result, seven factors were selected that allowed one to create a feature vector. These are: number of inbound tourists, currency exchange rate, per capita GDP, total import and export of goods, population, per capita GDP of destination countries, and virtual variables, that can reflect the situation of special events and improve the prediction model. The vector is then used in the neural network algorithm to forecast the inbound tourism demand. Demand function forecasting is also the subject of [6], where authors consider a panel of over 100 hotels in Milan (Italy), over a time interval of 274 days to determine the demand function for these hotels. The price index was a leading indicator in the set of explanatory variables. In economic theory, with other factors being constant, demand is inversely proportional to price. The close relationship between price and demand may suggest that other factors influencing demand may also influence price. The authors took into account the following factors related mainly to the properties of the hotel and its surroundings: Appl. Sci. 2022, 12, 12938 4 of 31 occupation rate, number of rooms, meeting rooms, restaurant seats, distance from city center, distance fom from airport, holidays, and events. Most of these determinants were also taken into account in the discussed project. It is worth noting that, apart from the price and occupation rate, the rest of these factors are constant. Using the collected data, the authors proposed a method of forecasting tourist demand by improving the set of available information. Another topic of research that takes into account tourism determinants are travel recommendation systems. From the point of view of tour operators, data collected by such a system allows for the study of customer preferences, and thus for price regulation. It is therefore important what factors customers consider as they may affect the price of the trip. In [7], the authors have listed a set of factors that are used to investigate customer preferences. Some of them are global, such as weather, season, weekday, transport, crowdedness and distance, while others are related to specific client preferences, such as budget, companion, feeling, travel goal and knowledge of the travel area. For many customers, the weather is the primary factor in choosing trips, especially leisure ones. However, “good weather” can mean different things for different people and different locations. It can be assumed that customers going to Greece, Italy or Croatia in the summer count on a lot of sunny days, warm water and accept the heat, while visitors to Iceland or Norway expect a few days without rain and skiers expect snow. In paper [8], the authors examined various weather factors for point of interest recommendations. They took into account the following factors: visibility, precipitation intensity, humidity, cloud cover, pressure, wind speed, temperature, and moon phase. The conducted research confirmed that the influence of factors differs depending on the location. For example, in Honolulu, the best performing feature is precipitation intensity, while in Minneapolis it is visibility. Likewise, the phases of the moon, which generally have little influence on the choice of destination, are crucial in certain specific places. In most publications on tourism, price is a factor (or one of the factors) for determining or forecasting other values related to tourism, e.g., a demand mentioned above. However, in [9], the author presents research on the price effects of demand and the results of interviews with tourists. Three variables were identified and used as factors in this study: airfare, hotel tariffs, and the exchange rate. However, this paper was published in 1994, and since then there have been many changes in tourism itself, as well as in information technology, data size, access to various offers, and tourists’ expectations. There are applications that can predict the prices of certain components of trips. Most of the time, it concerns searching for flights with the history of prices and information about the forecast price in the future. Google Flights [10] and Momondo [11] are examples of such search engines. The most straightforward approach to the problem of finding good predictors is feature selection. It is a process of reducing the number of original attributes to a subset good enough to predict the value of a target variable. There are some examples of applying feature selection methods in tourism. In [12], for example, the authors managed to select a number of keywords extracted from web search data to predict monthly statistics of China inbound foreign visitors. They applied random forest to estimate the importance of individual features, which were keyword frequencies. A similar idea of selecting keywords has been proposed in [13] in order to forecast tourist demand and hotel occupancy from search query data. In this case, the features were filtered in various ways, i.e., on the basis of correlation coefficient, information gain or random forest importance, by running the recursive elimination or genetic algorithm. Removing redundant and extraneous information enabled us to improve the forecasting accuracy. Tourist reviews deliver lots of valuable data, which can be used to infer tourists’ satisfaction, as for example in [14], where reviews of tourist agencies were analyzed. A number of keywords used as features were processed. The performed feature selection based on LASSO regression model revealed top factors affecting the satisfaction. The interesting fact was the discovery of several new factors that became essential after COVID-19, i.e., refunds, Appl. Sci. 2022, 12, 12938 5 of 31 bad reviews, assurance, and comparison. Satisfaction predictors were also analysed in [15], where results obtained by a feature selection filter were used as weights for traditional tf-idf feature values. This leads to the improvement of performance of sentiment analysis of online reviews of tourist attractions. Another interesting application of feature selection has been presented in [16]. In this study, several methods were implemented to find sets of features good for the classification of Polish voivodeships according to their tourist attractiveness. One of the main conclusions of this paper was that different selection methods lead to significant differences in classification results. To estimate the influence of selected variables on prices regression, models trained to predict prices are often implemented. Although there are a lot of studies on predicting prices of various products, there are no studies on predicting the prices of trip packages. However, the regression approach is worth taking into account. In [17], the regression approach has been applied to predict the prices of a set of products sold online. The authors implemented classical linear regression and least squares SVM with an artificial bee colony to optimize its parameters. The latter method let us achieve higher prediction accuracy. The method was implemented in a web application both to alert users when the price of a product changed and to predict the price for the next day. It might be useful for customers, as it enables monitoring product pricing. House prices have always been of particular interest. In [18], the authors analyzed a number of papers on significant factors influencing house prices. Among various determinants grouped into locational, structural, and neighbourhood, the locational ones turned out to be the most significant in fixing the prices of houses. Various methods were applied to create models able to predict prices of houses, e.g., support vector regression, neural networks, and gradient boosting. The hedonic model, which applies multivariate regression analysis, is the simplest way to estimate the significance of various factors. The obtained regression coefficients may be treated as factor weights indicating their influence on the target variable [19]. Interesting findings were presented in [20]. It has been demonstrated that partitioning the data on the basis of selected factors and applying a multi-task learning algorithm may improve the performance of house price prediction. There are also some research studies where dynamic factor approach, which is a tool commonly used for analyzing economic data, is applied in forecasting some tourism indices. Classical dynamic factor models represent the whole cross-section dynamics by a few common factors found, for example, by PCA and then perform forecasting for a target variable on the basis of these common factors using a simple linear regression model. It has been applied, for example, in [21] to predict checking-in and overnight stays of travellers in Spain on the basis of the numbers of queries on several traveling related topics. The authors managed to obtain good results for short-term forecasts. To relax the limitation of the classical dynamic factor model, which does not allow for correlations between idiosyncratic components, a generalised dynamic factor model (GDFM) [22] was proposed. This model was applied by the authors of [23]. They created a time-varying parameter factor vector autoregressive model based on GDFM to analyze the influence of six factors (dining, transportation, attractions, shopping, tours, and lodging) on tourist demand in different periods of time. The model investigated the time-varying characteristics of the relationship between variables. The original six factors were extracted from search queries and represented by selected keyword frequencies. They found that tourism demand responded more intensely to shock in dining, tours, and lodging than to the other three factors. Dining, attractions, and shopping had a positive effect on tourism demand, whereas transportation, tours, and lodging had negative effect. The presented analysis is rather unique, as it is a simultaneous quantitative analysis of several factors. Appl. Sci. 2022, 12, 12938 6 of 31 As already mentioned in the Introduction, time varying factors (see Table 1), as well as the price of the trip itself, change over time. Any change to the trip price can be treated as an event in the price change process. This event may be the consequence or cause of other events. This allows for the use of business process models to implement the price change process. The authors of [24] presented the process model in the form of Colored Petri Net (CPN) for determining activities related to changes in the prices of stocks. They analyzed trends of change, rather than changes in individual prices, as the prices of stocks fluctuate on almost every stock quote. Trends influence decisions to buy, sell, or hold stocks, and the presented CPN helps users make that decision. The model is predefined and immutable. Price changes at tour operators occur much less frequently, often without clear trends, so each increase or decrease can be analyzed. The use of process mining to automatically determine price change processes is a new application of these techniques. This paper presents an idea of applying several feature selection methods and the process mining technique to evaluate the influence of various factors on the changes of trip prices. The novelty of our study appears in several aspects. The first is the application area, as it analyzes the factors influencing the prices of tourist offers, which are not individual products, but packages containing transportation, accommodation, and meals. To our knowledge, such analysis has not been performed before. Moreover, the number of parameters considered in this study is also high if compared to other studies, e.g., those mentioned in this section. There are both time-varying and constant parameters analysed together, which is not a common approach. Our data examples are thousands of trips, which are offered at different times and for a different number of days, but usually no longer than for a few weeks, which is much shorter than the lengths of time series usually used for training price prediction models. This fact disables applying the methods that require observations at the same moments. Most regression models, e.g., predicting demand, apply to single time series or a number of parallel time series. Finally, applying process mining for attribute evaluation is an original idea. 3. Selecting Optimal Price Predictors This section describes a machine learning approach applied to evaluate the impact of various factors on the changes of trip prices. The aim of this stage of the project was to identify the optimal set of parameters to be used to predict whether the price of a trip would change in future. The task was defined as a classification problem aiming at predicting whether the price of a trip would rise, fall or remain unchanged in n days. It is a three-class classification problem, which was solved for several values of n, i.e., n = 7, 14, 21, 28. It means the predictions applied to 1, 2, 3, 4 weeks forward. The classification problem defined this way simplifies the problem to be solved in the whole project, i.e., the prediction of trip prices, which is to be solved in future stages, where several time series prediction algorithms would be implemented and tested. However, this simplified task is sufficient to determine the optimal set of factors or a set of factor weights, which could be applied in the next stage. In order to evaluate the influence of input parameters on price dynamics, several feature selection algorithms were applied. Figure 2 shows a diagram presenting the whole process implemented to evaluate the impact of various factors on the changes of trip prices. The following subsections describe in detail the stages of that procedure and present the obtained results. Appl. Sci. 2022, 12, 12938 7 of 31 Data frames Trip representation (feature extraction) Feature vectors Splitting data into training and testing subsets for n =7,14,21,28 Training and testing subsets Feature selection Selected value of k Mutual information (MI) MI values Random forest impurity decrease (ID) ID values Random forest accuracy decrease (AD) after feature permutation AD values Decision tree + sequential forward selection Sequence of features and accuracies Random forest + sequential backward elimination Sequence of features and accuracies Preliminary analysis of method parameters Results aggregation Final evaluation of all features obtained for different values of n Figure 2. The process of feature evaluation. 3.1. Trip Representation The aim of the data set preparation stage, which was a highly time-consuming process, was to generate a set of feature vectors representing trips. The available raw data required a complex processing. The data originated from two tour operators identified as T1 and T2 in subsequent sections. Two types of trips, depending on the transport type, were taken into account, i.e., flight and own transport. Evaluation of features was performed independently for these two trip types. Due to huge amounts of available data, the first step was to sample a set of trips for future analysis. A few restrictions were imposed during the sampling process: no missing values were accepted, a trip was supposed to be offered at least 30 times and at least one price change should have been observed during the time the trip was offered. In this way, 51,419 (61% from T1) trips with flight transport and 34,839 (98% from T1) with their own transport were sampled. Each trip was a source of a number of feature vectors. The available raw data enabled us to define a set of handcrafted features representing a trip at a given time. Such a representation vector of a trip in a given moment contained a set of constant parameters equal for all vectors generated on the basis of that trip and a set of varying parameters different for each vector originating from the given trip, depending on the day. Appl. Sci. 2022, 12, 12938 8 of 31 The constant parameters included accommodation and meal attributes, characteristics of local attractions, average weather parameters, season attributes indicating, for example, any holidays at the time of the trip, length of the trip, distance between the starting and destination locations and GDP per capita being a constant factor, because data were gathered during one year. Moreover, for trips with flight transport, several flight and airport attributes were incorporated. Time-varying variables included trip price per person per day, macroeconomic indices, and current temperature, both in the starting and destination location. Moreover, a few additional features were extracted on the basis of raw data. It was delta denoting the number of days from the first day a trip was offered, changes denoting the number of times the trip’s price was already changed, days_till_departure denoting the number of days from the current day until the departure, and month denoting the current month. For some of the time-varying parameters, seven new features were extracted to represent the rate of change of these variables. To do it, the best fit slope was calculated on the basis of k last values of the corresponding parameter. The new features representing the slope are called by adding a _serie suffix to the original name, e.g., barrel_price_serie was calculated on the basis of k last values of barrel_price. It was performed for k = 2, 3, 4, 5, 6, 7 to observe the influence of k on further results, which is shown further in Section 3.3.1. To sum up, 56 features for trips with own transport and 60 for flight transport were extracted. The four flight attributes present only in the case of trips with flight transport were as follows: flight_type_name, airport_distance, destination_from_airport_passengers, and direct_flight. The full list of features divided into groups is presented in Table 1. Depending on the value of n and the values of k, training data sets of different sizes were created, as it is shown in Table 2. If, for example, a trip was offered from 1 April to 30 April, then at most 23 feature vectors for n = 7 could be extracted on the basis of that trip, provided the trip was offered every day in April. Usually some gap days appeared during the lifetime of a trip. Therefore, the possible number of extracted vectors was usually lower. Table 2 presents numbers of feature vectors for k = 3. For higher values of k, the sizes of data sets were lower, because higher values of k required longer sequences of days when a trip was offered continually, which was less likely. The final stage of the preprocessing was transforming categorical features to numerical ones and normalizing all parameters. Table 1. Feature set. Features Description Constant parameters length Length of the trip in days tour_distance Distance between the starting and destination location start_month, high_season, affected_by_holiday Attributes of the season of the trip hotel_chain, category, categorized, number_of_restaurants, child_attractiveness, hygiene, health_services, swimming_pool, golf_course, fitness Hotel attributes room_type, room_attractiveness, meal_code, persons_number, max_persons, room_kitchenette Room attributes heritage, historic, museum-gallery, attraction, UNESCO, protected_area, hiking, coastline, lake Characteristics of local attractions Appl. Sci. 2022, 12, 12938 9 of 31 Table 1. Cont. Features Description destination_to_departure_date_temp, destination_from_departure_date_temp, destination_to_avg_temp, destination_to_avg_rainfall, destination_to_avg_number_of_sunny_days, destination_to_avg_water_temp destination_from_airport_passengers, airport_distance, flight_type_name, direct_flight GDP per capita Weather parameters the climate describing Flight and airport attributes, taken into account only for trips with flight transport GDP per capita in destination location, classified as a constant attribute, because data were originated from one year Time varying parameters personday_price Trip price per person, per day usd_avg barrel_price Unemployment_Rate Inflation_Rate Inflation_Rate_PL USD average exchange rate in zlotys Oil price per barrel Unemployment rate in destination location Inflation rate in destination location Inflation rate in Poland, where the trips are offered destination_to_current_temp destination_from_current_temp Temperature in the destination location Temperature in the starting location destination_to_current_temp_serie, destination_from_current_temp_serie, usd_avg_serie, barrel_price_serie, Inflation_Rate_serie, Inflation_Rate_PL_serie, Unemployment_Rate_serie delta Parameters representing best fit slope for the selected time-varying parameters Number of days from the first day a trip was offered Number of times the trip’s price was already changed Current month Number of days from the current day until the departure changes month days_till_departure Table 2. Sizes of data sets for various n and transport types, for k = 3. Touroperator Transport n=7 n = 14 n = 21 n = 28 Features T1 Own Flight 1,758,117 1,237,725 1,683,516 1,204,965 1,547,542 1,067,636 1,406,752 971,438 56 60 T2 Own Flight 23,890 621,135 21,078 545,807 17,784 514,031 15,340 417,984 56 60 3.2. Splitting Data into Training and Testing Subsets The classification tasks defined, as it has been described, and constrained some requirements on the way of dividing data into training and testing subsets. Standard crossvalidation was not appropriate in this case. The task corresponds to predicting price changes in future. To obtain unbiased error estimates, it was necessary to ensure that training samples referred to periods earlier than the testing ones. Therefore, several data splits have been applied and the results were averaged over these splits. Table 3 presents the time ranges covering successive splits of the data set into training and testing subsets. Appl. Sci. 2022, 12, 12938 10 of 31 Table 3. Splitting data into training and testing subsets. Test Data Split Training Data 1 2 3 1 January–30 April 1 January–31 May 1 January–30 June 4 1 January–31 July 5 1 January–31 August 6 1 January–30 September n=7 n = 14 n = 21 n = 28 7 May–6 June 7 June–7 July 7 July–6 August 7 August–6 September 7 September–7 October 7 October–6 November 14 May–13 June 14 June–14 July 14 July–13 August 14 August–13 September 14 September–14 October 14 October–13 November 21 May–20 June 21 June–21 July 21 July–20 August 21 August–20 September 21 September–21 October 21 October–20 November 28 May–27 June 28 June–28 July 28 July–27 August 28 August–27 September 28 September–28 October 28 October–27 November 3.3. Preliminary Analysis of Method Parameters Before a thorough feature investigation, some preliminary analysis was performed in order to fix a value of series length k and to confirm that the selected classification methods were adequate for future analysis. 3.3.1. Series Length for Time Varying Features The aim of this step was to evaluate the influence of parameter k on the quality of time-varying features, calculated as the best fit slopes on the basis of the last k values. Mutual information coefficient was chosen as a measure of usefulness of these features in the defined classification task. It measures the amount of information shared by the two variables together and is defined as follows [25]: I ( X; Y ) = ∑ ∑ x i ∈ X y i ∈Y P( xi , yi )log P ( xi , yi ) , P ( xi ) P ( yi ) (1) where P( xi , yi ) is the joint probability of xi and yi . In this case, variables X and Y are the feature and the class. Figure 3 presents the values of the mutual coefficient for the seven features calculated on the basis of series of values depending on series length k. The dependency has been demonstrated for each value of n. It can be observed that MI for barrel_price_serie and usd_avg_serie almost does not change for different values of k. For other features based on the series, higher values of k lead to higher MI, suggesting that longer series lead to better features in our classification problem. This regularity is less clear only for destination_from_current_temp_serie. However, it should be noted that the inflation and unemployment indices are updated no more than once a month. It means that the best fit slope calculated on the basis of values from the last several days is usually 0. The longer the series, the higher chance that any change in inflation or unemployment rate is observed. The low variance of these features based on the short series is the reason for lower MI. Another important observation is that usually lower values of n are associated with higher MI values, which means that the proposed features seem to be better predictors in the case of shorter forecast horizons. Appl. Sci. 2022, 12, 12938 11 of 31 Figure 3. Mutual information values obtained for time varying features for different values of series lengths. 3.3.2. Performance of Selected Classifiers In the case of some feature selection methods, their effectiveness in the classification task is evaluated using a classifier. Before selecting features, the classifiers were trained on the basis of a full set of features in order to verify whether the classifiers chosen as evaluation Appl. Sci. 2022, 12, 12938 12 of 31 models might achieve satisfying results. This step is necessary, because evaluating features on the basis of a weak classifier would not lead to finding good predictors. A classifier chosen for this study was a random forest. According to the no free lunch theorem, no machine learning algorithm is universally better than any other [26]. The choice of method depends on the training goal, the type and amount of available training data. Random forests are known to achieve high accuracies in various application and they successfully cope with the large number of features of any types. The numbers of samples in the splits were not equal, so any averages over splits were weighted averages. In each split, the training and test sets contained an equal number of samples per class. The training process was repeated six times for the splits defined in Table 3. Classification accuracies for each split, each data set and various series lengths are presented in Figure 4. The accuracies averaged over the splits and weighted by the number of feature vectors in the test sets, are shown in Figure 5. It has been performed for data from each tour operator separately and for the data set containing all vectors. More detailed results achieved for series length 3 are presented in Tables 4–6, where the values of metrics such as precision, recall and F1-score, calculated separately for each class and averaged over classes, are presented. Figure 4. Classification accuracy obtained for various data splits. Appl. Sci. 2022, 12, 12938 13 of 31 Table 4. Classification results for data from both tour operators and series length 3. n Metric Down No Change Up Avg 7 Precision [%] Recall [%] F1 score[%] 90.05 74.14 80.9 76.98 87.24 81.34 80.13 80.15 79.35 82.39 80.51 80.53 14 Precision [%] Recall [%] F1 score [%] 83.51 70.97 76.31 69.04 66.07 65.32 61.61 68.72 63.67 71.39 68.59 68.43 21 Precision [%] Recall [%] F1 score [%] 74.29 59.47 65.15 59.02 51.17 52.59 52.05 63.89 55.63 61.79 58.18 57.79 28 Precision [%] Recall [%] F1 score [%] 70.9 56.74 62.53 54.38 59.16 55.3 56.38 58.52 54.15 60.55 58.14 57.33 Figure 5. Average classification accuracy. Appl. Sci. 2022, 12, 12938 14 of 31 Table 5. Classification results [%] for data from tour operator T1 and series length 3. n Metric Down No Change Up Avg 7 Precision [%] Recall [%] F1 score [%] 89.05 75.4 81.19 82.03 85.25 83.07 78.67 84.21 80.6 83.25 81.62 81.62 14 Precision [%] Recall [%] F1 score [%] 77.66 64.69 69.45 81.67 56.44 63.12 60.05 80.23 66.8 73.13 67.12 66.46 21 Precision [%] Recall [%] F1 score [%] 63.64 56.48 57.76 57.71 33.59 40.22 48.03 69.96 55.31 56.46 53.34 51.1 28 Precision [%] Recall [%] F1 score [%] 52.78 57.03 53.87 49.56 43.43 44.83 53.74 53.18 50.96 52.03 51.21 49.89 The presented results demonstrated that the smaller n, the higher classification accuracy, which can be observed especially for data from tour operator T1 and for the combined set of feature vectors. It is not equally clear for tour operator T2, but it should be noted that the number of data from T2 is lower than from T1. It can be also said that some splits, i.e., split 4 and 5, show much lower accuracies. In the case of data from tour operator T2, there were very little samples for split 5 and n = 28, which resulted in low accuracy, and can be observed in Figure 4; for split 6, there were not enough data samples from T2 to train the models at all. The highest scores obtained for n = 7 exceed 80% for each metric. The lowest results obtained for n = 28 are slightly below 60%, which is still much higher than a random guess in the case of three classes. Such results demonstrate that the classifiers might be chosen as evaluation models during the feature selection process. Table 6. Classification results [%] for data from tour operator T2 and series length 3. n Metric Down No Change Up Avg 7 Precision [%] Recall [%] F1 score [%] 99.6 64.35 77.01 55.27 99.14 70.4 98.55 49.78 62.05 84.47 71.09 69.82 14 Precision [%] Recall [%] F1 score [%] 95.59 84.76 89.64 56.88 93.49 70.32 87.85 38.36 50.73 80.11 72.2 70.23 21 Precision [%] Recall [%] F1 score [%] 93.97 69.92 79.66 53.92 84.87 65.03 69.59 46.12 53.89 72.49 66.97 66.19 28 Precision [%] Recall [%] F1 score[%] 92.68 64.32 75.08 64.53 76.27 68.96 61.95 63.35 58.22 73.05 67.98 67.42 The other important observation is that there are no significant differences between the results for different series lengths k, even if some relation between k and mutual information was discovered for inflation and unemployment rates, as it was shown in Section 3.3.1. This observation led us to choose k = 3 for further experiments on feature selection. Lower value of k let us to achieve larger data sets, as it has already been mentioned. 3.4. Feature Selection Feature selection is the process of reducing the size of the set containing all original attributes describing the data. The dimensionality reduction achieved in this way not only reduces the computational and space complexity, but may also influence the accuracy of Appl. Sci. 2022, 12, 12938 15 of 31 the trained model, which may have better generalization ability. Feature selection methods fall into one of the following categories: • • • Filter methods apply an evaluation function measuring the dependence between a given feature and a target value. Example criteria used as the evaluation function are Pearson coefficient, Spearman coefficient, mutual information. The advantage of filter methods is their low computational cost, but the drawback is the fact that they treat the predictors independently, neglecting potential correlations between them. A feature that is useless alone may be significant in combination with other features [27]. Wrapper methods apply a trained model to evaluate the effectiveness of a selected feature subset. Depending on a given task, the model may be either a classifier or a regression model. These methods require choosing a feature space search strategy, leading to an iterative process. In each iteration, a new subset of features is generated, a model is trained and the subset is evaluated according to the model’s performance. Searching the whole space of feature subsets is infeasible; therefore, some heuristics are applied. One of the most popular ones are sequential methods, where in each iteration, features are either added to or removed from the current subset. The computational cost of wrapper methods is high, due to the need of training a model whenever a new subset of features has to be evaluated. To reduce the computational complexity, the recursive feature elimination proposed in [28] may be applied. This algorithm reduces the necessary number of repetitions of the model training procedure. In each stage, after creating a model, features are ranked and the least important predictor is eliminated. Then, the model is rebuilt on the basis of the reduced set of features. Embedded methods are feature selection algorithms constituting integral parts of the model training procedures. These are, for example, decision trees, where at each step of the decision tree construction, a feature is selected on the basis of a criterion, e.g., Gini index; or LASSO, which is able to find a linear regression model reducing the coefficients of some parameters to zero. One might say that deep learning models applied nowadays do not require any feature selection procedure before training the model. However, even in this case it is a good idea to remove irrelevant features before training the model to reduce space and time complexity. Moreover, neural networks are blackbox models, which do not enable one to interpret the meaning of particular inputs. There are applications where some interpretation of the obtained knowledge representation is valuable or even required. Finally, knowledge about irrelevant parameters might prevent one from collecting unnecessary data in future, which might result not only in reducing the required space but also costs the data collection process. During the performed experiments, the following feature selection methods have been implemented: 1. 2. 3. 4. 5. Filter method with mutual information criterion to evaluate the dependence between a feature’s values and classes; Embedded method with a random forest enabling to assign weights to the features on the basis of a mean decrease in impurity; Random forest letting one assign weights to the features on the basis of a decrease in the forest’s accuracy after a random permutation of a feature’s value; Wrapper method with sequential forward selection as a search strategy and a decision tree as a classification model; Wrapper method with recursive feature elimination as a search strategy and a random forest as a classification model. Is is worth mentioning that all implemented methods are supervised methods, i.e., class labels are taken into account in the process of feature selection, either by calculating some correlation between features and classes or by applying a classification model to inspect the power of features in the given classification task. In general, feature selection might Appl. Sci. 2022, 12, 12938 16 of 31 also be implemented in an unsupervised manner, but this type of selection is designed for clustering problems, which is not in this case [25]. In the case of embedded methods, decision trees or random forests, which are ensembles of decision trees, were selected [29]. These ensembles are sets of decision trees, that differ thanks to applying two types of randomness. The first one is bagging, which means that the training set for each tree is drawn, with replacement, from the original data. The second is the random selection of features at each node to define the splitting rule. Random forests are known to achieve a low generalization error, especially in classification tasks. In the case of sequential forward selection, we chose the decision tree instead of the random forest to reduce the time-complexity of this procedure. 3.5. Results’ Aggregation Although the aim of all methods mentioned above was the same, the obtained results might vary more or less. Therefore, it was necessary to propose an algorithm able to summarize the results generated by the five methods and present a final feature ranking. In the case of the first three feature selection methods, the features are assigned values, which may be treated as weights, i.e., higher mutual information or impurity reduction mean better discriminative power of a given feature. These values have been normalized to a range of [0, 1]. In the case of sequential methods, no weights have been extracted, but the wrapper approach lets us select a subset of features that enables for creating a prediction model demonstrating satisfying accuracy. We assign 1 to the features selected by a sequential method and 0 to all the others. Figures 6 and 7 present the way an optimal subset of features is selected during the sequential procedure. The graphs are generated for an example subset of flight trips data. The proposed method is as follows: 1. 2. 3. 4. 5. 6. For each iteration of the sequential feature selection, the algorithm estimates the accuracy of the wrapper model on the basis of a separate test set (blue lines on Figures 6 and 7). Smooth the series obtained in the previous step using the moving average of width 5 (orange lines). In the case of the sequential forward selection (Figure 6), identify the initial segment of the series, where the accuracy increases by at least 1 pp in each subsequent iteration. In the case of the sequential backward selection (Figure 7), identify the ending fragment of the series, where the accuracy decreases by at least 1 pp in each iteration. Omit the fragment of the series identified in the previous step and calculate the average value on the basis of the remaining part. These values are marked with green dashed lines on Figures 6 and 7. In the case of sequential forward selection (Figure 6), find the fifth feature, which makes the accuracy exceed the average value determined in the previous step. In the case of the sequential backward selection (Figure 7), find the feature, after removing which five more can be removed without dropping the accuracy below the average value determined in the previous step. The feature (iteration) identified in this way is marked with red dashed lines. In the case of sequential forward selection, select the features from the first to the one identified in the previous step. In the case of the sequential backward selection, select the features starting from the one identified in the previous step to the last one. Appl. Sci. 2022, 12, 12938 17 of 31 Figure 6. The accuracy of a model trained during the sequential forward selection (blue), smoothed accuracy (orange), accuracy averaged over the series excluding the segment of significant increase (green), identified iteration (red). Figure 7. The accuracy of a model trained during the sequential backward selection (blue), smoothed accuracy (orange), accuracy averaged over the series excluding the segment of significant decrease (green), identified iteration (red). As it has been mentioned in Section 3.4, five feature selection methods have been applied in order to evaluate the features. In the results, each method assigned a weight value Wi to every predictor, indicating its ability to predict whether the trip price would go up, down or remain unchanged. The weights are: • • • • • W1—weight assigned according to mutual information value; W2—weight assigned on the basis of a mean decrease in the impurity of a random forest; W3—weight assigned according to the decrease in a random forest’s accuracy after feature values permutation; W4—weight assigned on the basis of the results of the sequential forward selection method; W5—weight assigned on the basis of the results of the sequential backward elimination method. Weight W1 was calculated only once for the whole set of data. The other four weights required model training and testing to evaluate accuracy. In these cases, the evaluation was performed for all five splits and the resulting weight values were averaged over the Appl. Sci. 2022, 12, 12938 18 of 31 splits. Split 6 was omitted, because there were not enough data samples from T2 in the corresponding subset. To obtain the final evaluation, the sum of the weights was taken into account. Table A1 presents the summary values for each feature obtained for each value of n and for both sets of data, i.e., those with flight and their own transport type. The summary results have been also presented in Figures 8–11 for n = 7, 14, 21, 28, respectively. Features on these bar plots are sorted according to the results obtained for trips with flight transport. Due to large amounts of results, the detailed values obtained by applying individual methods are presented only for top 10 features identified for each value of n both for the flight (Table A2) and their own transport (Table A3) transport type. Figure 8. Final evaluation of features for n = 7. Blue and orange bars present the sum of the weights obtained by all five methods. Black bars indicate the result obtained using sequential backward elimination. Appl. Sci. 2022, 12, 12938 19 of 31 Figure 9. Final evaluation of features for n = 14. Blue and orange bars present the sum of the weights obtained by all five methods. Black bars indicate the result obtained using sequential backward elimination. Figure 10. Final evaluation of features for n = 21. Blue and orange bars present the sum of the weights obtained by all five methods. Black bars indicate the result obtained using sequential backward elimination. Appl. Sci. 2022, 12, 12938 20 of 31 Figure 11. Final evaluation of features for n = 28. Blue and orange bars present the sum of the weights obtained by all five methods. Black bars indicate the result obtained using sequential backward elimination. 3.6. Discussion First of all, it should be mentioned that the two types of features, i.e., time-varying and constant ones, play different roles in solving the stated classification problem. Time-varying parameters are obviously those that may have an impact on the price changes. Constant parameters, on the other hand, may cluster data into various groups, where the prices follow different rules. In our experiments, we analyzed all of them together, as most of the applied methods are tree-based methods, where the feature space is sequentially partitioned on the basis of selected features. In that way, data from various space regions may traverse various decision paths on the basis of all available parameters. Another possible approach could be applying a clustering algorithm only on the basis of constant attributes and then train a separate classifier for each cluster on the basis of time-varying features. Feature rankings obtained for the different values of n do not vary much. The current price per oil barrel (barrel_price) and USD average exchange rate (usd_avg) are at the top in each case of n, both for the flight and own transport type. An interesting observation is made for features representing the rate of changes of the above two parameters, i.e., barrel_price_serie and usd_avg_serie. They are also significant predictors, as they usually appear in the top 10 subsets, but it can be observed that the lower n, the higher the position of these two features. It means they are better in shorter time predictions. Another essential predictor is delta, representing the number of days a trip is already offered. In each case, it Appl. Sci. 2022, 12, 12938 21 of 31 takes places from three to five, no matter the transport type and the value of n. Among features that are not time-varying, the tour_distance is the most significant, no matter the transport type. Weather features did not turn out to be important. They usually fall at the end of the ranking list. The only exceptions are current temperatures in the destination and the starting location (destination_to_current_temp and destination_from_current_temp) in the case of flight trips for n = 7. This means that changes of weather are not essential for more predictions in the longer horizon. Comparisons between trips with flight and own transport indicate strong similarities. The same parameters are on the top of the ranking lists. There are several features that are often higher in the case of their own transport. These are fitness, attraction, and Inflation_rate_PL. On the other hand, month and changes appear to be better predictors in the case of flight transport. Apart from the fitness, which has already been mentioned, other hotel attributes do not demonstrate a high significance in solving the classification task. It is also interesting to analyze the results of the sequential backward elimination, which is one of the wrapper methods that lets us find a subset optimal from the point of view of the accuracy of the applied model. The weights assigned to the features using this algorithm have been demonstrated using black bars in Figures 8–11. To indicate an optimal m-element subset of features, it is important to make sure that the set of the top m predictors presented on the obtained ranking list contain a complete subset of features identified by the sequential method. There might be a feature, which has the discriminative power in combination with others, but individually it does not appear to be significant. Such a feature would not achieve high results using the methods that treat features independently, but it would using the method that takes into account feature correlations, e.g., sequential backward elimination. It can be observed that in the case of our data, the subsets selected by sequential backward elimination and on the basis of all methods together usually overlap to a great extent, especially for lower values of n. For larger n, this correlation is lower. For n = 28, there are several features achieving low results with the sequential method, but are quite high in total. In the case of flight transport, these parameters in are: Unemployment_rate, usd_avg_serie, barrel_price_serie, Inflation_rate_PL, protected_area and catgorized. In the case of own transport these are: Unemployment_rate, month, barrel_price_serie, categorized and, destination_to_current_temp. It should be noted that the two tour operators, which were the source of data sets, offered selected types of trips; therefore, any conclusions that are drawn from the presented analysis might refer to those trip types. However, a similar methodology might be applied for another data set. 4. Model of the Price Change Process As it is known from the previous sections, a trip is a basic data unit, and the price is its attribute that can change over time. The rest of the trip attributes are created based on factors that hypothetically affect the price, which can be constant or changing over time, within a single trip lifetime. The first step of modeling business processes is the ability to define the sequence of distinguishable events. The event has a specified beginning and an end, and after its completion, another event can be performed according to a workflow defined a priori or ad hoc. Events can be performed sequentially or in parallel, and can also describe a single action (e.g., click “send”) as well as some task to be performed (e.g., send a package to a customer). Hence, in order to model processes, it is necessary to change the attributes over a time, which causes the workflow to shift from one state to another. The solution described in this section is to model the price change process using process mining techniques [4]. The price is expressed by two factors of the trip: baseprice and personday_price. From both of these factors, it is possible to calculate whether the price has increased or decreased. The increase and decrease in the price—not the value of the price difference—are recorded as events. The personday_price was selected for further Appl. Sci. 2022, 12, 12938 22 of 31 research as, according to experts from the tourism industry, it is more reliable. Both prices change proportionally and can therefore be used interchangeably. As it has been already mentioned in Section 1, raw data collected from tourist industry databases and publicly available external databases were first formatted to predefined structures, saved on the HDSF and they are then available in the form of Data Frames. The first step needed to use process mining techniques is the appropriate transformation of the Data Frame into an Event Log (observe arrow no. 1 in Figure 12). This transformation is discussed in Section 4.1. Based on the Event Log, the entire set of process mining tools may be used to model and analyze business processes (see arrow no. 2 in Figure 12). Data Frames Event Log 1 Process Model 2 Figure 12. Steps to convert the Data Frames into a process model. The purpose of these transformations is to analyze the price change process in order to discover the influence of changing factors on the price and find other interesting relationships. An outline of the process mining experiments is in Section 4.2. 4.1. Event Log for Price Changes The event log design for trips is based on the eXtensible Event Stream (XES) format [30]. Its main element—the root—is log, and it contains all realized flows of the analyzed process. A log element contains a number of trace elements, each of which is a representation of a single process flow. A trace element consists of a number of event elements, where an event can be understood as a specific activity performed at a given time as part of the single process flow. The XES format allows for adding an unlimited number of attributes at different levels of the tree (for log, trace, and event elements). These attributes allow for adjusting the Event Log syntax to the needs of a specific project. Figure 13 shows the simplified UML diagram of the Event Log with an overview of what trip data have been assigned to the specific elements. The PM4Py [31] library was used to generate Event Logs and model business processes. This library allows the creation of an object-oriented representation of the event log. Since a lot of data was processed in the project, dedicated methods were implemented that transformed tourist data from the Data Frame directly into Event Log objects, bypassing its XML representation. This data transformation process takes the longest time; of course, the longer it is, the more data there is to transform. Therefore, before the transformation, the data were pre-filtered and sampled. For example, only those trips where the price changes at least once were selected. Trips with less than 30 days (last minute) and more than 30 days until departure are separated into two Event Logs. When it is known in advance what data are expected in the log, pre-filtering the data and then generating the Event Log is much more efficient than creating a very large Event Log and then filtering it. Appl. Sci. 2022, 12, 12938 23 of 31 log Contains all trips selected for analysis. Its attributes are global for all trips, eg. number of trips, time frames, etc. 0..n trace 0..n event 0..n Represents a single trip, its name is the trip ID, and its attributes are constant factors. There are two types of event: „basic”, created from the daily records of the trip; „change”, created whenever price or any of the changing factors have changed their value. attribute Figure 13. The simplified UML diagram of the Event Log. 4.2. Process Model Process models can be discovered based on the entire Event Log or filtered by attributes. It allows for analyzing the data at different levels of detail or dividing it into groups in relation to one of the constant factors. Filtering can be conducted on the level of whole traces or individual events. There are many process mining tools that could be used to analyze the Event Logs. There were mainly four methods used: 1. 2. 3. Directly-Follows Graph (DFG)—a simple representation of the process models. Each node represents an activity with the frequency of occurrences and the arcs describe the relationship between various activities. The weight of the edges is the number of transitions from one activity to the next. The MAX_NO_EDGES_IN_DIAGRAM parameter allows for adjusting the detail of the graph. Heuristics Miner—use a representation similar to DFG with frequencies of events and sequences, called a heuristics net. The basic idea is that infrequent paths are omitted from the model. Whether an arc will be added to the model is determined by the dependency value greater than the value of the DEPENDENCY_THRESH parameter. Transition Matrix (SM)—from the heuristics net object, the activities_occurrences and dfg_matrix can be read. Based on these data, Transition Matrices were calculated. In the matrix cells, there are values corresponding to the problem: what percentage are the situations in which the change of one factor immediately precedes the change of the other factor. The formula is: TM A,B = 4. n( A − → B) · 100% n( A) (2) where: A, B—activities denoting the change of the factor, e.g., OPEC up n( A)—number of occurrences of an activity A n( A − → B)—number of direct transitions from an activity A to B Cortado Variant Explorer [32] is one of the views generated by the Cortado tool. It clearly presents variants of the occurrence of activities with their actual sequence preserved. This tool also allows for generating process models based on sequences of activities, but in the problem under consideration, these models did not give the expected results. Using the above-mentioned methods, tourist data were analyzed in many different variants, and thus many models were generated that cannot be discussed in detail in this paper. One changing factor was chosen to demonstrate the process mining example: the barrel of oil price (OPEC). Other Event Log settings are presented in Table 7. The Event Log was created for one tour operator. Appl. Sci. 2022, 12, 12938 24 of 31 Table 7. Event Log settings. offers start day Offers end day Trips start day Trips end day Days until departure condition Traces 1 March 2021 30 November 2021 1 March 2021 30 November 2021 <30 8514 The fuel price change factor is recorded when the absolute value of the difference of the last two prices is greater than 0.5$. The activity “OPEC up” means a fuel price increase and similarly “OPEC down” means a fuel price decrease. The same naming rule has been applied to the trip prices. For trips, every price change is recorded, without any thresholds. Figure 14 presents two DFGs with different levels of detail. Figure 14 shows the number of occurrences of a given change and the transitions between them. It is quite general, with no dates or individual sequences. It can noticed that the vast majority of trip prices have increased in the last 30 days before departure and considering the relation from OPEC to PRICE, the most numerous transitions are from “OPEC up” to “PRICE up”. This model is used to compute the Transition Matrix shown in Table 8. Figure 14 shows a fragment of DFG with dates. It can be observed that the decline in OPEC prices took place on 17 June and 18 June, while on 19 June, 707 trip prices dropped. On the other hand, on 22 June and on 23 June, the fuel prices increased, while on 30 June, the prices of 1215 trips increased. (a) The whole graph without dates (b) A fragment of the graph with dates Figure 14. DFG with OPEC and PRICE factors. Table 8 shows that after the increase in OPEC prices, about 15% of trip prices increased, and this is the greatest relationship. However, also after the decline in OPEC prices, around 9% of the trip prices rose. This situation may be due to the fact that prices are mostly rising 30 days before departure and OPEC is just one of many factors that can influence it. Moreover, the table does not take into account the series of OPEC price changes. Table 8. TM for relation with OPEC − → PRICE. A: OPEC Up OPEC Down A− → PRICE up 14.94% 9.05% A− → PRICE down 4.14% 6.79% Appl. Sci. 2022, 12, 12938 25 of 31 The series of changes can be analyzed, for example, using the Cortado Variant Explorer tool. Figure 15 presents the 29 most common sequences of price changes. A frequent situation (circled in the Figure) is an increase in the trip prices after the series of OPEC price increases and a fall in the trip prices after a series of OPEC price drops. This may indicate that with frequently changing factors, the trend of these changes, not a single change, may contribute to a change in the trip prices. Figure 15. Series of changes generated by Cortado Variant Explorer. These types of process mining actions were performed for different factors and filters, allowing many different conclusions to be drawn. However, they are too numerous and detailed to list in this paper. There are, however, a few general conclusions worth mentioning here. Firstly, trip prices generally also increase in the last 30 days before departure—so in most cases, the cheapest trips are at the beginning of the offer (first minute). Prices rarely change and there are some single days on which many prices change, e.g., at the beginning of the month. A series of factor changes show a greater impact than single changes. Economic factors demonstrate a great influence, but they rarely change (e.g., once a month). In Poland, a drop in air temperature affects the increase in the prices of the last minute trips to warm countries. 5. Conclusions The presented results confirmed that time-varying factors have the greatest impact on changes of trips prices. Among them, currency rates and oil barrel price and their rates of change are the most significant ones. The number of days a trip has been already offered also turned out to be important. However, some constant variables are also worth being taken into account. Among them, the tour distance seems to be essential. The results of our work is a part of the innovative solutions enabling the effective selection of travel offers by consumers based on information provided by price prediction mechanisms implemented as meta-search engines. Moreover, these solutions enable ad- Appl. Sci. 2022, 12, 12938 26 of 31 vanced analysis of historical data for the travel market (i.e., tour operators, travel agents, brokers of tourist offers, and owners of hotels in Poland) available as a Software as a Service. Thanks to these solutions, participants of the tourism market, including primarily smaller entities that do not have their own technological facilities, will be able to compete with large players on the market in terms of pricing policy, more effective distribution of tourism products and services, and revenue management. The obtained results might be biased by the time data were gathered, which was pandemic. However, the proposed methodology might be applied to any period. Moreover, when more training data are gathered, it is advisable to rerun the implemented procedures to update the results. Feature rankings might change, especially if training data from several years are available. Several ideas come into mind to try to improve the selection of best predictors in future works. First of all, it is worth taking into account the heterogeneity among trips and across time. Although the tree-based models applied in the presented study are able to partition feature space and make examples follow different decision paths in different regions of the space, there are also other approaches. It is possible to perform data clustering prior to feature selection. Two types of clustering are worth investigating. It may be performed on the basis of expert knowledge, e.g., trips might be grouped depending on the region or on the season. Clustering may be also performed automatically by applying some of the well known algorithms [33–35]. After clustering, the data feature selection could be performed separately in each cluster, letting finding different feature subsets be optimal in different areas of the feature space. Another approach to incorporate some sources of heterogeneity while creating a model was presented in [36], where the authors assumed that individuals living in a particular location have homogeneous preferences. They applied geographically weighted multinomial logit models to estimate preferences over management options for national forest area. In this method, a set of local models together with location weights that incorporate distances between observations and locations was obtained. In this way, it was possible to take into account the fact that the relation between variables varied across space. It is also possible to account not only for the spatial heterogeneity but any other, including unobserved variable heterogeneity, by applying the mixed logit model. In [37], it was even demonstrated that model performance might be improved if the heterogeneity was modeled among cohorts, i.e., groups of individuals sharing common characteristics at a time or experienced a common event, rather than individuals. Another idea to improve the power of price predictors is to investigate other features not taken into account in this study. One such feature could carry information on some events at the destination location. Obviously, any natural disasters or terrorist threats might reduce interest on a given location. On the other hand, some sport or cultural events may attract more tourist than usual. All of these may have an impact on trip prices. Information on the events should be extracted automatically from some available sources, e.g., Wikipedia Current events or social media platforms. This complex task requires implementing web mining and natural language processing techniques [38,39]. Mining the web could also deliver valuable information on current tourist destination preferences, which should be added as another parameter worth investigating. A very interesting research could be a thorough investigation of the idea presented in Section 4 and comparing the results generated by two different approaches, i.e., process mining and feature selection. All efforts made in order to eliminate irrelevant or redundant information would benefit the future system of price prediction, as it may improve the accuracy of the final prediction model. Moreover, it would reduce the time and space complexity of the implemented algorithms, which require training the models on the basis of large volumes of data. Finally, the elimination of some parameters may bring economic benefits during the data collection stage, because of the high costs of gathering and integrating data, both in the sense of time and money. Appl. Sci. 2022, 12, 12938 27 of 31 Author Contributions: Conceptualization, A.K. and M.G.; feature selection (methodology, software, experiments, and analysis), A.K.; process mining (methodology, software, experiments, and analysis), M.G.; writing, reviewing and editing, A.K. and M.G. All authors have read and agreed to the published version of the manuscript. Funding: This research was supported by the National Centre for Research and Development, Poland under the contract no POIR.01.01.01-00-1252/19-00, as well as by funds of the ETI Faculty, Gdansk University of Technology. Institutional Review Board Statement: Not applicable. Data Availability Statement: Data sets were obtained from the Q&Q travel agency and are not publicly available. Conflicts of Interest: The authors declare no conflict of interest. Appendix A. Summary Results Obtained for the Features Identified in Classification Problems Table A1. Features’ weights assigned for four classification problems and two types of transport. Transport Type: Flight Feature direct_flight room_type meal_code persons_number max_persons length flight_type_name personday_price room_attractiveness airport_distance destination_from_airport_passengers high_season destination_to_current_temp destination_to_departure_date_temp destination_from_current_temp destination_from_departure_date_temp destination_to_avg_temp destination_to_avg_rainfall destination_to_avg_number_of_sunny_days destination_to_avg_water_temp days_till_departure affected_by_holiday tour_distance usd_avg barrel_price GDP per capita destination_to_current_temp_serie destination_from_current_temp_serie usd_avg_serie barrel_price_serie Inflation_Rate_serie Unemployment_Rate_serie Inflation_Rate_PL_serie delta hotel_chain category categorized Transport Type: Own n=7 n = 14 n = 21 n = 28 n=7 n = 14 n = 21 n = 28 0.87 0.63 0.69 0.9 0.33 0.4 0.77 1.34 0.39 1.77 0.69 0.85 2.39 0.12 1.82 0.13 0.13 0.33 0.15 0.14 1.2 0.04 2.46 3.48 4.79 0.49 1.04 1.16 2.99 3.16 0.38 0.39 0.16 2.44 0.04 0.54 0.76 0.72 0.32 0.24 0.77 0.47 0.24 0.73 1.3 0.18 1.75 0.38 0.53 2.13 0.15 2.24 0.17 0.17 0.32 0.15 0.18 0.87 0.08 2.41 4.08 4.6 0.26 0.35 0.58 1.99 2.4 0.43 0.4 0.24 2.45 0.28 0.48 0.4 0.9 1.0 0.65 0.9 0.79 0.56 0.75 1.75 0.56 2.1 0.82 0.8 1.43 0.34 2.38 0.53 0.36 0.47 0.6 0.35 1.51 0.37 2.88 3.64 4.13 0.99 0.76 0.53 2.33 2.75 0.57 0.52 0.48 3.11 0.36 0.79 1.05 0.96 1.27 1.08 1.21 1.07 0.84 1.21 2.01 0.56 2.36 0.91 0.99 0.9 0.52 1.56 0.69 0.76 0.66 0.56 0.48 1.21 0.3 2.79 3.89 3.57 0.74 1.11 0.67 1.96 1.91 0.57 0.49 0.7 3.53 0.7 1.04 1.35 0.42 0.28 0.7 0.75 0.2 1.31 0.14 0.4 2.02 0.19 2.48 0.15 0.2 0.11 0.17 0.2 1.24 0.05 2.49 3.6 4.08 1.57 0.28 2.18 3.79 2.88 0.15 0.52 0.35 2.72 0.07 0.73 0.92 0.39 0.3 0.67 0.55 0.58 1.55 0.18 0.78 1.14 0.69 3.17 0.24 0.7 0.14 0.22 0.28 1.14 0.09 2.52 3.36 4.24 0.58 0.23 0.82 2.64 2.2 0.43 0.36 0.47 2.53 0.49 0.33 0.69 0.73 0.43 1.04 0.86 1.75 2.12 0.22 0.91 1.36 1.03 2.36 0.77 0.96 0.15 0.29 0.33 1.51 0.08 2.65 3.24 4.19 0.66 0.45 0.92 2.6 2.33 0.4 0.13 0.2 3.21 0.86 0.67 0.66 0.79 0.69 0.69 0.51 2.01 2.16 0.25 0.76 1.17 1.24 1.62 0.82 0.99 0.19 0.34 0.37 1.53 0.32 2.63 3.9 4.23 0.69 0.26 0.48 1.96 1.68 0.85 0.37 0.47 3.13 0.12 0.72 1.53 Appl. Sci. 2022, 12, 12938 28 of 31 Table A1. Cont. Transport Type: Flight Feature room_kitchenette number_of_restaurants child_attractiveness hygiene health_services swimming_pool golf_course fitness heritage historic museum-gallery attraction UNESCO protected_area hiking coastline lake Inflation_Rate Unemployment_Rate Inflation_Rate_PL changes month start_month Transport Type: Own n=7 n = 14 n = 21 n = 28 n=7 n = 14 n = 21 n = 28 0.46 0.48 0.69 0.24 0.62 0.31 0.59 0.87 0.28 1.71 1.13 0.47 0.84 0.52 0.31 1.32 1.73 1.95 1.57 1.55 1.77 2.23 0.16 0.11 0.37 0.46 0.28 0.41 0.86 0.18 1.06 0.41 0.87 0.29 0.46 0.4 0.44 0.47 1.25 0.88 1.65 0.65 1.12 2.0 1.73 0.16 0.41 0.87 1.18 0.36 0.46 1.09 0.55 0.9 0.53 1.51 1.26 0.7 0.49 0.63 0.64 1.52 1.07 2.05 1.83 2.64 2.51 2.63 0.32 0.82 1.01 1.8 0.5 0.88 1.33 0.73 0.69 0.69 1.45 0.83 0.82 0.85 1.42 0.84 1.88 1.7 2.19 2.06 1.67 3.46 2.11 0.48 0.48 0.68 0.69 0.24 0.67 0.5 0.87 1.91 0.61 1.52 0.93 1.33 0.18 0.43 0.44 1.1 0.5 1.5 1.09 2.17 0.41 1.42 0.42 0.33 0.65 0.47 0.3 1.07 0.92 0.5 1.59 0.45 1.58 1.34 1.2 0.22 0.25 0.68 0.54 0.5 2.27 0.75 1.92 0.95 0.69 0.26 0.11 0.61 0.78 0.05 0.75 0.63 0.37 1.67 0.49 1.57 0.62 1.77 0.24 0.32 1.39 1.05 0.61 2.51 1.41 2.29 1.49 1.08 0.33 0.18 0.87 0.85 0.3 0.38 0.68 0.64 1.7 0.34 1.85 0.88 1.59 0.29 0.35 0.45 1.32 0.86 2.1 1.38 1.74 1.67 0.96 0.4 Table A2. Results obtained for top 10 features identified in each classification problem for trips with flight transport. n 7 14 Feature W1 W2 W3 W4 W5 ∑ barrel_price usd_avg barrel_price_serie usd_avg_serie tour_distance delta destination_to_current_temp month Inflation_Rate destination_from_current_temp 1.0 0.99 0.93 0.92 0.15 0.56 0.34 0.38 0.46 0.3 1.0 0.7 0.49 0.4 0.23 0.7 0.57 0.38 0.22 0.4 0.99 0.59 0.14 0.27 0.08 0.18 0.28 0.28 0.07 0.13 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 0.8 0.2 0.6 0.4 1.0 0.0 0.2 0.2 0.2 0.0 4.79 3.48 3.16 2.99 2.46 2.44 2.39 2.23 1.95 1.82 barrel_price usd_avg delta tour_distance barrel_price_serie destination_from_current_temp destination_to_current_temp changes usd_avg_serie airport_distance 1.0 1.0 0.61 0.13 0.91 0.37 0.34 0.32 0.92 0.11 1.0 0.76 0.57 0.19 0.31 0.5 0.44 0.47 0.34 0.15 0.8 0.72 0.27 0.08 0.18 0.17 0.16 0.21 0.14 0.09 1.0 1.0 1.0 1.0 0.8 1.0 0.8 1.0 0.6 1.0 0.8 0.6 0.0 1.0 0.2 0.2 0.4 0.0 0.0 0.4 4.6 4.08 2.45 2.41 2.4 2.24 2.13 2.0 1.99 1.75 Appl. Sci. 2022, 12, 12938 29 of 31 Table A2. Cont. n 21 28 Feature W1 W2 W3 W4 W5 ∑ barrel_price usd_avg delta tour_distance barrel_price_serie Inflation_Rate_PL month changes destination_from_current_temp usd_avg_serie 1.0 0.98 0.75 0.44 0.98 0.54 0.54 0.43 0.44 0.84 1.0 0.65 0.77 0.39 0.39 0.45 0.41 0.62 0.68 0.38 0.73 0.41 0.38 0.26 0.17 0.26 0.28 0.46 0.27 0.31 1.0 1.0 1.0 1.0 0.6 0.8 1.0 1.0 1.0 0.6 0.4 0.6 0.2 0.8 0.6 0.6 0.4 0.0 0.0 0.2 4.13 3.64 3.11 2.88 2.75 2.64 2.63 2.51 2.38 2.33 usd_avg barrel_price delta changes tour_distance airport_distance Inflation_Rate month Unemployment_Rate personday_price 0.95 0.96 0.8 0.47 0.53 0.43 0.8 0.5 1.0 0.19 0.71 0.82 0.72 0.95 0.57 0.44 0.35 0.53 0.25 0.4 0.63 0.79 0.61 0.65 0.29 0.3 0.25 0.28 0.22 0.43 1.0 1.0 1.0 1.0 1.0 1.0 0.6 0.8 0.2 1.0 0.6 0.0 0.4 0.4 0.4 0.2 0.2 0.0 0.4 0.0 3.89 3.57 3.53 3.46 2.79 2.36 2.19 2.11 2.06 2.01 Table A3. Results obtained for top 10 features identified in each classification problem for trips with own transport. n 7 14 21 Feature W1 W2 W3 W4 W5 ∑ barrel_price usd_avg_serie usd_avg barrel_price_serie delta tour_distance destination_from_current_temp destination_from_current_temp_serie Inflation_Rate_PL destination_to_current_temp 1.0 0.91 0.99 0.92 0.41 0.1 0.4 0.28 0.33 0.15 1.0 0.74 0.8 0.62 0.68 0.29 0.63 0.47 0.48 0.44 0.68 0.53 0.61 0.13 0.23 0.1 0.24 0.23 0.16 0.43 1.0 1.0 1.0 1.0 1.0 1.0 1.0 0.8 1.0 1.0 0.4 0.6 0.2 0.2 0.4 1.0 0.2 0.4 0.2 0.0 4.08 3.79 3.6 2.88 2.72 2.49 2.48 2.18 2.17 2.02 barrel_price usd_avg destination_from_current_temp usd_avg_serie delta tour_distance Inflation_Rate barrel_price_serie Inflation_Rate_PL fitness 1.0 0.98 0.46 0.88 0.45 0.15 0.5 0.92 0.32 0.06 1.0 0.75 0.78 0.48 0.68 0.28 0.19 0.35 0.32 0.17 0.44 0.62 0.92 0.28 0.21 0.09 0.18 0.13 0.28 0.16 1.0 1.0 1.0 0.8 1.0 1.0 0.6 0.6 0.6 0.6 0.8 0.0 0.0 0.2 0.2 1.0 0.8 0.2 0.4 0.6 4.24 3.36 3.17 2.64 2.53 2.52 2.27 2.2 1.92 1.59 barrel_price usd_avg delta tour_distance usd_avg_serie Inflation_Rate destination_from_current_temp barrel_price_serie Inflation_Rate_PL personday_price 1.0 0.95 0.59 0.28 0.81 0.88 0.49 0.96 0.53 0.13 0.99 0.58 0.83 0.48 0.47 0.29 0.51 0.49 0.5 0.77 1.0 0.52 0.39 0.09 0.13 0.14 0.16 0.07 0.26 0.23 1.0 1.0 1.0 1.0 1.0 1.0 1.0 0.6 0.8 1.0 0.2 0.2 0.4 0.8 0.2 0.2 0.2 0.2 0.2 0.0 4.19 3.24 3.21 2.65 2.6 2.51 2.36 2.33 2.29 2.12 Appl. Sci. 2022, 12, 12938 30 of 31 Table A3. Cont. n 28 Feature W1 W2 W3 W4 W5 ∑ barrel_price usd_avg delta tour_distance personday_price Inflation_Rate length usd_avg_serie historic Inflation_Rate_PL 1.0 0.96 0.61 0.3 0.13 0.86 0.07 0.85 0.26 0.53 1.0 0.85 0.71 0.4 0.75 0.26 0.49 0.33 0.28 0.25 0.83 0.89 0.41 0.12 0.27 0.18 0.25 0.18 0.11 0.17 1.0 1.0 1.0 1.0 1.0 0.8 1.0 0.6 1.0 0.6 0.4 0.2 0.4 0.8 0.0 0.0 0.2 0.0 0.2 0.2 4.23 3.9 3.13 2.63 2.16 2.1 2.01 1.96 1.85 1.74 References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. Juszczak, A. Trendy Rozwojowe Turystyki w Polsce Przed i w Trakcie Pandemii COVID-19; Instytut Turystyki w Krakowie sp. z o.o.: Cracow, Poland, 2020. (In Polish) Borthakur, D. HDFS Architecture Guide. Available online: https://hadoop.apache.org/docs/r1.2.1/hdfs_design.html (accessed on 5 July 2022). Apache Spark. Spark Overview. Available online: https://spark.apache.org/docs/3.3.0/ (accessed on 5 July 2022). van der Aalst, W. 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[CrossRef] Ankerst, M.; Breunig, M.M.; Kriegel, H.P.; Sander, J. OPTICS: Ordering Points to Identify the Clustering Structure. In Proceedings of the 1999 ACM SIGMOD International Conference on Management of Data, Philadelphia, PL, USA, 1–3 June 1999; Association for Computing Machinery: New York, NY, USA, 1999; pp. 49–60. [CrossRef] Budziński, W.; Campbell, D.; Czajkowski, M.; Demšar, U.; Hanley, N. Using Geographically Weighted Choice Models to Account for the Spatial Heterogeneity of Preferences. J. Agric. Econ. 2018, 69, 606–626. [CrossRef] Salvo, M.D.; Cucuzza, G.; Prato, C.; Signorello, G. Modeling preference heterogeneity in recreation random utility models when relevant information about users is limited. Aestinum 2020, 75, 5–17. [CrossRef] Foley, J.; Bendersky, M.; Josifovski, V. Learning to Extract Local Events from the Web. In Proceedings of the 38th International ACM SIGIR Conference on Research and Development in Information Retrieval, Santiago, Chile, 9–13 August 2015; Association for Computing Machinery: New York, NY, USA, 2015; pp. 423–432. [CrossRef] Wang, R.; Zhou, D.; He, Y. Open Event Extraction from Online Text using a Generative Adversarial Network. In Proceedings of the 2019 Conference on Empirical Methods in Natural Language Processing and the 9th International Joint Conference on Natural Language Processing, Hong Kong, China, 3–7 November 2019; pp. 282–291.
https://openalex.org/W4318066396	https://www.qeios.com/read/2J6APY/pdf	English	null	Review of: "How many papers are published each week reporting on trials of interventions involving behavioural aspects of health?"	null	2,023	cc-by	173	Qeios, CC-BY 4.0 · Review, January 26, 2023 Qeios ID: 2J6APY · https://doi.org/10.32388/2J6APY Review of: "How many papers are published each week reporting on trials of interventions involving behavioural aspects of health?" Nurdan Sezgin Potential competing interests: No potential competing interests to declare. This is a systematic examination worth reading and quoting. It does, however, have some weak points that can be repaired. This is a systematic examination worth reading and quoting. It does, however, have some weak points that can be repaired. I assume that the study's introduction is insufficient. Although the method is well explained, I would have expected Scopus and WOS to be included in addition to PubMed. Furthermore, there is no data comparison with directly related or similar publications in the discussion section, which makes this section slightly weak. It would be more accurate to change the title to "discussion and conclusion" when you add the conclusion section at the end of the discussion. Otherwise, readers may characterize it as a missing section. Qeios ID: 2J6APY · https://doi.org/10.32388/2J6APY 1/1
https://openalex.org/W2805226047	https://iris.unimore.it/bitstream/11380/1162691/1/Article%20post_print.pdf	English	null	Comprehensive evaluation of phenolic profile in dark chocolate and dark chocolate enriched with Sakura green tea leaves or turmeric powder	Food research international	2,018	cc-by	22,246	This is the peer reviewd version of the followng article: This is the peer reviewd version of the followng article: Comprehensive evaluation of phenolic profile in dark chocolate and dark chocolate enriched with Sakura green tea leaves or turmeric powder / Martini, Serena; Conte, Angela; Tagliazucchi, Davide. - In: FOOD RESEARCH INTERNATIONAL. - ISSN 0963-9969. - 112:(2018), pp. 1-16. [10.1016/j.foodres.2018.06.020] Terms of use: The terms and conditions for the reuse of this version of the manuscript are specified in the publishing policy. For all terms of use and more information see the publisher's website. The terms and conditions for the reuse of this version of the manuscript are specified in the publishing policy. For all terms of use and more information see the publisher's website. 24/10/2024 06:35 1 dark chocolate and dark chocolate enriched with Sakura green tea leaves or turmeric powder Serena Martini, Angela Conte, Davide Tagliazucchi Department of Life Sciences, University of Modena and Reggio Emilia, Via Amendola 2, 42100 Reggio Emilia, Italy Corresponding author. Tel.: +39-0522-522060; fax: +39-0522-522053 E-mail address: serena.martini@unimore.it (S. Martini) * Corresponding author. Tel.: +39-0522-522060; fax: +39-0522-522053 E-mail address: serena.martini@unimore.it (S. Martini) 1 1 Abstract 1 Recently, a huge number of studies have confirmed the important role of chocolate polyphenols in 2 human health, underlining its beneficial effects especially in the treatment of cardiovascular 3 diseases. However, a thorough evaluation of chocolate phenolic profile is still lacking. This study 4 aimed at a comprehensive characterisation of dark chocolate phenolic profile, using non-targeted 5 mass spectrometry identification. This approach allowed a tentative identification of 158 individual 6 phenolic compounds: 67 were newly detected in dark chocolate, among these 38 were observed for 7 the first time in chocolate as well as in cocoa beans or products. Ellagitannins, which have never 8 been reported in cocoa or chocolate, represented about the 10% of the phenolic profile of dark 9 chocolate. The enrichment of dark chocolate with Sakura green tea leaves or turmeric powder 10 influenced and modified the phenolic profile, resulting in a phenolic concentration increase. In this 11 way, this functional chocolate might maximize the beneficial effect of chocolate consumption, 12 combining the positive health effects of chocolate, turmeric and green tea and, at the same time, 13 reducing the amount of sugars and calories introduced with chocolate. 14 15 Keywords: epicatechin, curcuminoids, ellagitannins, mass spectrometry, polyphenols, 16 metabolomics, functional foods 17 Recently, a huge number of studies have confirmed the important role of chocolate polyphenols in 2 human health, underlining its beneficial effects especially in the treatment of cardiovascular 3 diseases. However, a thorough evaluation of chocolate phenolic profile is still lacking. This study 4 aimed at a comprehensive characterisation of dark chocolate phenolic profile, using non-targeted 5 mass spectrometry identification. This approach allowed a tentative identification of 158 individual 6 phenolic compounds: 67 were newly detected in dark chocolate, among these 38 were observed for 7 the first time in chocolate as well as in cocoa beans or products. Ellagitannins, which have never 8 been reported in cocoa or chocolate, represented about the 10% of the phenolic profile of dark 9 chocolate. The enrichment of dark chocolate with Sakura green tea leaves or turmeric powder 10 influenced and modified the phenolic profile, resulting in a phenolic concentration increase. In this 11 way, this functional chocolate might maximize the beneficial effect of chocolate consumption, 12 combining the positive health effects of chocolate, turmeric and green tea and, at the same time, 13 reducing the amount of sugars and calories introduced with chocolate. 14 15 2 2 1. Introduction 18 Western lifestyle built-around a highly refined diet rich in saturated fat and sugars but low in 19 complex plant carbohydrates, phytochemicals and vitamins is a hot research topic in the field of 20 nutrition. It is widely known that diet is the cause of many pathogenic age-related conditions. The 21 intake of certain dietary components is plays an essential role in the prevention or management of 22 these diseases (Del Rio et al., 2013). Increasing interest has pointed to naturally occurring 23 compounds, which have been considered non-nutritive for a long time. Polyphenols are a 24 representative class of these compounds and can be summarised into several groups, i.e. 25 hydroxybenzoic acids, hydroxycinnamic acids, flavan-3-ols, flavonols, flavones, flavanones, 26 isoflavones, anthocyanins, ellagitannins, stilbenes, and lignan. They occur in all fruits, vegetables, 27 nuts, seeds, flowers, bark, beverages and processed food. As reviewed by Wollgast, & Anklam 28 (2000a; 2000b) polyphenols are characterised by several beneficial effects including anti- 29 carcinogenic, anti-atherogenic, anti-inflammatory, immunomodulating and vasodilatory activities. 30 They can exert their protective effects through several mechanisms such as plasma cholesterol 31 reduction, modulation of lipid and lipoprotein metabolism, modulation of enzymes (phase I and 32 phase II) and apoptosis as well as their activity against reactive oxygen species (Del Rio et al., 33 2013). 34 Cocoa (Theobroma cacao) is known as a rich source of dietary phenolic compounds. Cocoa-derived 35 products such as dark chocolate are widely studied for their beneficial effects ascribed to 36 polyphenols. There is good evidence to suggest that cocoa derived polyphenols may have beneficial 37 effects on cardiovascular disease risk factors (Del Rio et al., 2013). Short-term dark chocolate 38 intake has been shown to reduce blood pressure in hypertensive subjects, to improve endothelial 39 function and insulin resistance as well as to inhibit platelet activation (Del Rio et al., 2013). As 40 reported by Rusconi, & Conti (2010), cocoa beans are characterised by phenolic compounds of the 41 Western lifestyle built-around a highly refined diet rich in saturated fat and sugars but low in 19 complex plant carbohydrates, phytochemicals and vitamins is a hot research topic in the field of 20 nutrition. It is widely known that diet is the cause of many pathogenic age-related conditions. The 21 intake of certain dietary components is plays an essential role in the prevention or management of 22 these diseases (Del Rio et al., 2013). 1. Introduction 18 Increasing interest has pointed to naturally occurring 23 compounds, which have been considered non-nutritive for a long time. Polyphenols are a 24 representative class of these compounds and can be summarised into several groups, i.e. 25 Western lifestyle built-around a highly refined diet rich in saturated fat and sugars but low in 19 complex plant carbohydrates, phytochemicals and vitamins is a hot research topic in the field of 20 nutrition. It is widely known that diet is the cause of many pathogenic age-related conditions. The 21 intake of certain dietary components is plays an essential role in the prevention or management of 22 these diseases (Del Rio et al., 2013). Increasing interest has pointed to naturally occurring 23 compounds, which have been considered non-nutritive for a long time. Polyphenols are a 24 representative class of these compounds and can be summarised into several groups, i.e. 25 3 3 procyanidins, anthocyanins (cyanidin glycosides) and flavonol glycosides such as quercetin-3-O- 43 rutinoside, quercetin-3-O-arabinoside, quercetin-3-O-glucoside, quercetin-3-O-glucuronide and 44 quercetin (Sanbongi et al., 1998). So far, only few studies have investigated the phenolic 45 composition of dark chocolate, focusing on flavan-3-ols as the major class in chocolate phenolic 46 profile (Ortega et al., 2008; Wollgast, & Anklam, 2000a). This lack of information is also due to the 47 great interest addressed to the study of phenolic profile of cocoa, intended as raw material in 48 chocolate production, without considering the impact of processing temperature, microbial 49 fermentation or oxidative phenomena on the phenolics structure during cocoa processing in 50 chocolate production. The majority of published researches were aimed at analyzing the impact of 51 processing on the polyphenol content and antioxidant properties of cocoa more than that of 52 chocolate (Di Mattia, Sacchetti, Mastrocola, & Serafini, 2017; Dorota, Oracz, Sosnowska, & 53 Nebesny, 2016). Concerning this, it was considered purposeful to investigate the comprehensive 54 phenolic profile of commercial dark chocolate (70%), using an un-targeted mass spectrometry 55 approach, in order to fill the gap of information about dark chocolate phenolic composition. 2.1. Materials 66 Phenolic compounds standard, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox), 67 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,4,6-tri(2-pyridyl)-S-triazine 68 (TPTZ), Folin-Ciocalteau phenol reagent were purchased from Sigma (Milan, Italy). Methanol and 69 formic acid were obtained from Carlo Erba (Milan, Italy). Three different types of chocolate (dark 70 70% cocoa (DC), dark 70% cocoa and 8% turmeric (TDC), dark 70% cocoa and 2% Sakura green 71 tea (GTDC)) were bought from a local shop in Modena (Italy). 72 1. Introduction 18 Materials 66 Phenolic compounds standard, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox), 67 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,4,6-tri(2-pyridyl)-S-triazine 68 (TPTZ), Folin-Ciocalteau phenol reagent were purchased from Sigma (Milan, Italy). Methanol and 69 formic acid were obtained from Carlo Erba (Milan, Italy). Three different types of chocolate (dark 70 70% cocoa (DC), dark 70% cocoa and 8% turmeric (TDC), dark 70% cocoa and 2% Sakura green 71 tea (GTDC)) were bought from a local shop in Modena (Italy). 72 73 2.2. Extraction of phenolic compounds 74 Polyphenols were extracted as reported in Martini, Conte, & Tagliazucchi (2017) with minor 75 modifications. Ten grams of chocolate were melted at 50°C for 10 minutes and homogenized with 76 20 mL of water/methanol/formic acid solution (28:70:2, v/v/v). The mixtures were stirred and 77 maintained at 37°C for 30 minutes. The homogenates were centrifuged (5000 rpm, 10 min, 4°C), 78 after that the floating cocoa butter layers were removed and the supernatants collected. Pellets were 79 then used for a second extraction step with acetone. Each pellet was added with 20 mL of acetone, 80 kept in agitation at 37°C, for 30 minutes and then centrifuged for 20 minutes at 5000 rpm, 4°C. The 81 supernatants were collected. Both methanol and acetone extractions were performed twice. The 82 methanolic and acetone extracts were diluted 8 and 2 times, respectively, using MilliQ water and 83 further used for the MS analysis. 84 85 2.3. Identification and quantification of phenolic compounds by liquid chromatography mass 86 spectrometry (LC-ESI-QTOF-MS/MS) 87 Chocolate methanolic and acetone extracts were analysed on Agilent HPLC 1200 Infinity (Agilent 88 Technologies, Santa Clara, CA) equipped with a C18 column (HxSil C18 Reversed phase, 250×4.6 89 1. Introduction 18 Finally, 56 the last task was to evaluate a possible polyphenolic enrichment of dark chocolate recipe, by adding 57 widely studied polyphenol-rich ingredients (Sakura green tea leaves and turmeric powder) in order 58 to obtain potential functional food, which can combine the above-mentioned chocolate properties 59 and those of green tea leaves and turmeric powder (Del Rio et al., 2013; Kunnumakkara et al., 60 2017). 61 Therefore, the aim of the present study was to identify, quantify and compare phenolic compounds 62 from three different types of dark chocolate using liquid chromatography-electrospray ionization 63 mass spectrometry (LC-ESI-QTOF-MS/MS). 64 procyanidins, anthocyanins (cyanidin glycosides) and flavonol glycosides such as quercetin-3-O- 43 rutinoside, quercetin-3-O-arabinoside, quercetin-3-O-glucoside, quercetin-3-O-glucuronide and 44 quercetin (Sanbongi et al., 1998). So far, only few studies have investigated the phenolic 45 composition of dark chocolate, focusing on flavan-3-ols as the major class in chocolate phenolic 46 profile (Ortega et al., 2008; Wollgast, & Anklam, 2000a). This lack of information is also due to the 47 great interest addressed to the study of phenolic profile of cocoa, intended as raw material in 48 chocolate production, without considering the impact of processing temperature, microbial 49 fermentation or oxidative phenomena on the phenolics structure during cocoa processing in 50 chocolate production. The majority of published researches were aimed at analyzing the impact of 51 processing on the polyphenol content and antioxidant properties of cocoa more than that of 52 chocolate (Di Mattia, Sacchetti, Mastrocola, & Serafini, 2017; Dorota, Oracz, Sosnowska, & 53 Nebesny, 2016). Concerning this, it was considered purposeful to investigate the comprehensive 54 phenolic profile of commercial dark chocolate (70%), using an un-targeted mass spectrometry 55 approach, in order to fill the gap of information about dark chocolate phenolic composition. Finally, 56 the last task was to evaluate a possible polyphenolic enrichment of dark chocolate recipe, by adding 57 widely studied polyphenol-rich ingredients (Sakura green tea leaves and turmeric powder) in order 58 to obtain potential functional food, which can combine the above-mentioned chocolate properties 59 and those of green tea leaves and turmeric powder (Del Rio et al., 2013; Kunnumakkara et al., 60 2017). 61 Therefore, the aim of the present study was to identify, quantify and compare phenolic compounds 62 from three different types of dark chocolate using liquid chromatography-electrospray ionization 63 mass spectrometry (LC-ESI-QTOF-MS/MS). 64 4 2. Materials and methods 65 2.1. 2.2. Extraction of phenolic compounds 74 MS operating 98 conditions (negative mode) were: a capillary temperature of 350°C, a dry gas flow rate of 10 L/min, 99 a nebulizer pressure of 35 psi, potential of the ESI source, 3.5 kV. 100 mm, 5 μm particle size, Hamilton Company, Reno, Nevada, USA). The mobile phase consisted of 90 (A) H2O/formic acid (99:1, v/v) and (B) acetonitrile/formic acid (99:1, v/v). The gradient started at 91 4% B for 0.5 min then linearly ramped up to 30% B in 60 min. The mobile phase composition was 92 raised up to 100% B in 1 min and maintained for 5 min in order to wash the column before 93 returning to the initial condition. The flow rate was set at 1 mL/min. The chocolate extracts were 94 injected in the amount of 20 µL. After passing to the column, the eluate was split, and 0.3 mL/min 95 was direct to a 6520 accurate-mass Q-TOF mass spectrometer (Agilent Technologies, Santa Clara, 96 CA). Identification of phenolic compounds in all samples was carried out using full scan, data- 97 dependent MS2 scanning from m/z 100 to 1700 and selected reaction monitoring. MS operating 98 conditions (negative mode) were: a capillary temperature of 350°C, a dry gas flow rate of 10 L/min, 99 a nebulizer pressure of 35 psi, potential of the ESI source, 3.5 kV. 100 The quantification of single phenolic compounds was carried out by integrating the area under the 101 peak from the extracted ion chromatograms (EICs). To obtain an accurate quantification the EICs 102 were obtained by centering a narrow mass window (± 5 ppm) on the theoretical m/z value of each 103 phenolic compound. For each standard compound, the calibration curve was built using seven 104 concentration points in the range of 0.2-50 ng. Hydroxycinnamic acids, hydroxybenzoic acids, 105 flavan-3-ols and ellagitannins were quantified as p-coumaric or ferulic acid, protocatechuic acid, (- 106 )-epicatechin and ellagic acid equivalents, respectively. Flavonols and flavones were quantified as 107 quercetin-3-rutinoside equivalents. Finally, curcuminoids were quantified as curcumin equivalent. 108 Quantitative results were expressed as mg of compounds per 100 g of chocolate. Calibration curve 109 equations, linearity ranges and limit of quantification (LOQ) for the different standards are given in 110 supplementary materials (Table S1). Folin-Ciocalteau assay was also performed to quantify the 111 total phenolic compounds as reported by Singleton, Orthofer, & Lamuela-Raventós (1999). 2.2. Extraction of phenolic compounds 74 f p p Polyphenols were extracted as reported in Martini, Conte, & Tagliazucchi (2017) with minor 75 modifications. Ten grams of chocolate were melted at 50°C for 10 minutes and homogenized with 76 20 mL of water/methanol/formic acid solution (28:70:2, v/v/v). The mixtures were stirred and 77 maintained at 37°C for 30 minutes. The homogenates were centrifuged (5000 rpm, 10 min, 4°C), 78 after that the floating cocoa butter layers were removed and the supernatants collected. Pellets were 79 then used for a second extraction step with acetone. Each pellet was added with 20 mL of acetone, 80 kept in agitation at 37°C, for 30 minutes and then centrifuged for 20 minutes at 5000 rpm, 4°C. The 81 supernatants were collected. Both methanol and acetone extractions were performed twice. The 82 methanolic and acetone extracts were diluted 8 and 2 times, respectively, using MilliQ water and 83 further used for the MS analysis. 84 85 2.3. Identification and quantification of phenolic compounds by liquid chromatography mass 86 spectrometry (LC-ESI-QTOF-MS/MS) 87 Chocolate methanolic and acetone extracts were analysed on Agilent HPLC 1200 Infinity (Agilent 88 Technologies, Santa Clara, CA) equipped with a C18 column (HxSil C18 Reversed phase, 250×4.6 89 2.3. Identification and quantification of phenolic compounds by liquid chromatography mass 86 spectrometry (LC-ESI-QTOF-MS/MS) 87 Chocolate methanolic and acetone extracts were analysed on Agilent HPLC 1200 Infinity (Agilent 88 Technologies, Santa Clara, CA) equipped with a C18 column (HxSil C18 Reversed phase, 250×4.6 89 5 5 mm, 5 μm particle size, Hamilton Company, Reno, Nevada, USA). The mobile phase consisted of 90 (A) H2O/formic acid (99:1, v/v) and (B) acetonitrile/formic acid (99:1, v/v). The gradient started at 91 4% B for 0.5 min then linearly ramped up to 30% B in 60 min. The mobile phase composition was 92 raised up to 100% B in 1 min and maintained for 5 min in order to wash the column before 93 returning to the initial condition. The flow rate was set at 1 mL/min. The chocolate extracts were 94 injected in the amount of 20 µL. After passing to the column, the eluate was split, and 0.3 mL/min 95 was direct to a 6520 accurate-mass Q-TOF mass spectrometer (Agilent Technologies, Santa Clara, 96 CA). Identification of phenolic compounds in all samples was carried out using full scan, data- 97 dependent MS2 scanning from m/z 100 to 1700 and selected reaction monitoring. 2.2. Extraction of phenolic compounds 74 The 112 results were expressed as mg of gallic acid per 100 g of chocolate. 113 The quantification of single phenolic compounds was carried out by integrating the area under the 101 peak from the extracted ion chromatograms (EICs). To obtain an accurate quantification the EICs 102 were obtained by centering a narrow mass window (± 5 ppm) on the theoretical m/z value of each 103 phenolic compound. For each standard compound, the calibration curve was built using seven 104 concentration points in the range of 0.2-50 ng. Hydroxycinnamic acids, hydroxybenzoic acids, 105 flavan-3-ols and ellagitannins were quantified as p-coumaric or ferulic acid, protocatechuic acid, (- 106 )-epicatechin and ellagic acid equivalents, respectively. Flavonols and flavones were quantified as 107 quercetin-3-rutinoside equivalents. Finally, curcuminoids were quantified as curcumin equivalent. 108 Quantitative results were expressed as mg of compounds per 100 g of chocolate. Calibration curve 109 equations, linearity ranges and limit of quantification (LOQ) for the different standards are given in 110 supplementary materials (Table S1). Folin-Ciocalteau assay was also performed to quantify the 111 total phenolic compounds as reported by Singleton, Orthofer, & Lamuela-Raventós (1999). The 112 results were expressed as mg of gallic acid per 100 g of chocolate. 113 The quantification of single phenolic compounds was carried out by integrating the area under the 101 peak from the extracted ion chromatograms (EICs). To obtain an accurate quantification the EICs 102 were obtained by centering a narrow mass window (± 5 ppm) on the theoretical m/z value of each 103 phenolic compound. For each standard compound, the calibration curve was built using seven 104 concentration points in the range of 0.2-50 ng. Hydroxycinnamic acids, hydroxybenzoic acids, 105 114 6 3. Result and discussion 129 3. Result and discussion 129 3.1. Non-targeted LC-MS profiling of phenolic compounds in different types of dark chocolate 130 This study aimed to identify and quantify the phenolic profile and content of three different types o 131 dark chocolate (dark chocolate 70% cocoa, dark chocolate 70% cocoa and 8% turmeric, dark 132 chocolate 70% cocoa and 2% Sakura green tea). The phytochemical composition focused on the 133 phenolic fraction, was investigated using a non-targeted procedure through LC-ESI-MS/MS 134 experiments, representative base peak chromatograms (BPCs) are shown in Figure 1. Within the 37 135 resolved peaks, 158 individual phenolic compounds were tentatively identified. Among them, 67 136 were firstly identified in dark chocolate and of these 38 were identified for the first time in 137 chocolate, cocoa beans and cocoa products. The structure of the newly identified phenolic 138 compounds is depicted in Figure 2. Peaks annotated with letters from a to g in Figure 1 did not 139 contain phenolic compounds and were not further investigated in this study. Two additional non- 140 phenolic compounds were recognised in peaks 28 and 30 and identified as 12-hydroxy jasmonic 141 acid sulphate as already described in raw fermented cocoa beans by Patras, Milev, Vrancken, & 142 Kuhnert (2014). The description of the non-phenolic compounds is reported in supplementary 143 material (Table S2). Table 1, instead, shows mass spectrum data along with peak assignments and 144 retention time for the identified phenolic compounds. A total of 16 compounds were identified by 145 comparison with authentic standards. The remaining compounds were tentatively identified based 146 on the interpretation of their fragmentation patterns obtained from MS2 experiments and by 147 comparison with literature. The description of the MS fragmentation pattern of phenolic compound 148 already identified in cocoa beans or products will not be further described. All identified 149 compounds were found in the methanol extract. The subsequent extraction of the pellet with 150 acetone did not resulted in the recovery of new compounds. The acetone extract just contained low 151 amount of the same compounds found in methanol extract (data not shown). 152 153 2.4. Antioxidant activity assays 115 The antioxidant properties of chocolate were evaluated performing two different assays. The ABTS 116 (2,2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) and ferric reducing power (FRAP) assays 117 were performed according to the protocols described by Re et al. (1999) and Benzie, & Strain 118 (1996), respectively. The ABTS scavenging capacity and FRAP value were expressed as mmol of 119 trolox equivalent per 100 g of chocolate, by means of a calibration curve obtained with Trolox 50- 120 500 µmol/L, in the same assay conditions. The absorbances were read using a Jasco V-550 UV/Vis 121 spectrophotometer (Orlando, FL, USA). 122 123 2.5. Statistic 124 All data are presented as mean ± SD for three replicates for each prepared sample. One-way 125 analysis of variance (one-way ANOVA) with Tukey’s post-hoc test was applied using Graph Pad 126 prism 6.0 (GraphPad software, San Diego, CA, U.S.A.). The differences were considered 127 significant with P<0.05. 128 7 7 3.1. Non-targeted LC-MS profiling of phenolic compounds in different types of dark chocolate 130 153 8 154 3.1.1. Flavan-3-ols and derivatives 155 The high-resolution mass-spectrometry method used in this study enabled the characterization of 72 156 flavan-3-ol derivatives. According to their chemical structures, this group may be divided into 157 monomeric forms, A-type, and B-type oligomeric forms. 158 The high-resolution mass-spectrometry method used in this study enabled the characterization of 72 156 flavan-3-ol derivatives. According to their chemical structures, this group may be divided into 157 monomeric forms, A-type, and B-type oligomeric forms. 158 Among the monomeric flavan-3-ols, four compounds had been already reported in dark chocolate 159 (compounds 9.1, 13.2, 15.2 and 19.9) (Wollgast, 2004), seven compounds had been detected in 160 cocoa beans or products but not in dark chocolate (compounds 13.1, 14.1, 15.1, 16.2 , 21.4, 22.6 161 and 25.3) (D’Souza et al., 2017; Patras et al., 2014), whereas seven compounds were newly 162 9 amu, detailing the loss of a galloyl group), 289 ((epi)catechin-aglycone and 465-176 amu; i.e. the 179 loss of a glucuronic acid moiety), 435 (gallate group 3-O-binding site) and 261 (glucuronide 7-O- 180 binding site), and were tentatively identified as (epi)catechin-3-O-gallate-7-O-glucuronide 181 (Shrestha et al., 2012). Compounds 18.5 and 28.1 had the same precursor ion [M-H]- at m/z 182 613.1138 and their main MS2 fragment ions at m/z 289 ((epi)catechin aglycone), m/z 451 ([M-H]- - 183 162 amu, i.e. hexosyl moiety loss) and m/z 433 revealing that one sugar is attached to another and 184 not directly to the aglycone describing two hexose sequential losses, as suggested by Hvattum, & 185 Ekeberg (2003). Moreover, the presence of MS2 product ion at m/z 259 identified 3-O-glycosylation 186 site. Regarding this, they were tentatively identified as two isomers of (epi)catechin-3-O- 187 dihexoside. Finally, three compounds (compounds 28.3, 29.5, 20.6) with deprotonated ions at m/z 188 441.0892 and 457.0829 were only detected in dark chocolate with added Sakura green tea leaves 189 (Table 1). They had been classified as catechin-3-O-gallate, epicatechin-3-O-gallate and 190 epigallocatechin-3-O-gallate in comparison with authentic standards (Table 1). 191 epigallocatechin-3-O-gallate in comparison with authentic standards (Table 1). 191 Procyanidins are mostly flavan-3,4-diols, found as dimers, trimers or oligomers with epicatechin as 192 the main extension sub-unit. Table 1 shows six B-type procyanidin dimers (compounds 12.1, 17.1, 193 18.3, 18.9, 23.1 and 29.3) and four derivative forms (compounds 18.10, 16.3, 18.2 and 18.7). 3.1.1. Flavan-3-ols and derivatives 155 Among these, three compounds 210 (compounds 2.1, 23.4 and 27.2) had never been identified in dark chocolate and cocoa beans or 211 products, whereas compound 20.4 had been already detected in cocoa but never in dark chocolate 212 (Stark, & Hofmann, 2005). According to Bauer, Harbaum-Piayda, & Schwarz (2012), the precursor 213 ion at m/z 325.1004 can be tentatively classified as ferulic acid-4-O-pentoside, which MS2 yielded a 214 major fragment ion at m/z 193, corresponding to the loss of pentose ([M-H] – 132 amu). Using the 215 fragmentation pattern and literature comparison, di-hydro-caffeic acid (compound 2.1) and di- 216 hydro-coumaric acid (compound 23.4) were tentatively ascribed to deprotonated ions 181.0575 and 217 165.0470, respectively (Bresciani et al., 2017). These two compounds can be originated from the 218 microbial metabolism during cocoa beans fermentation. Finally, compounds 12.3, 14.3, 19.4 and 219 20.2 (Table 1), with negative charged ion ([M-H]-) at m/z 337.1006, were only detected in 220 chocolate with added Sakura green tea leaves (Table 1). Based on their fragmentation pattern, 221 elution profile and in comparison with the scheme proposed by Clifford, Johnston, Knight, & 222 Kuhnert (2003), they had been tentatively classified as coumaroyl-quinic acids (Martini et al., 223 2017). 224 225 3.1.3. Flavonols, flavones and other phenolics 226 A total of 25 hydroxycinnamic acids were tentatively identified. Among these, three compounds 210 (compounds 2.1, 23.4 and 27.2) had never been identified in dark chocolate and cocoa beans or 211 products, whereas compound 20.4 had been already detected in cocoa but never in dark chocolate 212 (Stark, & Hofmann, 2005). According to Bauer, Harbaum-Piayda, & Schwarz (2012), the precursor 213 ion at m/z 325.1004 can be tentatively classified as ferulic acid-4-O-pentoside, which MS2 yielded a 214 major fragment ion at m/z 193, corresponding to the loss of pentose ([M-H] – 132 amu). Using the 215 fragmentation pattern and literature comparison, di-hydro-caffeic acid (compound 2.1) and di- 216 hydro-coumaric acid (compound 23.4) were tentatively ascribed to deprotonated ions 181.0575 and 217 165.0470, respectively (Bresciani et al., 2017). These two compounds can be originated from the 218 microbial metabolism during cocoa beans fermentation. Finally, compounds 12.3, 14.3, 19.4 and 219 20.2 (Table 1), with negative charged ion ([M-H]-) at m/z 337.1006, were only detected in 220 chocolate with added Sakura green tea leaves (Table 1). 3.1.1. Flavan-3-ols and derivatives 155 According to Bauer 213 ion at m/z 325.1004 can be tentatively classified 214 major fragment ion at m/z 193, corresponding t 215 fragmentation pattern and literature comparison 216 hydro-coumaric acid (compound 23.4) were ten 217 165.0470, respectively (Bresciani et al., 2017). 218 microbial metabolism during cocoa beans ferm 219 20.2 (Table 1), with negative charged ion ([M- 220 chocolate with added Sakura green tea leaves ( 221 elution profile and in comparison with the sche 222 Kuhnert (2003), they had been tentatively class 223 2017). 224 225 3 1 3 Fl l fl d th h li 226 glycosides have been already isolated from chocolate and cocoa (Hatano et al. 2002). Dimeric 204 (compounds 21.3, 22.2, 23.7, 24.3, 30.2, 31.1, 31.5, 32.2 and 34.1), trimeric (compounds 20.8, 24.4 205 and 24.9), tetrameric (compounds 19.6, 22.1, 22.7 and 24.1) and hexameric (compound 22.9) 206 structures of A-type procyanidins were also found and listed in Table 1. 207 208 3.1.2 Hydroxycinnamic acids 209 A total of 25 hydroxycinnamic acids were tentatively identified. Among these, three compounds 210 (compounds 2.1, 23.4 and 27.2) had never been identified in dark chocolate and cocoa beans or 211 products, whereas compound 20.4 had been already detected in cocoa but never in dark chocolate 212 (Stark, & Hofmann, 2005). According to Bauer, Harbaum-Piayda, & Schwarz (2012), the precursor 213 ion at m/z 325.1004 can be tentatively classified as ferulic acid-4-O-pentoside, which MS2 yielded a 214 major fragment ion at m/z 193, corresponding to the loss of pentose ([M-H] – 132 amu). Using the 215 fragmentation pattern and literature comparison, di-hydro-caffeic acid (compound 2.1) and di- 216 hydro-coumaric acid (compound 23.4) were tentatively ascribed to deprotonated ions 181.0575 and 217 165.0470, respectively (Bresciani et al., 2017). These two compounds can be originated from the 218 microbial metabolism during cocoa beans fermentation. Finally, compounds 12.3, 14.3, 19.4 and 219 20.2 (Table 1), with negative charged ion ([M-H]-) at m/z 337.1006, were only detected in 220 chocolate with added Sakura green tea leaves (Table 1). Based on their fragmentation pattern, 221 elution profile and in comparison with the scheme proposed by Clifford, Johnston, Knight, & 222 Kuhnert (2003), they had been tentatively classified as coumaroyl-quinic acids (Martini et al., 223 2017). 224 225 y y A total of 25 hydroxycinnamic acids were tentatively identified. 3.1.1. Flavan-3-ols and derivatives 155 194 Among these, three glycosidic dimers of procyanidin B-type were newly identified in dark 195 chocolate but already reported in cocoa beans (D’Souza et al., 2017). As reported by Gu, House, 196 Wu, Ou, & Prior (2006) chocolate and cocoa products were also characterized by polymeric 197 procyanidins. Trimers (compounds 13.5, 18.1, 18.8, 20.7, 21.2, 22.5, 23.3 and 29.2), tetramers 198 (compounds 16.1, 19.5, 19.7 and 22.8), pentamers (compounds 19.13, 23.2 and 24.2) and hexamers 199 (compounds 23.8, 24.7 and 26.1) of B-type procyanidins were also found. Compound 12.2 (Table 200 1) with precursor ion at m/z 593.1345 was only detected in Sakura green tea dark chocolate and 201 tentatively identified as (epi)catechin-(epi)gallocatechin in accordance to Jiang et al. (2013). A-type 202 linkage is a less common feature in procyanidins, however A-type procyanidins and their 203 Procyanidins are mostly flavan-3,4-diols, found as dimers, trimers or oligomers with epicatechin as 192 the main extension sub-unit. Table 1 shows six B-type procyanidin dimers (compounds 12.1, 17.1, 193 18.3, 18.9, 23.1 and 29.3) and four derivative forms (compounds 18.10, 16.3, 18.2 and 18.7). 194 Among these, three glycosidic dimers of procyanidin B-type were newly identified in dark 195 chocolate but already reported in cocoa beans (D’Souza et al., 2017). As reported by Gu, House, 196 Wu, Ou, & Prior (2006) chocolate and cocoa products were also characterized by polymeric 197 procyanidins. Trimers (compounds 13.5, 18.1, 18.8, 20.7, 21.2, 22.5, 23.3 and 29.2), tetramers 198 (compounds 16.1, 19.5, 19.7 and 22.8), pentamers (compounds 19.13, 23.2 and 24.2) and hexamers 199 (compounds 23.8, 24.7 and 26.1) of B-type procyanidins were also found. Compound 12.2 (Table 200 1) with precursor ion at m/z 593.1345 was only detected in Sakura green tea dark chocolate and 201 tentatively identified as (epi)catechin-(epi)gallocatechin in accordance to Jiang et al. (2013). A-type 202 linkage is a less common feature in procyanidins, however A-type procyanidins and their 203 10 glycosides have been already isolated from cho 204 (compounds 21.3, 22.2, 23.7, 24.3, 30.2, 31.1, 3 205 and 24.9), tetrameric (compounds 19.6, 22.1, 2 206 structures of A-type procyanidins were also fou 207 208 3.1.2 Hydroxycinnamic acids 209 A total of 25 hydroxycinnamic acids were tenta 210 (compounds 2.1, 23.4 and 27.2) had never been 211 products, whereas compound 20.4 had been alr 212 (Stark, & Hofmann, 2005). 3.1.1. Flavan-3-ols and derivatives 155 Compound 25.2 and 26.3 with a [M−H]− precursor ion at m/z 771.2042, producing product 247 ions at m/z 609 (loss of 162 mass units, a hexosyl-moiety), 463 (quercetin-3-O-glucoside, loss of 248 308 amu, a rutinose moiety) and 301 (quercetin-aglycone), and according to Martini et al. (2017), 249 were tentatively identified as quercetin-7-O-hexoside-3-O-rutinoside. As far as we know, also 250 compounds 31.4, 18.6, 32.1, 28.2, 31.2, 22.3 and 22.4 have been described for the first time in dark 251 3.1.1. Flavan-3-ols and derivatives 155 Based on their fragmentation pattern, 221 elution profile and in comparison with the scheme proposed by Clifford, Johnston, Knight, & 222 Kuhnert (2003), they had been tentatively classified as coumaroyl-quinic acids (Martini et al., 223 2017). 224 3.1.3. Flavonols, flavones and other phenolics 226 11 A tentatively characterization of 22 flavonols, 6 flavones and 7 other phenolics has been enabled 227 thanks to LC-ESI-MS/MS experiments. Among these, 15 compounds were identified for the first 228 time in chocolate and cocoa beans or products. 229 A tentatively characterization of 22 flavonols, 6 flavones and 7 other phenolics has been enabled 227 thanks to LC-ESI-MS/MS experiments. Among these, 15 compounds were identified for the first 228 time in chocolate and cocoa beans or products. 229 A tentatively characterization of 22 flavonols, 6 flavones and 7 other phenolics has been enabled 227 thanks to LC-ESI-MS/MS experiments. Among these, 15 compounds were identified for the first 228 time in chocolate and cocoa beans or products. 229 time in chocolate and cocoa beans or products. 229 230 3.1.3.1. Flavonols and derivative forms 231 According to MS and MS/MS data, the elution proﬁle and literature (Andres-Lacueva et al., 2008; 232 Counet, Callemien, & Collin 2006; Ortega et al., 2008; Sanbongi et al., 1998; Wollgast, 2004), four 233 flavonols already reported in chocolate and cocoa beans or products were identified as quercetin at 234 m/z 301.0423 (compound 35.1) and its pentoside at m/z 433.0832 (compound 33.2) and hexoside at 235 m/z 463.0950 (compounds 30.1 and 31.3) derivatives. Concerning flavonols O-glycosides, 236 compounds 32.4, 34.3 and 34.5 had been already detected in cocoa but never in dark chocolate 237 (Ortega et al., 2008; Sánchez-Rabaneda et al., 2003) and were tentatively ascribed to kaempferol-3- 238 O-hexoside isomers and quercetin-3-O-rhamnoside. Two quercetin derivatives, quercetin-7-O- 239 rhamnoside-3-O-rutinoside and quercetin-7-O-hexoside-3-O-rutinoside isomers (m/z 755.2103 and 240 771.2042, respectively; compounds 27.4, 25.2 and 26.3) were detected for the first time in dark 241 chocolate and cocoa beans or products by tentatively identification, screening the fragmentation 242 pattern (Table 1) (Guimarães et al., 2013; Lin, Chen, & Harnly, 2008). Compound 27.4 with a [M– 243 H]− deprotonated ion at m/z 755.2103 and MS2 fragment ions at m/z 609 (quercetin-3-O-rutinoside, 244 by loss of rhamnose moiety, 146 amu), 301 (quercetin-aglycone, underlining the loss of a rutinose 245 moiety, 308 amu) was tentatively identified as quercetin-7-O-rhamnoside-3-O-rutinoside (Lin et al., 246 2008). 3.1.3.1. Flavonols and derivative forms 231 According to MS and MS/MS data, the elution proﬁle and literature (Andres-Lacueva et al., 2008; 232 Counet, Callemien, & Collin 2006; Ortega et al., 2008; Sanbongi et al., 1998; Wollgast, 2004), four 233 flavonols already reported in chocolate and cocoa beans or products were identified as quercetin at 234 m/z 301.0423 (compound 35.1) and its pentoside at m/z 433.0832 (compound 33.2) and hexoside at 235 m/z 463.0950 (compounds 30.1 and 31.3) derivatives. Concerning flavonols O-glycosides, 236 compounds 32.4, 34.3 and 34.5 had been already detected in cocoa but never in dark chocolate 237 (Ortega et al., 2008; Sánchez-Rabaneda et al., 2003) and were tentatively ascribed to kaempferol-3- 238 O-hexoside isomers and quercetin-3-O-rhamnoside. Two quercetin derivatives, quercetin-7-O- 239 rhamnoside-3-O-rutinoside and quercetin-7-O-hexoside-3-O-rutinoside isomers (m/z 755.2103 and 240 771.2042, respectively; compounds 27.4, 25.2 and 26.3) were detected for the first time in dark 241 chocolate and cocoa beans or products by tentatively identification, screening the fragmentation 242 pattern (Table 1) (Guimarães et al., 2013; Lin, Chen, & Harnly, 2008). Compound 27.4 with a [M– 243 H]− deprotonated ion at m/z 755.2103 and MS2 fragment ions at m/z 609 (quercetin-3-O-rutinoside, 244 by loss of rhamnose moiety, 146 amu), 301 (quercetin-aglycone, underlining the loss of a rutinose 245 moiety, 308 amu) was tentatively identified as quercetin-7-O-rhamnoside-3-O-rutinoside (Lin et al., 246 2008). Compound 25.2 and 26.3 with a [M−H]− precursor ion at m/z 771.2042, producing product 247 ions at m/z 609 (loss of 162 mass units, a hexosyl-moiety), 463 (quercetin-3-O-glucoside, loss of 248 308 amu, a rutinose moiety) and 301 (quercetin-aglycone), and according to Martini et al. (2017), 249 were tentatively identified as quercetin-7-O-hexoside-3-O-rutinoside. As far as we know, also 250 compounds 31.4, 18.6, 32.1, 28.2, 31.2, 22.3 and 22.4 have been described for the first time in dark 251 12 chocolate and cocoa in general. Compound 31.4 was speculated to be kaempferol-7-O-hexoside, 252 since the difference between the precursor ion (m/z 447.1028) and its major fragment ion (m/z 285, 253 i.e. kaempferol-aglycone) was 162 amu detailing the loss of a hexosyl-moiety; the presence of the 254 MS2 ion at m/z 257 identified 7-O-glycosylation site (Hvattum, & Ekeberg, 2003). According to 255 Mena et al. 3.1.3.1. Flavonols and derivative forms 231 (2012), compound 18.6 (m/z 449.1182) was tentatively identified as dihydrokaempferol- 256 7-O-hexoside, borne out by the presence of MS2 major fragment ions at m/z 287 ([M-H]-– 162 amu; 257 characteristic loss of O-hexoside) and m/z 259, confirming 7-O-glycosylation site (Hvattum, & 258 Ekeberg, 2003). Compounds 32.1, 28.2 and 31.2 had deprotonated ions at m/z 739.2182 and m/z 259 755.2103 and gave product ions in the MS2 spectra at m/z 593 and 285 characteristic of kaempferol- 260 3-O-rutinoside and kaempferol-aglycone (Sánchez-Rabaneda et al., 2003). The negative product ion 261 at m/z 593 was formed by the loss of rhamnose, glucose or galactose moiety from the glycosides. 262 The presence of the product ion in MS2 spectra at m/z 257 identified 7-O-glycosylation site 263 (Hvattum, & Ekeberg, 2003). The loss of 308 amu is typical of a rutinose moiety, and therefore 264 these compounds were tentatively identified as kaempferol-7-O-rhamnoside-3-O-rutinoside 265 (compound 32.1) and kaempferol-7-O-hexoside-3-O-rutinoside isomers (compounds 28.2 and 266 31.2). Finally, peaks 22.3, 22.4, 23.9, 24.5 and 24.8 were tentatively identified as myricetin- 267 derivative-compounds. All of the compounds gave MS2 product ion at m/z 317, corresponding to 268 myricetin aglycone. Compounds 24.5 and 24.8, identified as myricetin-3-O-hexosides, had already 269 been detected in cocoa but never in dark chocolate (Lin et al., 2008). Two myricetin-derivatives 270 (compounds 22.3 and 22.4) have been described for the first time in dark chocolate and cocoa beans 271 or products in this study. The MS2 spectra of the compound 22.3 (m/z 631.1039) was characterized 272 by fragment ions at m/z 479 and 317, depicting losses of 152 and 162 amu (i.e. galloyl and hexosyl 273 moiety). This behaviour is indicative of myricetin-3-O-(O-galloyl)-hexoside (Saldanha, Vilegas, & 274 Dokkedal, 2013). Compound 22.4 showed a deprotonated ion at m/z 787.2050, which fragmented in 275 the MS2 experiments giving product ions at m/z 625 and 317, suggesting the presence of a myricetin 276 13 aglycone, through the loss of 308 amu (rutinose moiety) and the loss of 162 amu (hexose group). 277 Concerning this, the compound was therefore tentatively identified as myricetin-7-O-hexoside-3-O- 278 rutinoside (Lin et al., 2008). Finally, compound 23.9 (Table 1), with negative precursor ion ([M-H]- 279 ) at m/z 625.1455, was only detected in chocolate with added Sakura green tea leaves (Table 1). 280 Basing on its fragmentation pattern (Table 1) and according to Lin et al. (2008) it had been 281 tentatively classified as myricetin-3-O-rutinoside. 3.1.3.1. Flavonols and derivative forms 231 282 283 3.1.3.2. Flavones 284 Six glycosylated apigenins were detected in dark chocolate (Table 1); among these, five apigenins 285 (compounds 19.8, 19.12, 22.10, 25.1 and 27.3) were newly detected in this study. Compound 27.3 286 was tentatively associated to apigenin-C-hexoside-2′′-O-rhamnoside isomer, m/z 577.1617, whose 287 MS2 spectrum gave main fragment ions at m/z 457, 413 and 293, arising from the loss of 120 amu 288 (suggesting 1-2 linking between rhamnosyl-glucosyl group), 164 amu (rhamnosyl group), and 284 289 amu (164 plus 120 amu, i.e. rhamnose-glucosyl residue) (Dou, Lee, Tzen, & Lee, 2007; Hvattum, & 290 Ekeberg, 2003; Waridel et al., 2001). The next two isomers (compounds 19.8 and 19.12) at m/z 291 593.1591 were tentatively identified as C-diglycosylated apigenins; according to Jiang et al. (2013) 292 they were pinpointed as apigenin-6,8-di-C-glucoside isomers. Compound 25.1 generated the same 293 deprotonated ion at m/z 593.1591 and MS2 fragment ions at m/z 473, 413 and 293 corresponding to 294 the loss of 120 amu (suggesting 1-2 linking between two glucosyl groups), 180 amu (glucosyl 295 moiety) and 300 amu (a glucose-glucosyl residue). Thus, compound 25.1 was speculated to be 296 apigenin-C-hexoside-2′′-O-hexoside isomer (Hvattum, & Ekeberg, 2003; Dou et al., 2007). Finally, 297 following the scheme proposed by Lin et al. (2008) for the negative precursor ion [M-H]- m/z 298 563.1462 and considering the losses of 90 amu (MS2 fragment at m/z 473) and 120 amu (MS2 299 fragment at m/z 443) proving the existence of C-pentosyl- and C-hexosyl groups, compound 22.10 300 aglycone, through the loss of 308 amu (rutinose moiety) and the loss of 162 amu (hexose group). 277 Concerning this, the compound was therefore tentatively identified as myricetin-7-O-hexoside-3-O- 278 rutinoside (Lin et al., 2008). Finally, compound 23.9 (Table 1), with negative precursor ion ([M-H]- 279 ) at m/z 625.1455, was only detected in chocolate with added Sakura green tea leaves (Table 1). 280 Basing on its fragmentation pattern (Table 1) and according to Lin et al. (2008) it had been 281 tentatively classified as myricetin-3-O-rutinoside. 282 aglycone, through the loss of 308 amu (rutinose moiety) and the loss of 162 amu (hexose group). 277 Concerning this, the compound was therefore tentatively identified as myricetin-7-O-hexoside-3-O- 278 rutinoside (Lin et al., 2008). Finally, compound 23.9 (Table 1), with negative precursor ion ([M-H]- 279 ) at m/z 625.1455, was only detected in chocolate with added Sakura green tea leaves (Table 1). 3.1.3.1. Flavonols and derivative forms 231 280 Basing on its fragmentation pattern (Table 1) and according to Lin et al. (2008) it had been 281 tentatively classified as myricetin-3-O-rutinoside. 282 Six glycosylated apigenins were detected in dark chocolate (Table 1); among these, five apigenins 285 (compounds 19.8, 19.12, 22.10, 25.1 and 27.3) were newly detected in this study. Compound 27.3 286 was tentatively associated to apigenin-C-hexoside-2′′-O-rhamnoside isomer, m/z 577.1617, whose 287 MS2 spectrum gave main fragment ions at m/z 457, 413 and 293, arising from the loss of 120 amu 288 (suggesting 1-2 linking between rhamnosyl-glucosyl group), 164 amu (rhamnosyl group), and 284 289 amu (164 plus 120 amu, i.e. rhamnose-glucosyl residue) (Dou, Lee, Tzen, & Lee, 2007; Hvattum, & 290 Ekeberg, 2003; Waridel et al., 2001). The next two isomers (compounds 19.8 and 19.12) at m/z 291 593.1591 were tentatively identified as C-diglycosylated apigenins; according to Jiang et al. (2013) 292 they were pinpointed as apigenin-6,8-di-C-glucoside isomers. Compound 25.1 generated the same 293 deprotonated ion at m/z 593.1591 and MS2 fragment ions at m/z 473, 413 and 293 corresponding to 294 the loss of 120 amu (suggesting 1-2 linking between two glucosyl groups), 180 amu (glucosyl 295 moiety) and 300 amu (a glucose-glucosyl residue). Thus, compound 25.1 was speculated to be 296 apigenin-C-hexoside-2′′-O-hexoside isomer (Hvattum, & Ekeberg, 2003; Dou et al., 2007). Finally, 297 following the scheme proposed by Lin et al. (2008) for the negative precursor ion [M-H]- m/z 298 563.1462 and considering the losses of 90 amu (MS2 fragment at m/z 473) and 120 amu (MS2 299 fragment at m/z 443) proving the existence of C-pentosyl- and C-hexosyl groups, compound 22.10 300 was tentatively identified as apigenin-C-hexoside-C-pentoside isomer. 301 14 302 3.1.3.3. Other phenolics 303 Compounds 29.1 and 34.2, showing the same negative precursor ion [M-H]- at m/z 451.1103, were 304 tentatively ascribed to cinchonain isomers, already detected in cocoa powder (Cádiz-Gurrea et al., 305 2014) but never before in dark chocolate. Four glycosidic forms of naringenin (compound 19.3), 306 eriodictyol (compounds 18.4 and 19.2) and phloretin (compound 32.3) were tentatively identified 307 for the first time in dark chocolate and cocoa products and listed in Table 1. 3.1.3.1. Flavonols and derivative forms 231 Compound 19.3 at m/z 308 593.1591, was tentatively identified as naringeni-C-hexoside-7-O-hexoside isomer, confirmed by 309 the characteristic loss of 120 amu (C-glycosylation site) and 162 amu (O-glycosylation site) 310 from m/z 473 and 413, respectively, which pinpointed the presence of two hexose units attached to 311 the flavonoid aglycone in different positions (Hvattum, & Ekeberg, 2003; Waridel et al., 2001). The 312 7-O-glycosylation site was proved by the presence of MS2 fragment ion at m/z 283. Compound 18.4 313 displayed deprotonated ion at m/z of 449.1169 and showed MS2 major fragment ions at m/z 287 314 ([M−H]- -162; i.e. hexose moiety loss) and m/z 259, confirming 7-O-glycosylation site presence. 315 Based on these data, compound 18.4 was tentatively identified as eriodictyol-7-O-hexoside (De 316 Beer at al. 2012). Compound 19.2, characterized by the deprotonated ion at m/z 611.1662, gave 317 MS2 major fragment ions at m/z 449, 329 (corresponding to the loss of 120 amu (C-hexoside) and 318 162 amu (O-hexoside), respectively), 287 (eriodyctiol-aglycone) and 259 (7-O-glycosylation site). 319 Therefore, compound 19.2 was speculated to be eriodictiol-C-hexoside-7-O-hexoside isomer (De 320 Beer at al. 2012; Hvattum, & Ekeberg, 2003). The negative ionization mode of compound 32.3 321 exhibited a [M-H]- precursor ion at m/z 435.1376, with MS2 product ions at m/z 345 and 315, losing 322 90 and 120 amu, respectively. This fragmentation pattern has been previously described for 323 phloretin-C-hexoside isomer (Kazuno, Yanagida, Shindo, & Murayama, 2005). 324 325 Compounds 29.1 and 34.2, showing the same negative precursor ion [M-H]- at m/z 451.1103, were 304 tentatively ascribed to cinchonain isomers, already detected in cocoa powder (Cádiz-Gurrea et al., 305 2014) but never before in dark chocolate. Four glycosidic forms of naringenin (compound 19.3), 306 eriodictyol (compounds 18.4 and 19.2) and phloretin (compound 32.3) were tentatively identified 307 for the first time in dark chocolate and cocoa products and listed in Table 1. Compound 19.3 at m/z 308 593.1591, was tentatively identified as naringeni-C-hexoside-7-O-hexoside isomer, confirmed by 309 the characteristic loss of 120 amu (C-glycosylation site) and 162 amu (O-glycosylation site) 310 from m/z 473 and 413, respectively, which pinpointed the presence of two hexose units attached to 311 the flavonoid aglycone in different positions (Hvattum, & Ekeberg, 2003; Waridel et al., 2001). The 312 7-O-glycosylation site was proved by the presence of MS2 fragment ion at m/z 283. 3.1.3.1. Flavonols and derivative forms 231 Compound 18.4 313 displayed deprotonated ion at m/z of 449.1169 and showed MS2 major fragment ions at m/z 287 314 ([M−H]- -162; i.e. hexose moiety loss) and m/z 259, confirming 7-O-glycosylation site presence. 315 Based on these data, compound 18.4 was tentatively identified as eriodictyol-7-O-hexoside (De 316 Beer at al. 2012). Compound 19.2, characterized by the deprotonated ion at m/z 611.1662, gave 317 MS2 major fragment ions at m/z 449, 329 (corresponding to the loss of 120 amu (C-hexoside) and 318 162 amu (O-hexoside), respectively), 287 (eriodyctiol-aglycone) and 259 (7-O-glycosylation site). 319 Therefore, compound 19.2 was speculated to be eriodictiol-C-hexoside-7-O-hexoside isomer (De 320 Beer at al. 2012; Hvattum, & Ekeberg, 2003). The negative ionization mode of compound 32.3 321 exhibited a [M-H]- precursor ion at m/z 435.1376, with MS2 product ions at m/z 345 and 315, losing 322 90 and 120 amu, respectively. This fragmentation pattern has been previously described for 323 phloretin-C-hexoside isomer (Kazuno, Yanagida, Shindo, & Murayama, 2005). 324 325 3.1.4. Ellagitannins 326 15 Ellagitannins are known as polymeric structures including different numbers of galloyl and 327 hexahydroxydiphenoyl (HHDP) units esterified with glucose. Three ellagitannins were detected fo 328 the first time in cocoa. They were distinguished by their characteristic fragment ion spectra yieldin 329 sequential losses of galloyl (152 amu), gallate (170 amu), and HHDP residues (301 amu). 330 Following the ellagitannins fragmentation scheme pattern proposed by Mena et al. (2012), 331 compounds 16.5 (m/z 633.0796) and 37.1 (m/z 301.0054) can be tentatively identified as HHDP- 332 galloyl-hexose and ellagic acid, respectively. The ellagic acid was also confirmed by comparison 333 with the retention time of the standard and the MS2 spectrum. Compound 27.1, characterized by th 334 deprotonated ion at m/z 615.0723 and MS2 fragment ions at m/z 463, due to the loss of a galloyl 335 group from ([M–H]--152) and at m/z 301, due to the loss of one hexose moiety (162 amu), was 336 tentatively identified as ellagic acid-galloyl-hexoside (Teixeira, Bertoldi, Lajolo, Mariko, & 337 Hassimotto, 2015) 338 339 3.1.5. Hydroxybenzoic acids 340 A total of twenty hydroxybenzoic acids and derivatives were detected in this study. Three of these 341 (compounds 8.4, 9.2 and 9.3) were tentatively identified for the first time in dark chocolate and 342 cocoa beans and products. 3.1.3.1. Flavonols and derivative forms 231 Whereas 11 compounds (compounds 2.2, 5.1, 7.1, 8.1, 8.3, 9.4, 11.2, 343 13.4, 16.6, 16.7, 19.11) had been already identified in cocoa but never in dark chocolate (Ortega et 344 al., 2008). Compound 8.4 (m/z 315.0793) yielded MS2 fragment ions at m/z 153 and 109, displayin 345 the hexose moiety loss and the presence of protocatechuic-aglycone. It was tentatively identified a 346 protocatechuic acid-4-O-hexoside (Martini et al., 2017). Compounds 9.2 and 9.3, m/z 359.1073, 347 fragmented in the MS2 experiments giving major product ions at m/z 197, 182 and 153, suggesting 348 the presence of a syringic acid residue. The loss of 162 amu, proved by MS2 fragment ion at m/z 349 197, prompt us to tentatively identify this compound as syringic acid-4-O-hexoside. 350 351 Ellagitannins are known as polymeric structures including different numbers of galloyl and 327 hexahydroxydiphenoyl (HHDP) units esterified with glucose. Three ellagitannins were detected for 328 the first time in cocoa. They were distinguished by their characteristic fragment ion spectra yielding 329 sequential losses of galloyl (152 amu), gallate (170 amu), and HHDP residues (301 amu). 330 Following the ellagitannins fragmentation scheme pattern proposed by Mena et al. (2012), 331 compounds 16.5 (m/z 633.0796) and 37.1 (m/z 301.0054) can be tentatively identified as HHDP- 332 galloyl-hexose and ellagic acid, respectively. The ellagic acid was also confirmed by comparison 333 with the retention time of the standard and the MS2 spectrum. Compound 27.1, characterized by the 334 deprotonated ion at m/z 615.0723 and MS2 fragment ions at m/z 463, due to the loss of a galloyl 335 group from ([M–H]--152) and at m/z 301, due to the loss of one hexose moiety (162 amu), was 336 tentatively identified as ellagic acid-galloyl-hexoside (Teixeira, Bertoldi, Lajolo, Mariko, & 337 Hassimotto, 2015) 338 339 3.1.5. Hydroxybenzoic acids 340 A total of twenty hydroxybenzoic acids and derivatives were detected in this study. Three of these 341 (compounds 8.4, 9.2 and 9.3) were tentatively identified for the first time in dark chocolate and 342 cocoa beans and products. Whereas 11 compounds (compounds 2.2, 5.1, 7.1, 8.1, 8.3, 9.4, 11.2, 343 Following the ellagitannins fragmentation scheme pattern proposed by Mena et al. (2012), 331 A total of twenty hydroxybenzoic acids and derivatives were detected in this study. Three of these 341 (compounds 8.4, 9.2 and 9.3) were tentatively identified for the first time in dark chocolate and 342 cocoa beans and products. 3.1.3.1. Flavonols and derivative forms 231 Whereas 11 compounds (compounds 2.2, 5.1, 7.1, 8.1, 8.3, 9.4, 11.2, 343 13.4, 16.6, 16.7, 19.11) had been already identified in cocoa but never in dark chocolate (Ortega et 344 al., 2008). Compound 8.4 (m/z 315.0793) yielded MS2 fragment ions at m/z 153 and 109, displaying 345 the hexose moiety loss and the presence of protocatechuic-aglycone. It was tentatively identified as 346 protocatechuic acid-4-O-hexoside (Martini et al., 2017). Compounds 9.2 and 9.3, m/z 359.1073, 347 fragmented in the MS2 experiments giving major product ions at m/z 197, 182 and 153, suggesting 348 the presence of a syringic acid residue. The loss of 162 amu, proved by MS2 fragment ion at m/z 349 197, prompt us to tentatively identify this compound as syringic acid-4-O-hexoside. 350 351 351 16 3.1.6. Curcuminoids 352 3.1.6. Curcuminoids 352 From the extracts of turmeric dark chocolate sample, we detected the [M–H]- precursor ions at m/z 353 307.1043, 337.1164, and 367.1257 (compounds 36.1, 36.2 and 36.3). As reported by Jiang, 354 Somogyi, Jacobsen, Timmermann, & Gang (2006), product ions at m/z 187 or 217 were the typica 355 fragment ions in the MS2 spectra of deprotonated [M–H]- curcuminoids. In comparison to 356 fragmentation pattern proposed, compounds 36.1, 36.2 and 36.3 were tentatively identified as 357 bisdemethoxycurcumin, demethoxycurcumin and curcumin, respectively (Table 1). 358 359 3.2. Phenolic compounds in chocolate 360 Table 2 provides information about the amount of the 158 tentatively identified phenolic 361 compounds in the different types of chocolate. In order to quantify the amount of total phenolic 362 compounds in chocolates, seven calibration curves were prepared with the available authentic 363 standards: epicatechin, coumaric and ferulic acids, quercetin-3-O-rutinoside, ellagic acid, 364 protocatechuic acid and curcumin. In all cases, the linearity was better than 0.99. The other 365 compounds, for which no commercial standards were available, were tentatively quantified using 366 the standards with similar structural characteristics and considering the functional groups that may 367 affect the ionisation properties. As shown in Figure 3, even if flavan-3-ols were the most 368 representative class in each type of chocolate, the phenolic profile is thoroughly influenced by the 369 addition of Sakura green tea or turmeric powder. 370 371 3.2.1. Dark chocolate (DC) phenolic profile 372 From the extracts of turmeric dark chocolate sample, we detected the [M–H]- precursor ions at m/z 353 307.1043, 337.1164, and 367.1257 (compounds 36.1, 36.2 and 36.3). As reported by Jiang, 354 Somogyi, Jacobsen, Timmermann, & Gang (2006), product ions at m/z 187 or 217 were the typical 355 fragment ions in the MS2 spectra of deprotonated [M–H]- curcuminoids. In comparison to 356 fragmentation pattern proposed, compounds 36.1, 36.2 and 36.3 were tentatively identified as 357 bisdemethoxycurcumin, demethoxycurcumin and curcumin, respectively (Table 1). 358 359 From the extracts of turmeric dark chocolate sample, we detected the [M–H]- precursor ions at m/z 353 307.1043, 337.1164, and 367.1257 (compounds 36.1, 36.2 and 36.3). As reported by Jiang, 354 Somogyi, Jacobsen, Timmermann, & Gang (2006), product ions at m/z 187 or 217 were the typical 355 fragment ions in the MS2 spectra of deprotonated [M–H]- curcuminoids. 3.1.6. Curcuminoids 352 In comparison to 356 fragmentation pattern proposed, compounds 36.1, 36.2 and 36.3 were tentatively identified as 357 bisdemethoxycurcumin, demethoxycurcumin and curcumin, respectively (Table 1). 358 359 3.2.1. Dark chocolate (DC) phenolic profile 372 As determined by LC-MS/MS experiments, the total phenolic concentration in DC was 787.63 ± 373 10.90 mg/100 g of chocolate, representing about 30.0% of total phenolic compounds determined 374 with the Folin-Ciocalteau assay (2624.15 ± 112.36 mg/100 g of chocolate). The ABTS radical 375 scavenging and Fe3+-reducing ability of DC (Figure 4) were tested (11.00 ± 0.26 and 6.29 ± 0.13 376 17 mmol trolox equivalents/100 g of chocolate, respectively) resulting in line with the findings 377 proposed by Batista et al. (2016). As reported by Wollgast & Anklam (2000a), catechins and 378 procyanidins represent more than 90% of phenolic profile of cocoa beans and cocoa-products. We 379 found out that total flavan-3-ols amount in DC was 503.76 ± 8.98 mg/100 g of chocolate 380 representing the 64.0% of total polyphenols identified by MS experiments. Considering monomeric 381 structures, epicatechin and catechin were the major represented flavan-3-ols, whose estimated 382 concentrations were higher than those reported so far (Gu et al., 2006). Epicatechin alone 383 represented the 40.4 % of total flavan-3-ols and the 25.8% of total phenolic identified by MS 384 experiments, resulting the most present compounds in DC. Large amounts of oligomeric structures 385 were also found, displaying a total concentration value of 166.28± 4.13 mg/100 g of chocolate and 386 reaching approximately 33.0% of flavan-3-ols class. Epicatechin has been causally linked to the 387 reported cardiovascular effects observed after the consumption of cocoa (Schroeter et al., 2006). 388 The ingestion of flavanol-rich cocoa in healthy adult males was associated with acute elevations in 389 levels of circulating nitric oxide, an enhanced flow-mediated dilation response of conduit arteries, 390 and an augmented microcirculation in humans and the results were repeatable with pure epicatechin 391 intake (70 mg/day; equivalent to 35-40 g of DC). Indeed, elderly men with a median epicatechin 392 intake of 22 mg/day (equivalent to 10-15 g of DC) had a 38% lower risk of cardiovascular disease 393 mortality than that of subjects with a median intake of 8 mg/day (Dower, Geleijnse, 394 Hollman, Soedamah-Muthu, & Kromhout, 2016). 3.1.6. Curcuminoids 352 395 The hydroxycinnamic acids made up about 20.6% of DC phenolic profile, among these ferulic acid, 396 di-hydroxycinnamic aspartate and coumaroyl aspartate were the main hydroxycinnamic acids 397 Hollman, Soedamah-Muthu, & Kromhout, 2016). 395 The hydroxycinnamic acids made up about 20.6% of DC phenolic profile, among these ferulic acid, 396 di-hydroxycinnamic aspartate and coumaroyl aspartate were the main hydroxycinnamic acids 397 detected in DC. The largest contribution was given by ferulic acid, with a concentration of 61.23 ± 398 3.74 mg/100 g of chocolate. Among the N-phenylpropenoyl-L-amino acids, clovamide or caffeoyl- 399 tyrosine, described for the first time in cocoa by Sanbongi et al. (1998), was the main representative 400 with total concentration of its two isomers of 9.54 ± 0.54 mg/100 g of chocolate. Previous studies 401 18 found that clovamide exhibited antiradical properties (Locatelli et al., 2013; Sanbongi et al., 1998), 402 neuroprotective effects (Fallarini et al., 2009) and anti-inflammatory properties (Zeng et al., 2011). 403 Ellagitannins, which were identified for the first time in dark chocolate and cocoa in this study, 404 made up about 10% of DC phenolic profile. The higher amount was ascribed to ellagic acid (56.16 405 ± 3.58 mg/100 g of chocolate), followed by HHDP-galloyl-hexoside (15.79 ± 1.20 mg/100 g of 406 chocolate). Ellagic acid and ellagitannins can be metabolized by human microbiota in urolithins, 407 which are responsible for the health effects attributed to the consumption of ellagic acid and 408 ellagitannins-rich food (Tomás-Barberán et al., 2017). 409 410 3.2.2. Sakura green tea dark chocolate (GTDC) phenolic profile 411 The content of total polyphenolic compounds in GTDC displayed a significant increase (P value 412 <0.001) in respect to that of DC, recording a total concentration value of 1035.45 ± 14.81 mg/100 g 413 of chocolate (Figure 3). This value represented the 30.3% of total phenolic compounds determined 414 with the Folin-Ciocalteau assay (3417.81 ± 229.45 mg/100 g of chocolate). The increased phenolic 415 concentration resulted in increased antioxidant properties in comparison with DC, which gave rise 416 to 40% and 144% enhancements of GTDC ABTS radical scavenging and ferric-reducing power, 417 respectively (Figure 4). The major phenolics in GTDC were still flavan-3-ols accounting for about 418 70.1% of total phenolic compounds, displaying a concentration value of 726.03 ± 14.53 mg/100 g 419 of chocolate, significantly different from DC flavan-3-ols content (503.76 ± 8.98 mg/100 g of 420 chocolate, P value <0.001). 3.1.6. Curcuminoids 352 This flavan-3-ols increase was related to the Sakura green tea leaves 421 enrichment of dark chocolate formulation and was clearly reflected in the significant increase in 422 epicatechin (303.69 ± 11.65 mg/100 g of chocolate, P value <0.001, detailing about 30% of GTDC 423 phenolic profile), epigallocatechin (29.76 ± 1.74 mg/100 g of chocolate, P value <0.001) and total 424 procyanidins (230.76 ± 15.73 mg/100 g of chocolate, P value <0.001). The Sakura green tea 425 contribution was also confirmed by the presence of typical green tea gallate flavan-3-ols, especially 426 found that clovamide exhibited antiradical properties (Locatelli et al., 2013; Sanbongi et al., 1998), 402 neuroprotective effects (Fallarini et al., 2009) and anti-inflammatory properties (Zeng et al., 2011). 403 Ellagitannins, which were identified for the first time in dark chocolate and cocoa in this study, 404 made up about 10% of DC phenolic profile. The higher amount was ascribed to ellagic acid (56.16 405 ± 3.58 mg/100 g of chocolate), followed by HHDP-galloyl-hexoside (15.79 ± 1.20 mg/100 g of 406 chocolate). Ellagic acid and ellagitannins can be metabolized by human microbiota in urolithins, 407 which are responsible for the health effects attributed to the consumption of ellagic acid and 408 ellagitannins-rich food (Tomás-Barberán et al., 2017). 409 3.2.2. Sakura green tea dark chocolate (GTDC) phenolic profile 411 The content of total polyphenolic compounds in GTDC displayed a significant increase (P value 412 <0.001) in respect to that of DC, recording a total concentration value of 1035.45 ± 14.81 mg/100 g 413 of chocolate (Figure 3). This value represented the 30.3% of total phenolic compounds determined 414 with the Folin-Ciocalteau assay (3417.81 ± 229.45 mg/100 g of chocolate). The increased phenolic 415 concentration resulted in increased antioxidant properties in comparison with DC, which gave rise 416 to 40% and 144% enhancements of GTDC ABTS radical scavenging and ferric-reducing power, 417 respectively (Figure 4). The major phenolics in GTDC were still flavan-3-ols accounting for about 418 70.1% of total phenolic compounds, displaying a concentration value of 726.03 ± 14.53 mg/100 g 419 of chocolate, significantly different from DC flavan-3-ols content (503.76 ± 8.98 mg/100 g of 420 chocolate, P value <0.001). This flavan-3-ols increase was related to the Sakura green tea leaves 421 enrichment of dark chocolate formulation and was clearly reflected in the significant increase in 422 epicatechin (303.69 ± 11.65 mg/100 g of chocolate, P value <0.001, detailing about 30% of GTDC 423 phenolic profile), epigallocatechin (29.76 ± 1.74 mg/100 g of chocolate, P value <0.001) and total 424 procyanidins (230.76 ± 15.73 mg/100 g of chocolate, P value <0.001). The Sakura green tea 425 contribution was also confirmed by the presence of typical green tea gallate flavan-3-ols, especially 426 The content of total polyphenolic compounds in GTDC displayed a significant increase (P value 412 <0.001) in respect to that of DC, recording a total concentration value of 1035.45 ± 14.81 mg/100 g 413 of chocolate (Figure 3). This value represented the 30.3% of total phenolic compounds determined 414 with the Folin-Ciocalteau assay (3417.81 ± 229.45 mg/100 g of chocolate). The increased phenolic 415 concentration resulted in increased antioxidant properties in comparison with DC, which gave rise 416 to 40% and 144% enhancements of GTDC ABTS radical scavenging and ferric-reducing power, 417 respectively (Figure 4). The major phenolics in GTDC were still flavan-3-ols accounting for about 418 70.1% of total phenolic compounds, displaying a concentration value of 726.03 ± 14.53 mg/100 g 419 of chocolate, significantly different from DC flavan-3-ols content (503.76 ± 8.98 mg/100 g of 420 chocolate, P value <0.001). 3.2.2. Sakura green tea dark chocolate (GTDC) phenolic profile 411 These results may confirm a possible 431 polyphenols enrichment of dark chocolate profile which can lead to a potential combination of the 432 positive health effects and properties derived from both chocolate and green tea. LC-MS 433 experiments showed that GTDC contained 49% more epicatechin and 43% more flavan-3-ols than 434 DC. This can result in a lower intake to achieve the same biological effects. This seems a promising 435 way to maximise the potential beneficial effect of epicatechin consumption, contemporaneously 436 reducing the amount of sugars and calories introduced with chocolate. 437 438 3.2.3. Turmeric dark chocolate (TDC) phenolic profile 439 The TDC phenolic amount showed a significant increase (P value <0.001) respect to that of DC 440 which recorded a total concentration value of 1094.03 ± 10.15 mg/100 g of chocolate (Figure 3), 441 representing about 36% of total phenolic compounds assayed with the Folin-Ciocalteau method 442 (3043.81 ± 294.64 mg/100 g of chocolate). Despite that, single phenolic classes did not show a 443 significant and remarkable increase respect to those of DC. This higher concentration can be 444 ascribed to turmeric powder contribution as well as the related curcuminoids, which accounted for 445 about 25% of TDC total phenolic profile, displaying a concentration value of 272.73 ± 2.58 mg/100 446 g of chocolate (Figure 3). ABTS radical scavenging ability and ferric-reducing power were tested, 447 resulting in 12.30 ± 0.27 and 10.57 ± 0.2 mmol trolox equivalents/100 g of chocolate, respectively 448 (Figure 4). Bisdemethoxycurcumin was the most concentrated curcuminoid (115.55 ± 2.16 mg/100 449 g of chocolate), followed by demethoxycurcumin (82.64 ± 1.33 mg/100 g of chocolate) which are 450 considered to be curcumin natural analogues and were reported to have a similar biological activity 451 3.2.2. Sakura green tea dark chocolate (GTDC) phenolic profile 411 This flavan-3-ols increase was related to the Sakura green tea leaves 421 enrichment of dark chocolate formulation and was clearly reflected in the significant increase in 422 epicatechin (303.69 ± 11.65 mg/100 g of chocolate, P value <0.001, detailing about 30% of GTDC 423 phenolic profile), epigallocatechin (29.76 ± 1.74 mg/100 g of chocolate, P value <0.001) and total 424 procyanidins (230.76 ± 15.73 mg/100 g of chocolate, P value <0.001). The Sakura green tea 425 contribution was also confirmed by the presence of typical green tea gallate flavan-3-ols, especially 426 19 epigallocatechin gallate, showing a remarkable conc 427 chocolate. The hydroxycinnamic acids were still the 428 profile in GTDC, explaining about 15.3% of GTDC 429 showed a significant content increasing in GTDC re 430 chocolate, P value <0.001) with an incidence rate of 431 polyphenols enrichment of dark chocolate profile wh 432 positive health effects and properties derived from b 433 experiments showed that GTDC contained 49% mor 434 DC. This can result in a lower intake to achieve the 435 way to maximise the potential beneficial effect of ep 436 reducing the amount of sugars and calories introduce 437 438 3.2.3. Turmeric dark chocolate (TDC) phenolic pro 439 The TDC phenolic amount showed a significant incr 440 which recorded a total concentration value of 1094.0 441 representing about 36% of total phenolic compound 442 (3043.81 ± 294.64 mg/100 g of chocolate). Despite t 443 significant and remarkable increase respect to those 444 ascribed to turmeric powder contribution as well as t 445 about 25% of TDC total phenolic profile, displaying 446 g of chocolate (Figure 3). ABTS radical scavenging 447 resulting in 12.30 ± 0.27 and 10.57 ± 0.2 mmol trolo 448 (Figure 4). Bisdemethoxycurcumin was the most co 449 g of chocolate), followed by demethoxycurcumin (8 450 considered to be curcumin natural analogues and we 451 epigallocatechin gallate, showing a remarkable concentration value of 33.54 ± 2.16 mg/100 g of 427 chocolate. The hydroxycinnamic acids were still the second most representative class of phenolic 428 profile in GTDC, explaining about 15.3% of GTDC phenolic profile (Figure 2). Ellagitannins 429 showed a significant content increasing in GTDC respect to DC (89.12 ± 1.50 mg/100 g of 430 chocolate, P value <0.001) with an incidence rate of 8.6%. 3.2.3. Turmeric dark chocolate (TDC) phenolic profile 439 The TDC phenolic amount showed a significant increase (P value <0.001) respect to that of DC 440 which recorded a total concentration value of 1094.03 ± 10.15 mg/100 g of chocolate (Figure 3), 441 representing about 36% of total phenolic compounds assayed with the Folin-Ciocalteau method 442 (3043.81 ± 294.64 mg/100 g of chocolate). Despite that, single phenolic classes did not show a 443 significant and remarkable increase respect to those of DC. This higher concentration can be 444 ascribed to turmeric powder contribution as well as the related curcuminoids, which accounted for 445 about 25% of TDC total phenolic profile, displaying a concentration value of 272.73 ± 2.58 mg/100 446 g of chocolate (Figure 3). ABTS radical scavenging ability and ferric-reducing power were tested, 447 resulting in 12.30 ± 0.27 and 10.57 ± 0.2 mmol trolox equivalents/100 g of chocolate, respectively 448 (Figure 4). Bisdemethoxycurcumin was the most concentrated curcuminoid (115.55 ± 2.16 mg/100 449 g of chocolate), followed by demethoxycurcumin (82.64 ± 1.33 mg/100 g of chocolate) which are 450 considered to be curcumin natural analogues and were reported to have a similar biological activity 451 The TDC phenolic amount showed a significant increase (P value <0.001) respect to that of DC 440 which recorded a total concentration value of 1094.03 ± 10.15 mg/100 g of chocolate (Figure 3), 441 representing about 36% of total phenolic compounds assayed with the Folin-Ciocalteau method 442 (3043.81 ± 294.64 mg/100 g of chocolate). Despite that, single phenolic classes did not show a 443 significant and remarkable increase respect to those of DC. This higher concentration can be 444 ascribed to turmeric powder contribution as well as the related curcuminoids, which accounted for 445 about 25% of TDC total phenolic profile, displaying a concentration value of 272.73 ± 2.58 mg/100 446 g of chocolate (Figure 3). ABTS radical scavenging ability and ferric-reducing power were tested, 447 resulting in 12.30 ± 0.27 and 10.57 ± 0.2 mmol trolox equivalents/100 g of chocolate, respectively 448 (Figure 4). Bisdemethoxycurcumin was the most concentrated curcuminoid (115.55 ± 2.16 mg/100 449 g of chocolate), followed by demethoxycurcumin (82.64 ± 1.33 mg/100 g of chocolate) which are 450 considered to be curcumin natural analogues and were reported to have a similar biological activity 451 20 to curcumin itself (Kocaadam, & Şanlier, 2017). Curcumin was found at the lowest concentration of 452 74.55 ± 0.47 mg/100 g of chocolate. 3.2.3. Turmeric dark chocolate (TDC) phenolic profile 439 Normally, curcumin is present at a concentration higher or 453 similar to the demethoxylated analogue (Jayaprakasha, Rhao, & Sakariah, 2002). Since the phenolic 454 composition of spices (and of vegetable food in general) is greatly variable depending on the 455 cultivar and agro-climatic factors (such as growing, harvesting time, seasonal variability) as well as 456 technological processes, it is plausible that different turmeric powder preparation had different 457 phenolic composition. Moreover, in the case of dark chocolate enriched with turmeric powder a 458 possible food matrix effect should be considered since some macromolecules such as proteins and 459 polysaccharide may interact with curcuminoids reducing their extractability. It is important also to 460 note that only free and extractable phenolic compounds were considered and analysed in this study. 461 Curcuminoids are widely known for their healthy properties such as anti-inflammatory, antioxidant, 462 antimicrobial, anticoagulant, anticancer and antimutagenic properties (Kocaadam, & Şanlier, 2017; 463 Kunnumakkara et al., 2017). To date, over 100 different clinical trials have been successfully 464 carried out, showing their safety, tolerability and effectiveness against several chronic diseases in 465 humans such as various types of cancers, diabetes, obesity, cardiovascular and neurological diseases 466 (Kunnumakkara et al., 2017). Finally, the synergistic behaviour displayed by curcuminoids with 467 other nutraceuticals such as catechins and quercetin, resulting an increased effect against oxidative 468 stress in normal healthy adults, was demonstrated (Dominiak, McKinney, Heilbrun, & Sarkar, 469 2010). Therefore, an enhanced and strengthened health effect because of the union of polyphenol- 470 rich sources, combining the positive effects of dark chocolate phenolics and turmeric curcuminoids, 471 can be speculated. 472 473 4. Conclusions 474 Literature provides a lot of information about cocoa polyphenols and properties, but there is still a 475 big gap about the phenolic composition of chocolate. Few studies investigated the phenolic 476 to curcumin itself (Kocaadam, & Şanlier, 2017). Curcumin was found at the lowest concentration of 452 74.55 ± 0.47 mg/100 g of chocolate. Normally, curcumin is present at a concentration higher or 453 similar to the demethoxylated analogue (Jayaprakasha, Rhao, & Sakariah, 2002). Since the phenolic 454 composition of spices (and of vegetable food in general) is greatly variable depending on the 455 cultivar and agro-climatic factors (such as growing, harvesting time, seasonal variability) as well as 456 technological processes, it is plausible that different turmeric powder preparation had different 457 phenolic composition. 3.2.3. Turmeric dark chocolate (TDC) phenolic profile 439 Moreover, in the case of dark chocolate enriched with turmeric powder a 458 possible food matrix effect should be considered since some macromolecules such as proteins and 459 polysaccharide may interact with curcuminoids reducing their extractability. It is important also to 460 note that only free and extractable phenolic compounds were considered and analysed in this study. 461 Curcuminoids are widely known for their healthy properties such as anti-inflammatory, antioxidant, 462 antimicrobial, anticoagulant, anticancer and antimutagenic properties (Kocaadam, & Şanlier, 2017; 463 Kunnumakkara et al., 2017). To date, over 100 different clinical trials have been successfully 464 21 composition of dark chocolate, focusing on flavan-3-ols as the major class in chocolate phenolic 477 profile. The purpose of this study was to overcome this lack of information, providing an accurate 478 and comprehensive characterisation of the phenolic profile of dark chocolate (70%). The 479 quantitative metabolomics approach used in this study allowed a tentative identification of 158 480 individual phenolic compounds in dark chocolate. Among the detected compounds, 67 have been 481 reported for the first time in dark chocolate, 38 of whom were identified for the first time in 482 chocolate, cocoa beans and cocoa products. This characterization extends the current knowledge on 483 the phytochemistry of dark chocolate and is, to our knowledge, the broadest profiling of its phenolic 484 compounds to date. 485 Results reported in this study also showed that the addition of Sakura green tea leaves or turmeric 486 powder influenced and modified the phenolic profile of dark chocolate, resulting in a phenolic 487 concentration increase. Mass spectrometry confirmed that this increase was strictly connected to the 488 food matrix, showing typical compounds belonging to green tea and turmeric. In this way, this 489 functional chocolate might maximize the potential beneficial effect of polyphenols-rich food 490 consumption and, at the same time, reducing the amount of sugars and calories introduced with 491 chocolate, resulting in a lower intake to achieve the same biological effects. This work may revise 492 the concept of “optimal” dose of chocolate in the context of a balanced diet, which optimizes the 493 functional properties by avoiding potential side effects, such as high-calorie intake. 494 composition of dark chocolate, focusing on flavan-3-ols as the major class in chocolate phenolic 477 profile. 3.2.3. Turmeric dark chocolate (TDC) phenolic profile 439 The purpose of this study was to overcome this lack of information, providing an accurate 478 and comprehensive characterisation of the phenolic profile of dark chocolate (70%). The 479 quantitative metabolomics approach used in this study allowed a tentative identification of 158 480 individual phenolic compounds in dark chocolate. Among the detected compounds, 67 have been 481 reported for the first time in dark chocolate, 38 of whom were identified for the first time in 482 chocolate, cocoa beans and cocoa products. This characterization extends the current knowledge on 483 the phytochemistry of dark chocolate and is, to our knowledge, the broadest profiling of its phenolic 484 compounds to date. 485 Results reported in this study also showed that the addition of Sakura green tea leaves or turmeric 486 powder influenced and modified the phenolic profile of dark chocolate, resulting in a phenolic 487 concentration increase. Mass spectrometry confirmed that this increase was strictly connected to the 488 food matrix, showing typical compounds belonging to green tea and turmeric. In this way, this 489 functional chocolate might maximize the potential beneficial effect of polyphenols-rich food 490 consumption and, at the same time, reducing the amount of sugars and calories introduced with 491 chocolate, resulting in a lower intake to achieve the same biological effects. This work may revise 492 the concept of “optimal” dose of chocolate in the context of a balanced diet, which optimizes the 493 functional properties by avoiding potential side effects, such as high-calorie intake. 494 22 Financial support This work was supported by a grant from Department of Life Sciences, University of Modena and Reggio Emilia (research project FAR2016 “Dieta Mediterranea e salute: riduzione dei fenomeni ossidativi durante la digestione della carne”). 23 References Andres-Lacueva, C., Monagas, M., Khan, N., Izquierdo-Pulido, M., Urpi-Sarda, M., Permanyer, J., & Lamuela-Raventós, R. M. (2008). 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Representative negative ion mode base peak chromatograms (BPCs) of dark chocolate (A) green tea dark chocolate (B) and turmeric dark chocolate (C). The shown BPCs are representative of three independent experiments and represent the profile of the methanol extracts. Figure 2. Structures of newly identified dark chocolate phenolic compounds. Examples of some newly identified phenolic structures belonging to flavan-3-ols (A), flavonols (B), Figure 2. Structures of newly identified dark chocolate phenolic compounds. Examples of some newly identified phenolic structures belonging to flavan-3-ols (A), flavonols (B), hydroxybenzoic and hydroxycinnamic acids (C), flavones (D), flavanones (E) and dihydrochalcones (F). Gall: galloyl; gluc: glucuronide; hex: hexoside; pent: pentoside; rham: rhamnoside; rut: rutinoside; trihydroxy: trihydroxybenzene. hydroxybenzoic and hydroxycinnamic acids (C), flavones (D), flavanones (E) and dihydrochalcones (F). Gall: galloyl; gluc: glucuronide; hex: hexoside; pent: pentoside; rham: rhamnoside; rut: rutinoside; trihydroxy: trihydroxybenzene. Figure 3. Occurrence of phenolic classes in dark chocolates. Global percentage of flavan-3-ols, flavonols, hydroxybenzoic and hydroxycinnamic acids, ellagitannins, flavones and other phenolics in dark chocolate and dark chocolate enriched with Sakura green tea leaves or turmeric powder. In brackets are reported the total amounts of phenolic compounds quantified with mass spectrometry. 70% means the total percentage of cocoa in the dark chocolates. Figure 4. Antioxidant properties of dark chocolates. Antioxidant capacity (expressed as mmol trolox/100g of chocolate), measured by ABTS (A) and FRAP (B) assays. DC: dark chocolate; GTDC: dark chocolate enriched with Sakura green tea leaves; TDC: dark chocolate enriched with turmeric powder. Each sample was run in triplicate and results are reported as mean values ± SD. Values in the same graph with different lowercase letter are significantly different (P < 0.05). 31 Table 1. Mass spectral data of phenolic compounds identified in differe Peak Compound Formula Calc. (m/z) Exp. Figure 2. Structures of newly identified dark chocolate phenolic compounds. Examples of some newly identified phenolic structures belonging to flavan-3-ols (A), flavonols (B), (m/z) Error (ppm) 1 1.1 Coumaric acids C9H8O3 163.0473 163.0466 4.54 2 2.1 Di-hydro-caffeic acid C9H10O4 181.0579 181.0575 2.25 2.2 Hydroxybenzoic acid isomer C7H6O3 137.0317 137.0316 0.09 3 3.1 Gallic acida,s C7H6O5 169.0215 169.0221 -3.39 3.2 Galloyl glucose isomera C13H16O10 331.0744 331.0756 -3.77 1 4 4.1 Galloylquinic acid isomera C14H16O10 343.0744 343.0730 3.91 5 5.1 Vanillic acid-4-O-hexoside isomer C14H18O9 329.0951 329.0967 -4.90 6 6.1 Galloylquinic acid isomera C14H16O10 343.0744 343.0730 3.91 7 7.1 Hydroxybenzoic acid isomer C7H6O3 137.0317 137.0316 0.09 8 8.1 Vanillic acid-4-O-hexoside isomer C14H18O9 329.0951 329.0967 -4.90 1 8.2 Vanillic acid derivative C15H14N2O9 365.0699 365.0690 2.54 1 8.3 Vanillic acid isomer C8H8O4 167.0423 167.0417 3.33 1 8.4 Protocatechuic acid-4-O- hexoside C13H16O9 315.0794 315.0793 0.42 8.5 Di-hydroxycinnamic acid isomer C9H8O4 179.0423 179.0431 -4.67 8.6 Protocatechuic acids C7H6O4 153.0266 153.0267 -0.09 9 9.1 Gallocatechins C15H14O7 305.0740 305.0733 2.13 2 2 9.2 Syringic acid-4-O-hexoside isomer C15H20O10 359.1056 359.1073 -4.59 19 9.3 Syringic acid-4-O-hexoside isomer C15H20O10 359.1056 359.1073 -4.59 19 9.4 Syringic acid C9H10O5 197.0528 197.0532 -0.38 18 10 10.1 Gallocatechin-3-O-hexoside C21H24O12 467.1268 467.1270 -0.48 35 10.2 Di-hydroxycinnamic aspartate isomer C13H13NO7 294.0692 294.0692 0.00 1 10.3 Di-hydroxycinnamic acid isomer C9H8O4 179.0423 179.0431 -4.67 1 11 11.1 Epigallocatechin-3-O- hexoside C21H24O12 467.1268 467.1270 -0.48 35 11.2 Hydroxybenzoic acid isomer C7H6O3 137.0317 137.0316 0.09 12 12.1 Procyanidin dimer B type isomer C30H26O12 577.1424 577.1411 2.29 4 28 12.2 (Epi)catechin- (Epi)gallocatechina C30H26O13 593.1373 593.1345 4.78 4 30 12.3 3-O-Coumaroylquinic acid cisa C16H18O8 337.1002 337.1006 -1.28 19 13 13.1 (Epi)catechin-3-O-hexoside isomer C21H24O11 451.1319 451.1320 -0.31 2 2 13.2 Epigallocatechins C15H14O7 305.0740 305.0733 2.13 2 2 13.3 Caffeic acids C9H8O4 179.0423 179.0431 -4.67 13.4 Hydroxybenzoic acid isomer C7H6O3 137.0317 137.0316 0.09 ified in different dark chocolates. Figure 2. Structures of newly identified dark chocolate phenolic compounds. Examples of some newly identified phenolic structures belonging to flavan-3-ols (A), flavonols (B), Error (ppm) MS2 ion fragments (m/z) (relative abundance in %) 4.54 119.0492 (100), 93.0435 (5) 2.25 137.0234 (100) 0.09 93.0315 (100) -3.39 125.0287 (100) -3.77 169.0194 (100), 125.0252 (29) 3.91 191.0731 (100), 169.0275 (5) -4.90 167.0403 (100) 3.91 191.0676 (100), 169.0213 (5) 0.09 93.0390 (100) -4.90 167.0341 (100), 123.0494 (25) 2.54 167.0319 (100), 123.0463 (14) 3.33 151.0067 (100), 123.0479 (24) 0.42 153.0183, 109.0270 -4.67 163.0253 (5), 135.0475 (100) -0.09 109.0313 (100) 2.13 261.0800 (14), 221.0471 (28), 219.0695 (19), 179.0373 (35), 125.0225 (100) -4.59 197.0574 (100), 182.0310 (28), 153.0619 (51) -4.59 197.0463 (100), 182.0212 (18), 153.0541 (22) -0.38 182.0320 (100), 167.0073 (30), 153.0246 (13) -0.48 357.0558 (23), 305.0858 (100), 287.0436 (71), 125.0215 (79) 0.00 276.0682 (13), 250.0946 (3), 232.0868 (5), 206.0683 (3), 179.0190 (16), 132.0395 (100) -4.67 163.0348 (10), 135.0426 (100) -0.48 357.0588 (19), 305.0885 (100), 287.0722 (75), 125.0232 (83) 0.09 93.0351 (100) 2.29 425.0822 (11), 407.0788 (75), 289.0717 (100), 245.0784 (17), 205.0476 (6), 125.0279 (66) 4.78 425.1001 (33), 407.0958 (21), 305.0739 (74), 289.0857 (100), 205.0563 (26), 125.0238 (80) -1.28 191.0642 (40), 163.0460 (100), 119.0504 (27) -0.31 289.0797 (100), 259.1312 (3), 245.0956 (23), 205.0748 (11), 179.0377 (5) 2.13 261.0883 (19), 221.0467 (19), 219.0688 (28), 179.0355 (42), 125.0219 (100) -4.67 163.0244 (8), 135.0398 (100) 0.09 93.0328 (100) 13.5 Procyanidin trimer B type isomer C45H38O18 865.2058 865.2044 1.63 577.1764 (5), 425.0757 (10), 407.0839 (42), 289.0634 (64), 287.0617 (38), 245.0573 (14), 125.0264 (100) 14 14.1 (Epi)catechin-C-hexoside isomer C21H24O11 451.1319 451.1320 -0.31 361.0988 (25), 331.0879 (100) 14.2 Di-hydroxycinnamic aspartate isomer C13H13NO7 294.0692 294.0692 0.00 276.0573 (2), 250.0744 (2), 232.0617 (1), 206.0771 (2), 179.0318 (7), 132.0256 (100) 14.3 3-O-Coumaroylquinic acid transa C16H18O8 337.1002 337.1006 -1.28 191.0453 (4), 163.0307 (100), 119.0409 (15) 15 15.1 (Epi)catechin-7-O-hexoside C21H24O11 451.1319 451.1320 -0.31 289.0942 (100), 261.1150 (3), 245.1104 (42), 205.0561 (9), 179.0365 (3) 15.2 Catechins C15H14O6 289.0790 289.0791 -0.21 245.0878 (100), 205.0554 (38), 179.0372 (31), 125.0247 (40) 16 16.1 Procyanidin tetramer B type isomer C60H50O24 576.1346 576.1342 (2-) 0.69 425.1088 (5), 407.0948 (15), 289.0840 (100), 287.0653 (6), 245.1158 (43), 125.0282 (77) 16.2 (Epi)catechin-C-hexoside isomer C21H24O11 451.1319 451.1320 -0.31 361.0843 (16), 331.0787 (100) 16.3 Procyanidin dimer B type hexoside isomer C36H36O17 739.1952 739.1915 5.00 449.1175 (11), 407.0840 (8), 289.0595 (100), 245.0629 (16), 125.0197 (42) 16.4 Vanillin C8H8O3 151.0473 151.0481 -4.97 136.0181 (28), 123.0487 (100), 121.0315 (20), 109.0296 (24), 108.0201 (97) 16.5 HHDP-galloyl-hexoside C27H22O18 633.0806 633.0796 1.60 463.0687 (18), 301.0102 (100), 275.0315 (11), 257.0196 (4), 229.0331 (4) 16.6 Vanillic acid isomer C8H8O4 167.0423 167.0417 3.33 151.0050 (30), 123.0471 (100) 16.7 Vanillic acid-4-O-hexoside isomer C14H18O9 329.0951 329.0967 -4.90 167.0473 (100), 123.0524 (6) 16.8 5-O-Caffeoylquinic acid C16H18O9 353.0951 353.0952 -0.12 191.0133 (100), 173.0049 (36) 17 17.1 Procyanidin dimer B type isomer C30H26O12 577.1424 577.1411 2.29 425.0867 (30), 407.0750 (61), 289.0641 (100), 245.0792 (20), 205.0497 (25), 125.0158 (72) 17.2 3-O-Caffeoylquinic acids C16H18O9 353.0951 353.0952 -0.12 191.0667 (38), 179.0433 (100), 173.0504 (43), 135.0459 (8) 18 18.1 Procyanidin trimer B type isomer C45H38O18 865.2058 865.2044 1.63 577.1838 (8), 425.1135 (18), 407.1065 (48), 289.0844 (69), 287.0773 (73), 245.0947 (9), 125.0187 (100) 18.2 Procyanidin dimer B type hexoside isomer C36H36O17 739.1952 739.1915 5.00 449.1328 (86), 407.1113 (11), 289.0963 (100), 287.0829 (54), 245.0728 (10), 125.0276 (6) 18.3 Procyanidin dimer B type isomer C30H26O12 577.1424 577.1411 2.29 425.0945 (21), 407.0855 (72), 289.0800 (86), 245.0870 (11), 205.0547 (5), 125.0239 (100) 18.4 Eriodictyol-7-O-hexoside C21H22O11 449.1162 449.1169 -1.56 287.0722 (100), 269.0834 (69), 235.0379 (11), 167.0364 (10), 125.0268 (9) 18.5 (Epi)catechin-3-O- dihexoside isomer C25H26O18 613.1119 613.1138 -3.07 451.1279 (10), 433.0719 (19), 407.0998 (23), 289.0900 (100), 259.0581 (4), 245.0959 (6), 125.0317 (30) 18.6 Dihydro-kaempferol-7-O- hexoside C21H22O11 449.1162 449.1182 -4.42 287.0816 (43), 269.0714 (100), 259.0884 (57), 135.0554 (25) 18.7 Procyanidin dimer B type hexoside isomer C36H36O17 739.1952 739.1915 5.00 449.1097 (37), 289.0608 (48), 287.0725 (55), 245.0713 (25), 125.0129 (100) 18.8 Procyanidin trimer B type C45H38O18 865.2058 865.2044 1.63 577.1811 (8), 425.1213 (16), isomer isomer 407.1120 (34), 289.0958 (50), 287.0598 (26), 245.0738 (16), 125.0292 (100) 18.9 Procyanidin dimer B type isomer C30H26O12 577.1424 577.1411 2.29 425.1060 (8), 407.0974 (55), 289.0850 (100), 245.0920 (10), 205.0600 (3), 125.0251 (45) 18.10 Procyanidin dimer B type derivative C50H35NO13 856.2108 856.2104 0.51 577.1265 (10), 425.0996 (45), 407.0922 (40), 289.0847 (79), 278.0775 (100), 125.0281 (16) 18.11 Coumaroyl aspartate C13H13NO6 278.0743 278.0730 4.61 260.2382 (2), 234.0819 (14), 216.0670 (11), 190.0861 (10), 172.0810 (2), 163.0414 (56), 119.0481 (100) 19 19.1 Di-hydroxycinnamic acid isomer C9H8O4 179.0423 179.0431 -4.67 163.0337 (11), 135.0453 (100) 19.2 Eriodictyol-C-hexoside-7-O- hexoside isomer C27H32O16 611.1690 611.1662 4.63 449.1540 (41), 329.0991 (92), 287.0956 (17), 269.0810 (10), 235.0310 (5), 167.0427 (59), 149.0208 (100), 125.0245 (10) 19.3 Naringenin-C-hexoside-7-O- hexoside isomer C27H30O15 593.1585 593.1591 -1.86 473.0960 (100), 413.0732 (9), 383.0630 (46), 353.0540 (94), 311.0533 (8), 283.0433 (2) 19.4 4-O-Coumaroylquinic acid cisa C16H18O8 337.1002 337.1006 -1.28 191.0663 (10), 173.0557 (100), 163.0486 (34), 119.0476 (17) 19.5 Procyanidin tetramer B type isomer C60H50O24 576.1346 576.1342 (2-) 0.69 425.1141 (6), 407.0929 (11), 289.0732 (27), 287.0653 (17), 245.1158 (7), 125.0282 (100) 19.6 Procyanidin tetramer A type isomer C60H48O24 1151.2559 1151.2548 0.97 695.2148 (16), 575.1237 (21), 449.0797 (11), 425.0865 (64), 407.0734 (11), 289.0723 (61), 287.0585 (49), 125.0187 (100) 19.7 Procyanidin tetramer B type isomer C60H50O24 576.1346 576.1342 (2-) 0.69 425.0893 (37), 407.0788 (30), 289.0752 (100), 287.0605 (55), 245.0874 (5), 125.0205 (95) 19.8 Apigenin-6,8-di-C-hexoside isomer C27H30O15 593.1585 593.1591 -1.06 473.1283 (100), 383.0977 (46), 353.0818 (94), 149.0312 (9) 19.9 Epicatechins C15H14O6 289.0790 289.0791 -0.21 245.0877 (100), 205.0549 (34), 179.0368 (26), 125.0255 (34) 19.10 Di-hydroxycinnamic acid isomer C9H8O4 179.0423 179.0431 -4.67 163.0277 (10), 135.0421 (100) 19.11 Hydroxybenzoic acid isomer C7H6O3 137.0317 137.0316 0.09 93.0336 (100) 19.12 Apigenin-6,8-di-C-hexoside isomer C27H30O15 593.1585 593.1591 -1.06 473.0960 (100), 383.0630 (61), 353.0540 (54), 149.0.316 (6) 19.13 Procyanidin pentamer B type isomer C75H62O30 720.1663 720.1659 (2-) 0.56 449.0819 (10), 407.0718 (20), 289.0783 (100), 287.0583 (67), 245.0482 (28), 125.0230 (85) 20 20.1 Clovamide (caffeoyl- tyrosine) isomer C18H17NO7 358.1005 358.0996 2.51 222.0459 (69), 178.0540 (64), 161.0272 (61), 135.0441 (100) 20.2 4-O-Coumaroylquinic acid transa C16H18O8 337.1002 337.1006 -1.28 191.0553 (6), 173.0441 (100), 163.0369 (23), 119.0478 (2) 20.3 (Epi)catechin-C-pentoside isomer C20H22O10 421.1213 421.1223 -2.38 361.1138 (24), 331.1001 (100), 205.0614 (18), 123.0368 (11) 20.4 Feruloyl aspartate C14H15NO7 308.0849 308.0842 2.11 290.2986 (9), 264.1033 (74), 246.0733 (17), 220.1030 (9), 193.0557 (100), 149.0587 (65) 20.5 (Epi)catechin derivative isomer C43H28O10 703.1682 703.1676 0.92 533.1280 (14), 407.0992 (13), 289.0827 (100), 251.0643 (44), 125.0253 (48) 20.6 Epigallocatechin-3-O- gallatea,s C22H18O11 457.0849 457.0829 4.39 331.0620 (2), 305.0823 (9), 169.0315 (100), 125.0396 (24) 20.7 Procyanidin trimer B type isomer C45H38O18 865.2058 865.2044 1.63 577.1985 (33), 425.1465 (10), 407.1269 (35), 289.1131 (47), 287.0598 (26), 245.0738 (16), 125.0292 (100) 18.9 Procyanidin dimer B type isomer C30H26O12 577.1424 577.1411 2.29 425.1060 (8), 407.0974 (55), 289.0850 (100), 245.0920 (10), 205.0600 (3), 125.0251 (45) 18.10 Procyanidin dimer B type derivative C50H35NO13 856.2108 856.2104 0.51 577.1265 (10), 425.0996 (45), 407.0922 (40), 289.0847 (79), 278.0775 (100), 125.0281 (16) 18.11 Coumaroyl aspartate C13H13NO6 278.0743 278.0730 4.61 260.2382 (2), 234.0819 (14), 216.0670 (11), 190.0861 (10), 172.0810 (2), 163.0414 (56), 119.0481 (100) 19 19.1 Di-hydroxycinnamic acid isomer C9H8O4 179.0423 179.0431 -4.67 163.0337 (11), 135.0453 (100) 19.2 Eriodictyol-C-hexoside-7-O- hexoside isomer C27H32O16 611.1690 611.1662 4.63 449.1540 (41), 329.0991 (92), 287.0956 (17), 269.0810 (10), 235.0310 (5), 167.0427 (59), 149.0208 (100), 125.0245 (10) 19.3 Naringenin-C-hexoside-7-O- hexoside isomer C27H30O15 593.1585 593.1591 -1.86 473.0960 (100), 413.0732 (9), 383.0630 (46), 353.0540 (94), 311.0533 (8), 283.0433 (2) 19.4 4-O-Coumaroylquinic acid cisa C16H18O8 337.1002 337.1006 -1.28 191.0663 (10), 173.0557 (100), 163.0486 (34), 119.0476 (17) 19.5 Procyanidin tetramer B type isomer C60H50O24 576.1346 576.1342 (2-) 0.69 425.1141 (6), 407.0929 (11), 289.0732 (27), 287.0653 (17), 245.1158 (7), 125.0282 (100) 19.6 Procyanidin tetramer A type isomer C60H48O24 1151.2559 1151.2548 0.97 695.2148 (16), 575.1237 (21), 449.0797 (11), 425.0865 (64), 407.0734 (11), 289.0723 (61), 287.0585 (49), 125.0187 (100) 19.7 Procyanidin tetramer B type isomer C60H50O24 576.1346 576.1342 (2-) 0.69 425.0893 (37), 407.0788 (30), 289.0752 (100), 287.0605 (55), 245.0874 (5), 125.0205 (95) 19.8 Apigenin-6,8-di-C-hexoside isomer C27H30O15 593.1585 593.1591 -1.06 473.1283 (100), 383.0977 (46), 353.0818 (94), 149.0312 (9) 19.9 Epicatechins C15H14O6 289.0790 289.0791 -0.21 245.0877 (100), 205.0549 (34), 179.0368 (26), 125.0255 (34) 19.10 Di-hydroxycinnamic acid isomer C9H8O4 179.0423 179.0431 -4.67 163.0277 (10), 135.0421 (100) 19.11 Hydroxybenzoic acid isomer C7H6O3 137.0317 137.0316 0.09 93.0336 (100) 19.12 Apigenin-6,8-di-C-hexoside isomer C27H30O15 593.1585 593.1591 -1.06 473.0960 (100), 383.0630 (61), 353.0540 (54), 149.0.316 (6) 19.13 Procyanidin pentamer B type isomer C75H62O30 720.1663 720.1659 (2-) 0.56 449.0819 (10), 407.0718 (20), 289.0783 (100), 287.0583 (67), 245.0482 (28), 125.0230 (85) 20 20.1 Clovamide (caffeoyl- tyrosine) isomer C18H17NO7 358.1005 358.0996 2.51 222.0459 (69), 178.0540 (64), 161.0272 (61), 135.0441 (100) 20.2 4-O-Coumaroylquinic acid transa C16H18O8 337.1002 337.1006 -1.28 191.0553 (6), 173.0441 (100), 163.0369 (23), 119.0478 (2) 20.3 (Epi)catechin-C-pentoside isomer C20H22O10 421.1213 421.1223 -2.38 361.1138 (24), 331.1001 (100), 205.0614 (18), 123.0368 (11) 20.4 Feruloyl aspartate C14H15NO7 308.0849 308.0842 2.11 290.2986 (9), 264.1033 (74), 246.0733 (17), 220.1030 (9), 193.0557 (100), 149.0587 (65) 20.5 (Epi)catechin derivative isomer C43H28O10 703.1682 703.1676 0.92 533.1280 (14), 407.0992 (13), 289.0827 (100), 251.0643 (44), 125.0253 (48) 20.6 Epigallocatechin-3-O- gallatea,s C22H18O11 457.0849 457.0829 4.39 331.0620 (2), 305.0823 (9), 169.0315 (100), 125.0396 (24) 20.7 Procyanidin trimer B type isomer C45H38O18 865.2058 865.2044 1.63 577.1985 (33), 425.1465 (10), 407.1269 (35), 289.1131 (47), 19 20 287.0623 (51), 245.0887 (10), 125.0209 (100) 20.8 Procyanidin trimer A type C45H36O18 863.1902 863.1907 -0.62 407.0783 (20), 289.0767 (100), 287.0515 (24), 205.0193 (20), 125.0262 (67) 21 21.1 (Epi)catechin derivative isomer C43H28O10 703.1682 703.1676 0.92 533.1227 (15), 407.1080 (69), 289.0865 (99), 251.0703 (38), 245.0918 (32), 125.0245 (100) 21.2 Procyanidin trimer B type isomer C45H38O18 865.2058 865.2044 1.63 577.1513 (11), 425.0959 (26), 407.0869 (49), 289.0776 (71), 287.0667 (89), 245.0511 (9), 125.0209 (100) 21.3 Procyanidin dimer A type hexoside isomer C36H34O17 737.1796 737.1769 3.66 611.1895 (44), 539.1341 (67), 449.1191 (100), 407.1020 (13), 289.0952 (28), 287.0778 (4) 21.4 (Epi)catechin-3-O-hexoside isomer C21H24O11 451.1319 451.1320 -0.31 289.0709 (100), 245.0731 (21), 205.0599 (36), 179.0374 (3) 22 22.1 Procyanidin tetramer A type isomer C60H48O24 1151.2559 1151.2548 0.97 695.1732 (16), 575.1251 (67), 449.1017 (46), 425.0889 (59), 407.1007 (6), 289.0880 (9), 287.0664 (100), 125.0238 (13) 22.2 Procyanidin dimer A type hexoside isomer C36H34O17 737.1796 737.1769 3.66 611.2053 (61), 539.1105 (67), 449.1017 (100), 407.1065 (6), 289.0750 (18), 287.0661 (28) 22.3 Myricetin-3-O-(O-galloyl)- hexoside C28H24O17 631.1013 631.1039 -4.04 479.1152 (100), 317.0449 (20), 316.0441 (73), 271.0510 (10), 179.0122 (5) 22.4 Myricetin-7-O-hexoside-3- O-rutinoside C33H40O22 787.2011 787.2050 -4.92 625.1547 (8), 317.0379 (53), 316.0323 (100), 289.0464 (6), 287.0273 (4), 273.0646 (4), 271.0269 (4), 179.0066 (7) 22.5 Procyanidin trimer B type isomer C45H38O18 865.2058 865.2044 1.63 577.1857 (6), 425.1225 (18), 407.1117 (39), 289.0980 (49), 287.0489 (35), 245.0673 (16), 125.0291 (100) 22.6 (Epi)catechin-O-sulphate isomer C15H14O9S 369.0359 369.0361 -0.67 289.0763 (100), 245.0704 (53), 205.0674 (8), 137.0302 (16), 125.0271 (3), 79.9372 (2) 22.7 Procyanidin tetramer A type isomer C60H48O24 1151.2559 1151.2548 0.97 695.1376 (8), 575.1350 (21), 449.0887 (6), 425.1031 (51), 407.0806 (4), 289.0758 (44), 287.0622 (22), 125.0218 (100) 22.8 Procyanidin tetramer B type isomer C60H50O24 576.1346 576.1342 (2-) 0.69 425.1090 (3), 407.1229 (10), 289.0954 (63), 287.0800 (7), 245.0898 (10), 125.0276 (100) 22.9 Procyanidin hexamer A type C90H72O36 863.1900 863.1921 (2-) -2.43 449.1176 (22), 407.1026 (16), 289.1016 (85), 245.0984 (44), 125.0351 (100) 22.10 Apigenin-C-hexoside-C- pentoside isomer C26H28O14 563.1479 563.1462 3.02 503.1285 (25), 473.1501 (32), 443.1347 (84), 383.1096 (78), 353.0940 (100), 149.0467 (13) 23 23.1 Procyanidin dimer B type isomer C30H26O12 577.1424 577.1411 2.29 425.0943 (10), 407.0812 (53), 289.0761 (100), 245.0856 (64), 205.0536 (5), 125.0237 (33) 23.2 Procyanidin pentamer B type isomer C75H62O30 720.1663 720.1659 (2-) 0.56 449.0877 (12), 407.0726 (12), 289.0769 (49), 287.0730 (14), 245.0567 (5), 125.0234 (100) 23.3 Procyanidin trimer B type isomer C45H38O18 865.2058 865.2044 1.63 577.1938 (20), 425.1202 (18), 407.1051 (52), 289.0944 (35), 287.0653 (66), 245.0623 (14), 125.0276 (100) 23.4 Di-hydro-coumaric acid C9H10O3 165.0477 165.0470 4.45 147.0355 (100), 119.0399 (31) 23.5 Clovamide (caffeoyl- C18H17NO7 358.1005 358.0996 2.51 222.0593 (53), 178.0637 (100), 21 23 tyrosine) isomer 161.0339 (43), 135.0517 (54) 23.6 (Epi)catechin-3-O- trihydroxybenzene C21H20O9 415.1107 415.1111 -0.88 289.0775 (100), 259.0863 (3), 245.0961 (15), 205.0639 (32), 125.0295 (9) 23.7 Procyanidin dimer A type pentoside isomer C35H32O16 707.1690 707.1681 1.32 581.1333 (21), 539.1166 (67), 449.1031 (100), 407.0918 (8), 289.0797 (13), 287.0638 (7) 23.8 Procyanidin hexamer B type isomer C90H74O36 864.1980 864.1963 (2-) 1.97 449.0875 (14), 407.0865 (37), 289.0800 (63), 287.0566 (41), 245.0443 (13), 205.0100 (6), 125.0197 (100) 23.9 Myricetin-3-O-rutinosidea C27H30O17 625.1483 625.1455 4.47 317.0572 (28), 316.0519 (100), 287.0203 (5) 24 24.1 Procyanidin tetramer A type isomer C60H48O24 1151.2559 1151.2548 0.97 695.2385 (2), 575.1613 (34), 449.1279 (19), 425.1263 (12), 407.1210 (14), 289.0922 (42), 287.0837 (71), 125.0338 (100) 24.2 Procyanidin pentamer B type isomer C75H62O30 720.1663 720.1659 (2-) 0.56 449.1237 (8), 407.1024 (24), 289.0892 (53), 287.0730 (35), 245.0953 (18), 125.0266 (100) 24.3 Procyanidin dimer A type pentoside isomer C35H32O16 707.1690 707.1681 1.32 581.1413 (48), 539.0950 (30), 449.0884 (100), 407.0789 (16), 289.0629 (21), 287.0685 (5) 24.4 Procyanidin trimer A type hexoside isomer C51H48O23 1025.2430 1025.2416 1.35 407.0897 (16), 289.0675 (100), 285.0442 (22), 125.0300 (6) 24.5 Myricetin-3-O-galattoside C21H20O13 479.0904 479.0885 3.94 317.0394 (27), 316.0316 (100), 287.0228 (5) 24.6 Mono-deoxyclovamide (caffeoyl-DOPA / coumaroyl-tyrosine) isomer C18H17NO6 342.1056 342.1057 -0.33 222.0368 (18), 206.0630 (82), 178.1359 (14), 145.1558 (13), 135.0378 (100), 119.0446 (23) 24.7 Procyanidin hexamer B type isomer C90H74O36 864.1980 864.1963 (2-) 1.97 449.0767 (17), 407.0874 (24), 289.0824 (40), 287.0663 (19), 245.0553 (39), 205.0097 (8), 125.0255 (100) 24.8 Myricetin-3-O-glucoside C21H20O13 479.0904 479.0885 3.94 317.0736 (23), 316.0615 (100), 287.0571 (7) 24.9 Procyanidin trimer A type hexoside isomer C51H48O23 1025.2430 1025.2416 1.35 407.1011 (8), 289.0869 (100), 285.0645 (33), 245.1007 (16), 125.0224 (35) 25 25.1 Apigenin-C-hexoside-2'’-O- hexoside isomer C27H30O15 593.1585 593.1591 -1.06 473.0871 (10), 413.0978 (63), 311.0682 (12), 293.0528 (100), 149.0416 (4) 25.2 Quercetin-7-O-hexoside-3- O-rutinoside isomer C33H40O21 771.2062 771.2042 2.60 609.1837 (4), 463.1192 (3), 301.0584 (100), 300.0492 (60), 273.0559 (3), 271.0419 (8), 255.0513 (3), 179.0098 (5), 151.0028 (3) 25.3 (Epi)catechin-O-sulphate isomer C15H14O9S 369.0359 369.0361 -0.67 289.0755 (100), 245.0713 (33), 205.0686 (10), 137.0321 (58), 125.0288 (7), 79.9361 (11) 26 26.1 Procyanidin hexamer B type isomer C90H74O36 864.1980 864.1963 (2-) 1.97 449.0645 (12), 407.0668 (12), 289.0651 (70), 287.0580 (33), 245.0514 (43), 205.0494 (11), 125.0306 (100) 26.2 (Epi)catechin-3-O- trihydroxybenzene C21H20O9 415.1107 415.1111 -0.88 289.0705 (100), 259.0918 (2), 245.0913 (25), 205.0421 (21), 125.0283 (8) 26.3 Quercetin-7-O-hexoside-3- O-rutinoside isomer C33H40O21 771.2062 771.2042 2.60 609.1579 (6), 463.0993 (6), 301.0353 (100), 300.0283 (57), 273.0427 (3), 271.0222 (7), 255.0339 (4), 179.0062 (14), 151.0007 (9) 27 27.1 Ellagic acid-galloyl- C27H20O17 615.0700 615.0723 2.04 463.0607 (100), 301.0147 (37), tyrosine) isomer 161.0339 (43), 135.0517 (54) 23.6 (Epi)catechin-3-O- trihydroxybenzene C21H20O9 415.1107 415.1111 -0.88 289.0775 (100), 259.0863 (3), 245.0961 (15), 205.0639 (32), 125.0295 (9) 23.7 Procyanidin dimer A type pentoside isomer C35H32O16 707.1690 707.1681 1.32 581.1333 (21), 539.1166 (67), 449.1031 (100), 407.0918 (8), 289.0797 (13), 287.0638 (7) 23.8 Procyanidin hexamer B type isomer C90H74O36 864.1980 864.1963 (2-) 1.97 449.0875 (14), 407.0865 (37), 289.0800 (63), 287.0566 (41), 245.0443 (13), 205.0100 (6), 125.0197 (100) 23.9 Myricetin-3-O-rutinosidea C27H30O17 625.1483 625.1455 4.47 317.0572 (28), 316.0519 (100), 287.0203 (5) 24 24.1 Procyanidin tetramer A type isomer C60H48O24 1151.2559 1151.2548 0.97 695.2385 (2), 575.1613 (34), 449.1279 (19), 425.1263 (12), 407.1210 (14), 289.0922 (42), 287.0837 (71), 125.0338 (100) 24.2 Procyanidin pentamer B type isomer C75H62O30 720.1663 720.1659 (2-) 0.56 449.1237 (8), 407.1024 (24), 289.0892 (53), 287.0730 (35), 245.0953 (18), 125.0266 (100) 24.3 Procyanidin dimer A type pentoside isomer C35H32O16 707.1690 707.1681 1.32 581.1413 (48), 539.0950 (30), 449.0884 (100), 407.0789 (16), 289.0629 (21), 287.0685 (5) 24.4 Procyanidin trimer A type hexoside isomer C51H48O23 1025.2430 1025.2416 1.35 407.0897 (16), 289.0675 (100), 285.0442 (22), 125.0300 (6) 24.5 Myricetin-3-O-galattoside C21H20O13 479.0904 479.0885 3.94 317.0394 (27), 316.0316 (100), 287.0228 (5) 24.6 Mono-deoxyclovamide (caffeoyl-DOPA / coumaroyl-tyrosine) isomer C18H17NO6 342.1056 342.1057 -0.33 222.0368 (18), 206.0630 (82), 178.1359 (14), 145.1558 (13), 135.0378 (100), 119.0446 (23) 24.7 Procyanidin hexamer B type isomer C90H74O36 864.1980 864.1963 (2-) 1.97 449.0767 (17), 407.0874 (24), 289.0824 (40), 287.0663 (19), 245.0553 (39), 205.0097 (8), 125.0255 (100) 24.8 Myricetin-3-O-glucoside C21H20O13 479.0904 479.0885 3.94 317.0736 (23), 316.0615 (100), 287.0571 (7) 24.9 Procyanidin trimer A type hexoside isomer C51H48O23 1025.2430 1025.2416 1.35 407.1011 (8), 289.0869 (100), 285.0645 (33), 245.1007 (16), 125.0224 (35) 25 25.1 Apigenin-C-hexoside-2'’-O- hexoside isomer C27H30O15 593.1585 593.1591 -1.06 473.0871 (10), 413.0978 (63), 311.0682 (12), 293.0528 (100), 149.0416 (4) 25.2 Quercetin-7-O-hexoside-3- O-rutinoside isomer C33H40O21 771.2062 771.2042 2.60 609.1837 (4), 463.1192 (3), 301.0584 (100), 300.0492 (60), 273.0559 (3), 271.0419 (8), 255.0513 (3), 179.0098 (5), 151.0028 (3) 25.3 (Epi)catechin-O-sulphate isomer C15H14O9S 369.0359 369.0361 -0.67 289.0755 (100), 245.0713 (33), 205.0686 (10), 137.0321 (58), 125.0288 (7), 79.9361 (11) 26 26.1 Procyanidin hexamer B type isomer C90H74O36 864.1980 864.1963 (2-) 1.97 449.0645 (12), 407.0668 (12), 289.0651 (70), 287.0580 (33), 245.0514 (43), 205.0494 (11), 125.0306 (100) 26.2 (Epi)catechin-3-O- trihydroxybenzene C21H20O9 415.1107 415.1111 -0.88 289.0705 (100), 259.0918 (2), 245.0913 (25), 205.0421 (21), 125.0283 (8) 26.3 Quercetin-7-O-hexoside-3- O-rutinoside isomer C33H40O21 771.2062 771.2042 2.60 609.1579 (6), 463.0993 (6), 301.0353 (100), 300.0283 (57), 273.0427 (3), 271.0222 (7), 255.0339 (4), 179.0062 (14), 151.0007 (9) 27 27.1 Ellagic acid-galloyl- C27H20O17 615.0700 615.0723 2.04 463.0607 (100), 301.0147 (37), 24 25 26 27 hexoside 300.0047 (46), 229.0317 (11) 27.2 Ferulic acid-4-O-pentoside C15H18O8 325.1002 325.1004 -0.71 221.1495 (21), 193.1510 (100), 178.1361 (68) 27.3 Apigenin-C-hexoside-2''-O- rhamnoside isomer C27H30O14 577.1636 577.1617 3.21 457.1107 (10), 413.0843 (45), 341.0690 (17), 311.0552 (25), 293.0441 (100), 149.0494 (5) 27.4 Quercetin-7-O-rhamnoside- 3-O-rutinoside C33H40O20 755.2113 755.2103 1.31 609.1224 (10), 301.0486 (100), 300.0360 (87), 273.0601 (6), 271.0473 (5), 179.0037 (3), 151.0093 (3) 27.5 (Epi)catechin-7-O- trihydroxybenzene C21H20O9 415.1107 415.1111 -0.88 289.0802 (100), 261.0909 (4), 245.0993 (60), 205.0656 (17), 125.0213 (51) 27.6 (Epi)catechin-3-O-gallate-7- O-glucuronide isomer C25H30O18 617.1432 617.1413 3.10 465.0846 (11), 435.1642 (18), 327.0688 (100), 289.0852 (29), 261.0610 (3), 245.0965 (9), 205.0306 (21), 165.0270 (34), 125.0294 (6) 27.7 Apigenin-C-hexoside isomer C21H20O10 431.1056 431.1058 -0.35 341.0904 (26), 311.0739 (100), 283.0892 (40), 149.0201 (5), 117.0540 (3) 28 28.1 (Epi)catechin-3-O- dihexoside isomer C25H26O18 613.1119 613.1138 -3.07 451.1227 (11), 433.0755 (5), 407.1000 (56), 289.0864 (71), 259.0650 (3), 245.0938 (10), 125.0257 (100) 28.2 Kaempferol-7-O-hexoside- 3-O-rutinoside isomer C33H40O20 755.2113 755.2103 1.31 593.1490 (11), 285.0417 (100), 284.0356 (11), 257.0578 (9), 255.0186 (7), 151.0018 (2) 28.3 Catechin-3-O-gallatea,s C22H18O10 441.0900 441.0892 1.80 331.0496 (4), 289.0773 (42), 259.0667 (6), 245.0862 (10), 169.0148 (100), 125.0227 (19) 28.4 Quercetin-3-O-rutinosidea,s C27H30O16 609.1534 609.1536 -0.35 301.0422 (41), 300.0324 (55), 271.0281 (5), 179.0023 (8), 167.0391 (100), 151.0038 (7) 29 29.1 Cinchonain isomer C24H20O9 451.1107 451.1103 0.96 341.0565 (100), 217.0327 (21) 29.2 Procyanidin trimer B type isomer C45H38O18 865.2058 865.2044 1.63 577.1742 (11), 425.1189 (13), 407.1076 (64), 289.0963 (45), 287.0541 (28), 245.0653 (43), 125.0266 (100) 29.3 Procyanidin dimer B type isomer C30H26O12 577.1424 577.1411 2.29 425.0964 (18), 407.0861 (100), 289.0787 (97), 245.0527 (15), 205.0527 (6), 125.0234 (79) 29.4 Mono-deoxyclovamide (caffeoyl-DOPA / coumaroyl-tyrosine) isomer C18H17NO6 342.1056 342.1057 -0.33 222.0469 (35), 206.0538 (36), 178.0531 (78), 145.0297 (27), 135.0478 (100), 119.0507 (14) 29.5 Epicatechin-3-O-gallatea,s C22H18O10 441.0900 441.0892 1.80 331.0689 (2), 289.0930 (38), 259.0807 (3), 245.0673 (6), 169.0290 (100), 125.0351 (22) 30 30.1 Quercetin-3-O-galactoside C21H20O12 463.0955 463.0950 1.03 301.0508 (52), 300.0424 (100), 271.0377 (6), 255.0400 (3), 179.0056 (4), 151.0124 (3) 30.2 Procyanidin dimer A type hexoside isomer C36H34O17 737.1796 737.1769 3.66 611.1715 (50), 539.1170 (49), 449.1112 (100), 407.0973 (12), 289.0811 (33), 287.0649 (19) 30.3 Ferulic acids C10H10O4 193.0579 193.0573 3.14 178.0336 (100), 149.0597 (38) 31 31.1 Procyanidin dimer A type hexoside isomer C36H34O17 737.1796 737.1769 3.66 611.1492 (29), 539.0970 (32), 449.0885 (100), 407.0748 (16), 289.0705 (24), 287.0577 (31) 31.2 Kaempferol-7-O-hexoside- 3-O-rutinoside isomer C33H40O20 755.2113 755.2103 1.31 593.1553 (13), 285.0433 (100), 284.0385 (12), 257.0661 (6), 255.0492 (3), 151.0002 (2) 31.3 Quercetin-3-O-glucosides C21H20O12 463.0955 463.0950 1.03 301.0525 (56), 300.0461 (100), 271.0392 (8), 255.0425 (3), 28 29 30 31 179.0048 (5), 151.0086 (5) 31.4 Kaempferol-7-O-hexoside C21H20O11 447.1006 447.1028 5.00 285.0122 (100), 257.0463 (10) 31.5 Procyanidin dimer A type pentoside isomer C35H32O16 707.1690 707.1681 1.32 581.1482 (49), 539.1210 (86), 449.1154 (100), 407.0421 (12), 289.0778 (30), 287.0569 (13) 32 32.1 Kaempferol-7-O- rhamnoside-3-O-rutinoside C33H40O19 739.2164 739.2182 -2.46 593.1482 (14), 285.0483 (100), 257.0806 (11), 255.0809 (8) 32.2 Procyanidin dimer A type pentoside isomer C35H32O16 707.1690 707.1681 1.32 581.1394 (18), 539.1030 (39), 449.0942 (100), 407.0812 (9), 289.0800 (14), 287.0605 (13) 32.3 Phloretin-C-hexoside isomer C21H24O10 435.1369 435.1376 -1.50 345.1005 (24), 315.0928 (100), 285.0967 (25), 167.0404 (14), 137.0580 (14) 32.4 Kaempferol-3-O-galactoside C21H20O11 447.1006 447.1028 5.00 285.0517 (19), 284.0473 (100), 255.0328 (7), 179.0122 (16), 151.0080 (9) 33 33.1 Kaempferol-3-O-rutinoside C27H30O15 593.1585 593.1591 -1.06 285.0483 (100), 284.0386 (49), 255.0421 (5), 179.0336 (4), 151.0446 (4) 33.2 Quercetin-3-O-pentoside C20H18O11 433.0849 433.0832 3.94 301.0474 (21), 300.0406 (100), 271.0364 (6), 255.0390 (2), 179.0059 (2), 151.0101 (3) 34 34.1 Procyanidin dimer A type C30H24O12 575.1268 575.1274 -1.08 449.1241 (15), 407.1091 (42), 289.0966 (39), 287.0755 (14), 285.0567 (100), 125.0234 (7) 34.2 Cinchonain isomer C24H20O9 451.1107 451.1103 0.96 341.0921 (100), 217.0270 (18) 34.3 Kaempferol-3-O-glucoside C21H20O11 447.1006 447.1028 5.00 285.0462 (51), 284.0395 (100), 255.0390 (4), 179.0704 (12), 151.0920 (11) 34.4 Di-deoxyclovamide (coumaroyl-DOPA) C18H17NO5 326.1107 326.1109 -0.70 282.1414 (100), 206.0625 (28), 163.0502 (32), 147.0530 (22), 134.0671 (4), 119.0536 (63) 34.5 Quercetin-3-O-rhamnoside C21H20O11 447.1006 447.1028 5.00 301.0206 (90), 300.0135 (100), 271.0102 (10) 34.6 (Epi)catechin-3-O-gallate-7- O-glucuronide isomer C25H30O18 617.1432 617.1413 3.10 465.1064 (5), 435.1628 (11), 327.0704 (100), 289.0874 (25), 261.0623 (3), 245.0900 (26), 205.0379 (17), 165.0285 (78), 125.0294 (14) 35 35.1 Quercetins C15H10O7 301.0427 301.0423 1.17 179.0118 (100), 151.0087 (65) 36 36.1 Bisdemethoxycurcuminb C19H16O4 307.1049 307.1043 1.81 187.0351 (36), 145.0249 (14), 143.0462 (65), 119.0470 (100) 36.2 Demethoxycurcuminb C20H18O5 337.1154 337.1164 -2.89 217.0450 (33), 173.0556 (42), 149.0563 (32), 119.0466 (100) 36.3 Curcuminb,s C21H20O6 367.1260 367.1257 0.78 217.0426 (38), 175.0331 (50), 173.0540 (57), 149.0547 (100), 134.0316 (48) 37 37.1 Ellagic acids C14H6O8 301.0063 301.0054 2.87 257.0162 (100), 229.0307 (61) a and b are referred to the compounds detected only in Sakura green tea dark chocolate or turmeric dark chocolate, respectively, whereas s means identification by comparison with authentic standard. a and b are referred to the compounds detected only in Sakura green tea dark chocolate or turmeric dark chocolate, respectively, whereas s means identification by comparison with authentic standard. s is referred to the compounds detected with authentic standards. Figure 2. Structures of newly identified dark chocolate phenolic compounds. Examples of some newly identified phenolic structures belonging to flavan-3-ols (A), flavonols (B), s is referred to the compounds detected with authentic standards. 32 a and b are referred to the compounds detected only in Sakura green tea dark chocolate or turmeric dark chocolate, respectively, whereas s means identification by comparison with authentic standard. s is referred to the compounds detected with authentic standards. Table 2. Quantitative results (mg/100 g of chocolate) for phenolic compounds identified in the different types of chocolate. Values represent means ± standard deviation of triplicate determination. Compound Dark chocolate Green tea dark chocolate Turmeric dark chocolate Flavan-3-ols 15.2 Catechin 66.20 ± 1.99 a 69.62 ± 5.28 a 71.13 ± 2.15 a 19.9 Epicatechin 203.29 ± 10.68 a 303.69 ± 11.65 b 218.43 ± 8.08 a 9.1 Gallocatechin 1.88 ± 0.10 a 2.09 ± 0.14 a < l.o.q. b 13.2 Epigallocatechin 15.93 ± 0.10 a 29.76 ± 1.74 b 15.11 ± 0.08 a 22.6 (Epi)catechin-O-sulphate isomer 2.06 ± 0.13 a 1.73 ± 0.20 a 3.07 ± 0.31 b 25.3 (Epi)catechin-O-sulphate isomer 7.15 ± 0.35 a 7.16 ± 0.12 a 10.23 ± 0.83 b 23.6 (Epi)catechin-3-O-trihydroxybenzene 4.92 ± 0.50 a 4.66 ± 0.10 b 5.94 ± 0.56 ab 26.2 (Epi)catechin-3-O- trihydroxybenzene 3.61 ± 0.06 a 4.58 ± 0.26 b 4.84 ± 0.42 b 27.5 (Epi)catechin-7-O-trihydroxybenzene 2.60 ± 0.15 a 3.83 ± 0.56 b 3.39 ± 0.14 ab 20.3 (Epi)catechin-C-pentoside isomer 0.42 ± 0.05 a 0.30 ± 0.07 a 0.83 ± 0.11 a 28.3 Catechin-3-O-gallate* n.d. < l.o.q. a n.d. 29.5 Epicatechin-3-O-gallate * n.d. 9.12 ± 0.13 a n.d. 13.1 (Epi)catechin-3-O-hexoside isomer 12.64 ± 0.20 a 9.78 ± 1.44 a 13.51 ± 0.82 a 14.1 (Epi)catechin-C-hexoside isomer 1.91 ± 0.04 a 1.62 ± 0.10 a 1.32 ± 0.19 a 15.1 (Epi)catechin-7-O-hexoside 4.11 ± 0.29 a 4.26 ± 0.30 a 5.24 ± 0.43 a 16.2 (Epi)catechin-C-hexoside isomer 4.09 ± 0.21 a 3.48 ± 0.80 a 3.72 ± 0.01 a 21.4 (Epi)catechin-3-O-hexoside isomer 1.36 ± 0.21 a 1.81 ± 0.22 a 1.48 ± 0.08 a 20.6 Epigallocatechin-3-O-gallate * n.d. 33.54 ± 2.16 a n.d. Figure 2. Structures of newly identified dark chocolate phenolic compounds. Examples of some newly identified phenolic structures belonging to flavan-3-ols (A), flavonols (B), 10.1 Gallocatechin-3-O-hexoside 0.09 ± 0.01 a 0.06 ± 0.01 a 0.15 ± 0.01 a 11.1 Epigallocatechin-3-O-hexoside 0.09 ± 0.01 a 0.07 ± 0.01 a 0.17 ± 0.01 a 34.1 Procyanidin dimer A type 1.10 ± 0.06 a 1.31 ± 0.08 a 1.21 ± 0.07 a 12.1 Procyanidin dimer B type isomer 8.65 ± 0.84 a 8.39 ± 0.34 a 10.59 ± 1.48 a 17.1 Procyanidin dimer B type isomer 3.05 ± 0.22 a 2.69 ± 0.14 a 3.62 ± 0.19 a 18.3 Procyanidin dimer B type isomer 37.23 ± 3.44 a 52.45 ± 5.44 b 35.12 ± 0.04 a 18.9 Procyanidin dimer B type isomer 34.08 ± 1.98 a 44.99 ± 1.15 b 38.25 ± 2.76 a 23.1 Procyanidin dimer B type isomer 5.32 ± 0.30 a 6.66 ± 0.64 a 6.93 ± 0.74 a 23.3 Procyanidin dimer B type isomer 10.17 ± 0.13 a 12.35 ± 0.35 a 13.12 ± 0.89 a 12.2 (Epi)catechin-(Epi)gallocatechin * n.d. 0.12 ± 0.01 a n.d. 18.5 (Epi)catechin-3-O-dihexoside isomer 0.66 ± 0.04 a 0.73 ± 0.09 a 0.44 ± 0.03 a 28.1 (Epi)catechin-3-O-dihexoside isomer 1.65 ± 0.02 a 1.04 ± 0.51 a 1.34 ± 0.03 a 27.6 (Epi)catechin-3-O-gallate-7-O- glucuronide isomer 0.33 ± 0.01 a 0.18 ± 0.01 a 0.24 ± 0.03 a 34.6 (Epi)catechin-3-O- gallate-7-O- glucuronide isomer 0.31 ± 0.02 a 0.14 ± 0.01 a 0.23 ± 0.02 a 20.5 (Epi)catechin derivative isomer 1.16 ± 0.04 a 0.93 ± 0.10 a 1.30 ± 0.14 a 21.1 (Epi)catechin derivative isomer 1.02 ± 0.04 a 1.06 ± 0.04 a 0.77 ± 0.05 a 23.7 Procyanidin dimer A type pentoside isomer 1.42 ± 0.01 a 1.29 ± 0.24 a 1.68 ± 0.04 a 24.3 Procyanidin dimer A type pentoside 1 09 ± 0 01a 1 17 ± 0 08a 1 04 ± 0 04a Table 2. Quantitative results (mg/100 g of chocolate) for phenolic compounds identified in the different types of chocolate. Figure 2. Structures of newly identified dark chocolate phenolic compounds. Examples of some newly identified phenolic structures belonging to flavan-3-ols (A), flavonols (B), Values represent means ± standard deviation of triplicate determination isomer 21.3 Procyanidin dimer A type hexoside isomer 1.48 ± 0.06 a 1.52 ± 0.23 a 1.91 ± 0.07 a 22.2 Procyanidin dimer A type hexoside isomer 1.01 ± 0.09 a 1.24 ± 0.13 a 1.58 ± 0.18 a 30.2 Procyanidin dimer A type hexoside isomer 2.06 ± 0.09 a 3.09 ± 0.37 b 2.33 ± 0.05 a 31.1 Procyanidin dimer A type hexoside isomer 1.47 ± 0.03 a 2.58 ± 0.15 b 2.06 ± 0.03 a 16.3 Procyanidin dimer B type hexoside isomer 0.80 ± 0.04 a 0.89 ± 0.07 a 1.02 ± 0.10 a 18.2 Procyanidin dimer B type hexoside isomer 1.04 ± 0.02 a 1.06 ± 0.04 a 1.22 ± 0.04 a 18.7 Procyanidin dimer B type hexoside isomer 0.51 ± 0.08 a 0.55 ± 0.03 a 0.54 ± 0.04 a 18.10 Procyanidin dimer B type derivative 1.86 ± 0.13 a 1.88 ± 0.47 a 2.27 ± 0.13 a 20.8 Procyanidin trimer A type 0.70 ± 0.05 a 0.67 ± 0.04 a 0.97 ± 0.04 a 13.5 Procyanidin trimer B type isomer 0.93 ± 0.05 a 0.92 ± 0.03 a 1.11 ± 0.11 a 18.1 Procyanidin trimer B type isomer 4.30 ± 0.31 a 7.13 ± 0.51 b 4.96 ± 0.41 a 18.8 Procyanidin trimer B type isomer 3.21 ± 0.17 a 4.20 ± 0.25 b 3.65 ± 0.21 a 20.7 Procyanidin trimer B type isomer 11.02 ± 0.45 a 15.16 ± 0.90 b 11.60 ± 0.64 a 21.2 Procyanidin trimer B type isomer 8.90 ± 0.17 a 17.49 ± 1.34 b 9.64 ± 0.25 a 22.5 Procyanidin trimer B type isomer 3.60 ± 0.19 a 3.67 ± 0.18 a 3.50 ± 0.22 a 23.3 Procyanidin trimer B type isomer 2.05 ± 0.08 a 2.15 ± 0.07 a 2.91 ± 0.15 a 29.2 Procyanidin trimer B type isomer 1.11 ± 0.03 a 1.68 ± 0.08 a 1.38 ± 0.02 a 24.4 Procyanidin trimer A type hexoside isomer 0.70 ± 0.06 a 0.99 ± 0.03 a 0.84 ± 0.07 a 24.9 Procyanidin trimer A type hexoside isomer 0.54 ± 0.02 a 0.97 ± 0.08 a 0.70 ± 0.04 a 19.6 Procyanidin tetramer A type isomer 1.44 ± 0.01 a 1.38 ± 0.07 a 1.42 ± 0.08 a 22.1 Procyanidin tetramer A type isomer 2.02 ± 0.13 a 3.68 ± 0.57 b 2.04 ± 0.16 a 22.7 Procyanidin tetramer A type isomer 2.02 ± 0.03 a 4.04 ± 0.82 b 2.83 ± 0.08 a 24.1 Procyanidin tetramer A type isomer 0.79 ± 0.07 ab 1.11 ± 0.29 a 0.66 ± 0.06 b 16.1 Procyanidin tetramer B type isomer 0.37 ± 0.01 a 0.37 ± 0.05 a 0.37 ± 0.05 a 19.5 Procyanidin tetramer B type isomer 1.34 ± 0.04 a 1.81 ± 0.22 a 1.85 ± 0.05 a 19.7 Procyanidin tetramer B type isomer 0.93 ± 0.03 a 1.82 ± 0.07 a 1.71 ± 0.16 a 22.8 Procyanidin tetramer B type isomer 1.19 ± 0.08 a 2.58 ± 0.03 b 1.65 ± 0.07 a 19.13 Procyanidin pentamer B type isomer 0.60 ± 0.02 a 0.55 ± 0.04 a 0.67 ± 0.02 a 23.2 Procyanidin pentamer B type isomer 0.82 ± 0.04 a 1.55 ± 0.14 b 0.99 ± 0.01 a 24.2 Procyanidin pentamer B type isomer 1.17 ± 0.08 a 3.47 ± 0.28 b 1.75 ± 0.14 a 22.9 Procyanidin hexamer A type < l.o.q. Figure 2. Structures of newly identified dark chocolate phenolic compounds. Examples of some newly identified phenolic structures belonging to flavan-3-ols (A), flavonols (B), < l.o.q. < l.o.q. 23.8 Procyanidin hexamer B type isomer 0.31 ± 0.01 a 0.98 ± 0.02 b 0.37 ± 0.02 a 24.7 Procyanidin hexamer B type isomer 0.43 ± 0.04 a 1.35 ± 0.04 b 0.29 ± 0.01 a 26.1 Procyanidin hexamer B type isomer 0.22 ± 0.02 a 1.85 ± 0.20 b 0.46 ± 0.04 a Total flavan-3-ols 503.76 ± 8.98 a 726.03 ± 14.53 b 538.71 ± 8.99 c Hydroxycinnamic acids 1.1 Coumaric acid 0.34 ± 0.01 a 0.37 ± 0.05 a 0.55 ± 0.01 b 23.4 Di-hydro-coumaric acid 8.13 ± 0.14 a 4.57 ± 0.08 b 11.18 ± 0.41 c 8.5 Di-hydroxycinnamic acid isomer 0.92 ± 0.06 ab 1.14 ± 0.07 a 2.33 ± 0.03 b 10.3 Di-hydroxycinnamic acid isomer 0.48 ± 0.02 a 0.36 ± 0.01 a 0.57 ± 0.01 b 13.3 Caffeic acid 1.08 ± 0.01 a 1.68 ± 0.02 b 1.09 ± 0.02 a 29.2 Procyanidin trimer B type isomer 19.6 Procyanidin tetramer A type isomer 19.1 Di-hydroxycinnamic acid isomer 0.48 ± 0.05 a 0.34 ± 0.06 a 0.60 ± 0.04 b 19.10 Di-hydroxycinnamic acid isomer 0.58 ± 0.04 a 0.63 ± 0.03 a 0.58 ± 0.07 a 2.1 Di-hydro-caffeic acid 1.88 ± 0.16 a 0.24 ± 0.01 b 0.88 ± 0.04 c 30.3 Ferulic acid 61.23 ± 3.74 a 58.09 ± 2.33 a 55.30 ± 2.36 a 18.11 Coumaroyl aspartate 15.72 ± 0.23 a 14.07 ± 0.45 a 16.83 ± 0.17 a 10.2 Di-hydroxycinnamic aspartate isomer 8.59 ± 0.52 a 6.86 ± 0.16 b 6.92 ± 0.04 b 14.2 Di-hydroxycinnamic aspartate isomer 35.88 ± 2.89 a 33.48 ± 0.75 a 33.12 ± 1.58 a 20.4 Feruloyl aspartate 8.18 ± 0.26 a 9.11 ± 0.17 a 8.96 ± 0.41 a 27.2 Ferulic acid-4-O-pentoside 0.40 ± 0.06 a 0.48 ± 0.01 a 0.47 ± 0.02 a 34.4 Di-deoxyclovamide (Coumaroyl-DOPA) 4.66 ± 0.21 a 4.75 ± 0.02 a 4.57 ± 0.24 a 12.3 3-Coumaroylquinic acid cis * n.d. 1.51 ± 0.01 a n.d. 14.3 3-Coumaroylquinic acid trans * n.d. 0.68 ± 0.01 a n.d. 19.4 4-Coumaroylquinic acid cis * n.d. 2.05 ± 0.10 a n.d. 20.2 4-Coumaroylquinic acid trans * n.d. 3.52 ± 0.09 a n.d. Figure 2. Structures of newly identified dark chocolate phenolic compounds. Examples of some newly identified phenolic structures belonging to flavan-3-ols (A), flavonols (B), 24.6 Mono-deoxyclovamide (Caffeoyl-DOPA / Coumaroyl-tyrosine) isomer 0.58 ± 0.02 a 0.48 ± 0.04 a 0.50 ± 0.01 a 29.4 Mono-deoxyclovamide (Caffeoyl-DOPA / Coumaroyl-tyrosine) isomer 2.93 ± 0.02 a 2.25 ± 0.10 a 2.11 ± 0.13 a 16.8 5-Caffeoylquinic acid 0.23 ± 0.02 a 0.65 ± 0.02 b 0.25 ± 0.03 a 17.2 3-Caffeoylquinic acid 0.12 ± 0.02 a 0.31 ± 0.02 b 0.21 ± 0.01 c 20.1 Clovamide (caffeoyl-tyrosine) isomer 1.81 ± 0.05 a 1.42 ± 0.02 a 1.18 ± 0.07 a 23.5 Clovamide (caffeoyl-tyrosine) isomer 7.73 ± 0.54 a 8.94 ± 0.36 b 8.31 ± 0.01 b Total hydroxycinnamic acids 161.95 ± 4.80 a 157.98 ± 2.38 a 156.51 ± 1.62 a Flavonols 35.1 Quercetin 1.08 ± 0.01 a 1.62 ± 0.03 b 1.37 ± 0.01 a 33.2 Quercetin-3-O-pentoside 3.31 ± 0.21 a 2.80 ± 0.14 a 4.15 ± 0.40 b 31.4 Kaempferol-7-O-hexoside 0.14 ± 0.01 a 0.24 ± 0.01 b 0.15 ± 0.01 a 32.4 Kaempferol-3-O-galactoside 0.04 ± 0.01 a 0.28 ± 0.01 b < l.o.q. c 34.3 Kaempferol-3-O-glucoside 0.05 ± 0.01 a 0.37 ± 0.03 b < l.o.q. c 34.5 Quercetin-3-O-rhamnoside 0.05 ± 0.01 a 0.50 ± 0.02 b < l.o.q. c 18.6 Dihydro-kaempferol-7-O-hexoside 0.25 ± 0.01 a 0.21 ± 0.01 a 0.23 ± 0.01 a 30.1 Quercetin-3-O-galactoside 0.49 ± 0.01 a 1.67 ± 0.01 b 2.44 ± 0.04 c 31.3 Quercetin-3-O-glucoside 1.96 ± 0.02 a 2.29 ± 0.07 a 3.05 ± 0.27 b 24.5 Myricetin-3-O-galattoside 0.30 ± 0.01 a 2.35 ± 0.04 b < l.o.q. c 24.8 Myricetin-3-O-glucoside 0.46 ± 0.01 a 1.84 ± 0.14 b < l.o.q. c 33.1 Kaempferol-3-O-rutinoside 0.26 ± 0.01 a 0.65 ± 0.02 b < l.o.q. c 28.4 Quercetin-3-O-rutinoside * n.d. 4.20 ± 0.19 a n.d. 23.9 Myricetin-3-O-rutinoside * n.d. 0.58 ± 0.01 a n.d. 22.3 Myricetin-3-O-(O-galloyl) hexoside 0.11 ± 0.01 a 0.62 ± 0.02 b < l.o.q. c 31.1 Kaempferol-7-O-rhamnoside-3-O- rutinoside 0.03 ± 0.01 a 0.09 ± 0.01 a < l.o.q. c 27.4 Quercetin-7-O-rhamnoside-3-O- rutinoside 0.14 ± 0.01 a 0.20 ± 0.02 a < l.o.q. b 28.2 Kaempferol-7-O-hexoside-3-O-rutinoside isomer 0.57 ± 0.01 a 1.27 ± 0.02 b < l.o.q. c 31.2 Kaempferol-7-O-hexoside-3-O-rutinoside isomer 0.44 ± 0.01 a 1.38 ± 0.01 b < l.o.q. c 25.2 Quercetin-7-O-hexoside-3-O-rutinoside isomer 1.12 ± 0.01 a 2.24 ± 0.01 b < l.o.q. c 26.3 Quercetin-7-O-hexoside-3-O-rutinoside 2.09 ± 0.01 a 3.65 ± 0.26 b < l.o.q. Figure 2. Structures of newly identified dark chocolate phenolic compounds. Examples of some newly identified phenolic structures belonging to flavan-3-ols (A), flavonols (B), c isomer 22.4 Myricetin-7-O-hexoside-3-O-rutinoside 0.12 ± 0.01 a 0.23 ± 0.02 b < l.o.q. c Total flavonols 13.01 ± 0.21 a 29.28 ± 0.39 b 11.40 ± 0.48 a Other phenolics 16.4 Vanillin 1.33 ± 0.05 a 1.81 ± 0.11 b 1.85 ± 0.08 b 32.3 Phloretin-C-hexoside isomer 0.15 ± 0.01 a 0.16 ± 0.01 a 0.63 ± 0.03 b 18.4 Eriodictyol-7-O-hexoside 0.10 ± 0.01 a 0.19 ± 0.01 b 0.53 ± 0.03 c 29.1 Cinchonain isomer 1.81 ± 0.20 a 1.13 ± 0.01 a 2.09 ± 0.03 b 34.2 Cinchonain isomer 0.77 ± 0.66 a 0.79 ± 0.02 a 0.75 ± 0.02 a 19.3 Naringenin-C-hexoside-7-O-hexoside isomer 0.23 ± 0.01 a 0.63 ± 0.02 b < l.o.q. c 19.2 Eriodictyol-C-hexoside-7-O-hexoside isomer 0.15 ± 0.01 a 0.28 ± 0.01 b n.d. Total other phenolics 4.54 ± 0.69 a 4.99 ± 0.11 a 5.85 ± 0.10 b Flavones 27.7 Apigenin-C-hexoside isomer 0.10 ± 0.01 a 0.42 ± 0.01 b 0.19 ± 0.01 a 22.10 Apigenin-C-hexoside-C-pentoside isomer 0.95 ± 0.01 a 2.11 ± 0.07 b 0.83 ± 0.03 a 27.3 Apigenin-C-hexoside-2''-O-rhamnoside isomer 1.31 ± 0.32 ab 0.89 ± 0.13 a 1.71 ± 0.43 b 19.8 Apigenin-6,8-di-C-hexoside isomer 0.17 ± 0.01 a 0.47 ± 0.01 b 0.23 ± 0.01 a 19.12 Apigenin-6,8-di-C-hexoside isomer 0.20 ± 0.01 a 0.54 ± 0.01 b 0.33 ± 0.01 a 25.1 Apigenin-C-hexoside-2’'-O-hexoside isomer 0.25 ± 0.01 a 0.47 ± 0.01 b n.d. Figure 2. Structures of newly identified dark chocolate phenolic compounds. Examples of some newly identified phenolic structures belonging to flavan-3-ols (A), flavonols (B), Total flavones 2.98 ± 0.32 a 4.90 ± 0.15 b 3.29 ± 0.43 a Ellagitannins 37.1 Ellagic acid 56.16 ± 3.58 a 53.44 ± 1.02 a 50.50 ± 3.30 a 27.1 Ellagic acid-galloyl-hexoside 4.65 ± 0.27 a 11.13 ± 0.43 b 4.09 ± 0.15 a 16.5 HHDP-galloyl-hexose 15.79 ± 1.20 a 24.65 ± 1.01 b 14.11 ± 0.69 a Total ellagitannins 76.60 ± 3.78 a 89.12 ± 1.50 b 68.70 ± 3.37 a Hydroxybenzoic acids 2.2 Hydroxybenzoic acid isomer 0.21 ± 0.03 a 0.20 ± 0.04 a 0.27 ± 0.02 a 7.1 Hydroxybenzoic acid isomer 0.13 ± 0.01 a 0.11 ± 0.01 a 0.41 ± 0.02 b 11.2 Hydroxybenzoic acid isomer 5.47 ± 0.03 a 6.46 ± 0.06 ab 7.50 ± 0.47 b 13.4 Hydroxybenzoic acid isomer 0.31 ± 0.01 a 1.32 ± 0.02 b 0.33 ± 0.02 a 19.11 Hydroxybenzoic acid isomer 0.62 ± 0.04 a 0.94 ± 0.11 b 0.77 ± 0.03 a 8.6 Protocatechuic acid 10.76 ± 0.30 a 9.11 ± 0.09 a 18.31 ± 0.97 b 8.3 Vanillic acid isomer 0.39 ± 0.04 a 0.37 ± 0.01 a 0.45 ± 0.02 a 16.6 Vanillic acid isomer 0.75 ± 0.13 a 0.55 ± 0.15 a 1.12 ± 0.01 b 3.1 Gallic acid * n.d. 0.14 ± 0.01 a n.d. 9.4 Syringic acid 0.13 ± 0.02 a 0.26 ± 0.03 a 0.14 ± 0.02 a 8.4 Protocatechuic acid-4-O-hexoside 0.10 ± 0.01 a 0.31 ± 0.03 b 0.19 ± 0.01 a 5.1 Vanillic acid-4-O-hexoside isomer 0.25 ± 0.01 a 0.20 ± 0.02 a 0.30 ± 0.01 a 8.1 Vanillic acid-4-O-hexoside isomer 1.77 ± 0.06 a 1.67 ± 0.04 a 2.63 ± 0.01 b 16.7 Vanillic acid-4-O-hexoside isomer 1.92 ± 0.07 a 1.81 ± 0.03 a 1.73 ± 0.09 a 3.2 Galloyl glucose isomer * n.d. 0.29 ± 0.01 a n.d. 4.1 Galloylquinic acid isomer * n.d. 1.84 ± 0.08 a n.d. G * isomer 9.2 Syringic acid-4-O-hexoside isomer 0.94 ± 0.08 a 0.81 ± 0.04 a 1.07 ± 0.11 a 9.3 Syringic acid-4-O-hexoside isomer 0.74 ± 0.02 a 0.71 ± 0.04 a 1.04 ± 0.11 b 8.2 Vanillic acid derivative 0.31 ± 0.01 a 0.49 ± 0.03 b 0.56 ± 0.04 b Total hydroxybenzoic acids 24.80 ± 0.33 a 23.05 ± 0.20 a 36.82 ± 1.08 b Curcuminoids 36.1 Bisdemethoxycurcumin n.d. n.d. 115.55 ± 2.16 a 36.2 Demethoxycurcumin n.d. n.d. Figure 2. Structures of newly identified dark chocolate phenolic compounds. Examples of some newly identified phenolic structures belonging to flavan-3-ols (A), flavonols (B), 82.64 ± 1.33 a 36.3 Curcumin ** n.d. n.d. 74.55 ± 0.47 a Total curcuminoids n.d. n.d. 272.73 ± 2.58 a Total phenolic compounds 787.63 ± 10.90 a 1035.45 ± 14.81 b 1094.03 ± 10.15 c * compounds were detected only in green tea dark chocolate or turmeric dark chocolate, respectively. ** mean the compounds were detected only in green tea dark chocolate or turmeric dark chocolate, respectively. The data represent the sum of the quantities of a specific compound found in the methanol extract and in the subsequent acetone extract. p y g p y The data represent the sum of the quantities of a specific compound found in the methanol extract and in the subsequent acetone extract. Different superscript letters within the same row indicate that the values are significantly different (P<0.05). p p g y avan-3-ols as well as compounds 29.1 and 34.2 were quantified as epicatechin equivalent. Hydroxycinnamic acids were quantified as coumaric acid equivalent except compounds 20.4, 27.2 and 30.3 which were quantified as ferulic acid equivalent. Flavonols, flavones as well as compounds 18.4, 19.2, 19.3 and 32.3 were quantified as quercetin-3-O-rutinoside i l quantified as ferulic acid equivalent. Flavonols, flavones as well as compounds 18.4, 19.2, 19.3 and 32.3 were quantified as quercetin-3-O-rutinoside equivalent. q Ellagitannins were quantified as ellagic acid equivalent. Hydroxybenzoic acids as well as compound 16.4 were quantified as protocatechuic acid equivalent. Curcuminoids were quantified as curcumin equivalent. Hydroxybenzoic acids as well as compound 16.4 were quantified as protocatechuic acid equivalent. Curcuminoids were quantified as curcumin equivalent. The numbering of the compounds is referred to that used in Table 1. The numbering of the compounds is referred to that used in Table 1.
https://openalex.org/W1967308064	https://europepmc.org/articles/pmc2483290?pdf=render	English	null	Identification of a set of genes showing regionally enriched expression in the mouse brain	BMC neuroscience	2,008	cc-by	12,151	BioMed Central BioMed Central Published: 14 July 2008 Received: 24 December 2007 Accepted: 14 July 2008 Published: 14 July 2008 Received: 24 December 2007 Accepted: 14 July 2008 BMC Neuroscience 2008, 9:66 doi:10.1186/1471-2202-9-66 This article is available from: http://www.biomedcentral.com/1471-2202/9/66 This article is available from: http://www.biomedcentral.com/1471-2202/9/66 This article is available from: http://www.biomedcentral.com/1 © 2008 D'Souza 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. BMC Neuroscience Open Access Open Ac Research article Identification of a set of genes showing regionally enriched expression in the mouse brain Cletus A D'Souza†1, Vikramjit Chopra†1, Richard Varhol1, Yuan-Yun Xie2, Slavita Bohacec2, Yongjun Zhao1, Lisa LC Lee2, Mikhail Bilenky1, Elodie Portales-Casamar2, An He1, Wyeth W Wasserman2, Daniel Goldowitz2, Marco A Marra1, Robert A Holt2, Elizabeth M Simpson2 and Steven JM Jones1 Address: 1Genome Sciences Centre, British Columbia Cancer Agency, 570 West 7th Ave – Suite 100, Vancouver, BC, V5Z 4E6, Canada and 2Centre for Molecular Medicine and Therapeutics, Child and Family Research Institute, Department of Medical Genetics, University of British Columbia, 950 West 28th Ave., Vancouver, BC, V5Z 4H4, Canada Email: Cletus A D'Souza - cdsouza@bcgsc.ca; Vikramjit Chopra - vchopra@bcgsc.ca; Richard Varhol - rvarhol@bcgsc.ca; Yuan- Yun Xie - yyxie@cmmt.ubc.ca; Slavita Bohacec - slavita@cmmt.ubc.ca; Yongjun Zhao - yzhao@bcgsc.ca; Lisa LC Lee - Lisa.Lee@postgrad.manchester.ac.uk; Mikhail Bilenky - mbilenky@bcgsc.ca; Elodie Portales-Casamar - elodie@cmmt.ubc.ca; An He - ahe@bcgsc.ca; Wyeth W Wasserman - wyeth@cmmt.ubc.ca; Daniel Goldowitz - dang@cmmt.ubc.ca; Marco A Marra - mmarra@bcgsc.ca; Robert A Holt - rholt@bcgsc.ca; Elizabeth M Simpson - simpson@cmmt.ubc.ca; Steven JM Jones - sjones@bcgsc.ca * Corresponding author †Equal contributors Received: 24 December 2007 Accepted: 14 July 2008 Identification of brain region-enriched gene expression by LongSAGE g To identify regionally enriched gene expression within the brain of the adult mouse strain C57BL/6J, we used the pre- cision of Laser Capture Microdissection (LCM; Figure 1) [16] to isolate component tissues and construct SAGE libraries from 17 brain regions as well as the whole adult mouse brain for comparison (Methods). As shown in Table 1, these libraries have been sampled to a depth of > 100,000 tags each, a level shown to be adequate for the discovery of medium-to-high level transcripts [8]. Bioin- formatics analysis of differential gene expression was per- formed as described in Methods. Since the majority of transcripts were detected in multiple libraries, we employed a heuristic approach to identify and rank expression patterns (outlined in Table 2). For each brain region, we ranked genes from 1–91 based on the level and pattern of expression in descending order. Expression spe- cificity of a ranked list of 1999 SAGE-identified genes was then confirmed by examining related literature informa- tion and Allen Brain Atlas in situ hybridization data. Based on this collective information, region-specific or region- enriched genes were further considered. Selection of the most optimal genes for promoter design necessitates detailed assessment of gene expression pat- terns. An invaluable resource to identify genes expressed in the mammalian brain is the serial analysis of gene expression (SAGE) technique [4,5]. A modern improve- ment of tag-based expression analysis is LongSAGE, which produces longer transcript tags (21-bp) better suited to unique mapping onto cDNA and genome sequences [6]. As part of the Mouse Atlas of Gene Expression project [7], LongSAGE was used to profile transcriptomes of 72 tissues of mouse strain C57BL/6J at various stages of develop- ment [8]. For the Pleiades Promoter Project [9], a scion of the Mouse Atlas project, we have generated new Long- SAGE data on gene expression in the adult mouse central nervous system to identify genes that display enriched expression in key brain regions. Of the 237 genes identified as displaying regionally enriched expression in this study, 132 genes [see Addi- tional file 1] displayed expression patterns listed in Table 2. Only 22 genes were found in a single library and five of these (A930006D11Rik, Chrna6, Gdf10, Hcrt, and Hes3) were determined to be tissue-specific at a statistically sig- nificant level (tag counts > 5, P < 0.05). http://www.biomedcentral.com/1471-2202/9/66 http://www.biomedcentral.com/1471-2202/9/66 BMC Neuroscience 2008, 9:66 Background we report the successful utilization of a combination of gene-finding tools, including SAGE analysis, text mining and ABA expression data, to identify genes displaying regionally enriched expression in surrogate regions of therapeutic interest within the mouse brain. g The Pleiades Promoter Project (please see Availability & requirements for more information) addresses two major challenges identified in gene therapy – first, the delivery of DNA to specific cell types to reduce side effects from treat- ing healthy cells and second, controlled delivery of DNA to a specific locus in the genome to avoid insertional mutagenesis. The goal for the project is the generation of human DNA promoters less than 4 kb in length (mini- promoters) that drive gene expression in brain regions important in neurological conditions. To achieve this goal, we have first identified genes with enriched expres- sion in different regions of the adult mouse brain. Regional expression patterns within the brain tend to be conserved between orthologous human and mouse genes [1]. Additionally, as regulatory sequences in tissue-specific genes tend to be highly conserved [2], human mini-pro- moters are expected to drive regional gene expression in transgenic mice based on earlier studies [3]. Therefore, promoter regions from orthologous human genes will be assessed in the mouse brain for the ability to drive regional expression. Page 2 of 13 (page number not for citation purposes) Abstract Background: The Pleiades Promoter Project aims to improve gene therapy by designing human mini-promoters (< 4 kb) that drive gene expression in specific brain regions or cell-types of therapeutic interest. Our goal was to first identify genes displaying regionally enriched expression in the mouse brain so that promoters designed from orthologous human genes can then be tested to drive reporter expression in a similar pattern in the mouse brain. Results: We have utilized LongSAGE to identify regionally enriched transcripts in the adult mouse brain. As supplemental strategies, we also performed a meta-analysis of published literature and inspected the Allen Brain Atlas in situ hybridization data. From a set of approximately 30,000 mouse genes, 237 were identified as showing specific or enriched expression in 30 target regions of the mouse brain. GO term over-representation among these genes revealed co-involvement in various aspects of central nervous system development and physiology. Conclusion: Using a multi-faceted expression validation approach, we have identified mouse genes whose human orthologs are good candidates for design of mini-promoters. These mouse genes represent molecular markers in several discrete brain regions/cell-types, which could potentially provide a mechanistic explanation of unique functions performed by each region. This set of markers may also serve as a resource for further studies of gene regulatory elements influencing brain expression. Page 1 of 13 (page number not for citation purposes) Page 1 of 13 (page number not for citation purposes) Complexity of the adult mouse brain transcriptome and SAGE-based analysis of transcriptome similarity of brain regions of Genes Total Tagsb SM098 Whole braina 6893 108441 SM110 Hypothalamus 6676 108882 SM132 Ventral Thalamus 6441 105701 SM137 Hippocampus Dentate Gyrus, dorsal/anterior 5935 104322 SM139 Medial Thalamus 6608 105364 SM147 Visual Cortex Layers II/III/IV 6683 136039 SM152 Substantia Nigra 6584 115991 SM153 Basal Nucleus of Meynert 6581 120997 SM180 Locus Coeruleus 6282 102933 SM181 Raphe Nuclei 6434 104627 SM182 Cerebellum White Matter 5461 107335 SM183 Primary Motor Cortex 6543 115262 SM184 Hippocampus CA1, dorsal/anterior 6331 118198 SM193 Amygdala, basolateral complex 6396 109772 SM194 Amygdala, central nucleus 6451 110056 SM195 Dorsal striatum 6185 105509 SM196 Cerebellum, Purkinje Cell Layer 6604 104850 SM201 Ependymal and Subependymal Layers 6561 107041 aManually dissected; all others were laser capture microdissected bRepresents filtered data Table 1: List of adult brain region SAGE libraries aManually dissected; all others were laser capture microdissected bRepresents filtered data libraries provided about 8% (1,357/17,357) additional genes to the total number of genes detectable by ABA. Hierarchical clustering was done based on unweighted average distance between formed clusters (see description in Methods), the results of which are displayed in the form of a dendrogram (Figure 2). A pattern of divergent tissue clusters consistently emerges: a cluster of neuronal tissues and several discrete single tissue clusters including Ependymal Layers, Cerebellum White Matter and Cerebel- We also analyzed SAGE data to measure transcriptome similarity between selected tissues. The premise was that tissues would cluster together or diverge based on the degree to which their genes are differentially expressed. Complexity of the adult mouse brain transcriptome and SAGE-based analysis of transcriptome similarity of brain regions While LongSAGE provides a rich perspective on gene expression patterns, we extended our data mining efforts to include other large information sources. The PubMed database [10] provides an unparalleled compendium of text from the scientific literature. In order to facilitate extraction of key information from Medline abstracts or full-text articles in PubMed, natural language processing tools are routinely employed to semi-automate the proc- ess of literature mining [11,12]. In this study we investi- gated an approach to specifically and automatically identify associations between genes and brain regions from the literature. We further analysed expression data from the Allen Brain Atlas (ABA; [13]), a high-throughput in situ hybridization platform that has assayed expression for ~20,000 genes in the adult mouse brain [14,15]. Here, g As an indication of complexity of the adult mouse brain transcriptome, within the 18 Pleiades libraries (including whole adult brain library) expression was observed for 11,836 genes of the total 17,098 genes detectable within the Mouse Atlas (total number of tags mapped to the Mouse Atlas libraries was approximately 8.8 million including singletons). In contrast, the Allen Brain Atlas (ABA) contains expression patterns of approximately 16,000 genes across the entire adult C57BL/6J mouse brain (Susan Sunkin, ABA, personal communication); of these genes, roughly 65.5% (10,479/16,000) were detect- able in the 18 Pleiades libraries. Furthermore, the Pleiades Page 2 of 13 (page number not for citation purposes) Page 2 of 13 (page number not for citation purposes) http://www.biomedcentral.com/1471-2202/9/66 BMC Neuroscience 2008, 9:66 Table 1: List of adult brain region SAGE libraries Name Description No. * TL = Target library (brain region of interest), OTL = Off-target library (background region) Complexity of the adult mouse brain transcriptome and SAGE-based analysis of transcriptome similarity of brain regions Table 2: Rank order based on the level and pattern of gene expression Rank Order Expression Pattern 1 1 TL* and 0 OTL* (PTL-OTL < = 0.05) (TL tag count > = 5) 2–6 1 TL and 1–5 OTLs (PTL-OTL < = 0.05) 7–11 1 TL and 1–5 OTLs (PTL-OTL > 0.05) (TL tag count > = 5, OTL tag count: 1–4) 12–17 2 TLs and 0–5 OTLs (PTL-TL > 0.05; PTL-OTL < = 0.05) 18–22 2 TLs and 1–5 OTLs (PTL-TL > 0.05; PTL-OTL > 0.05) (TL tag count > = 5, OTL tag count: 1–4) 23–28 3 TLs and 0–5 OTLs (PTL-TL > 0.05; PTL-OTL < = 0.05) 29–33 3 TLs and 1–5 OTLs (PTL-TL > 0.05; PTL-OTL> 0.05) (TL tag count > = 5, OTL tag count: 1–4) 34–39 4 TLs and 0–5 OTLs (PTL-TL > 0.05; PTL-OTL< = 0.05) 40–44 4 TLs and 1–5 OTLs (PTL-TL > 0.05; PTL-OTL > 0.05) (TL tag count > = 5, OTL tag count: 1–4) 45–55 1 TL and 6–16 OTLs (PTL-OTL < = 0.05) 56–65 2 TLs and 6–15 OTLs (PTL-TL > 0.05; PTL-OTL < = 0.05) 66–74 3 TLs and 6–14 OTLs (PTL-TL > 0.05; PTL-OTL < = 0.05) 75–82 4 TLs and 6–13 OTLs (PTL-TL > 0.05; PTL-OTL < = 0.05) 83 1 TL with 4 tags 84 1 TL with 3 tags 85 2 TLs with 4 tags 86 2 TLs with 3 tags 87 1 TL with 2 tags 88 2 TLs with 2 tags 89 3 TLs with 3 tags 90 3 TLs with 2 tags 91 1 TL with 1 tag * TL = Target library (brain region of interest), OTL = Off-target library (background region) Table 2: Rank order based on the level and pattern of gene expression Page 3 of 13 (page number not for citation purposes) Page 3 of 13 (page number not for citation purposes) http://www.biomedcentral.com/1471-2202/9/66 BMC Neuroscience 2008, 9:66 Use of Laser Capture Microdissection to isolate the hippocampus dentate gyrus from an adult mouse Figure 1 Use of Laser Capture Microdissection to isolate the hippocampus dentate gyrus from an adult mouse. A) Intact coronal brain section at ~Bregma -1.35 stained with cresyl violet. B & C) dentate gyrus (DG) has been microdissected with laser. D) dentate gyrus has been isolated and captured for total RNA extraction and construction of SAGE libraries. Complexity of the adult mouse brain transcriptome and SAGE-based analysis of transcriptome similarity of brain regions Barrier, Barrington's Nucleus, Astroglia etc., for which SAGE libraries had not been constructed. Therefore, to expand our set of genes with regionally enriched expres- sion for all brain regions, we then scrutinized literature from PubMed. We obtained a list of Medline records using Boolean logic with search term combinations indicated in Table 3. To facilitate retrieval of publications from a large literature database such as PubMed, we also developed a semi-automated literature mining strategy (see Methods and Figure 3) based on natural language processing. In this approach we looked for the appearance of a gene name or synonym and a brain region in a sentence. Of the 99.7 million sentences searched, 314,515 occurrences of a brain region term were found; 4,395 mouse genes names, or the names of their human orthologs, were found to appear within the same sentence as a brain region (not shown). lum Purkinje Cell Layer. Among neuronal tissues, the Ventral and Medial Thalamus consistently clustered tightly together and had the lowest expression divergence between any two pairs of tissues. Additionally, Visual Cor- tex, Primary Motor Cortex, Amygdala (basolateral), Amy- gdala (central), and Dorsal Striatum also clustered together. Segregation of the Ependymal tissue into a sepa- rate single cluster makes sense given its non-neuronal nature [17], and the Cerebellar White Matter is composed of myelinated axonal processes. Clustering is usually sen- sitive to the specific expression divergence measure used. However, we tried several empirical measures, as well as different P values for selecting differentially expressed genes, and observed that the main pattern of clustering outlined above remains unchanged. Complexity of the adult mouse brain transcriptome and SAGE-based analysis of transcriptome similarity of brain regions ! # " # $ # $% # &' # ! !! !! ! " !! ! # " p pp p gy g Use of Laser Capture Microdissection to isolate the hippocampus dentate gyrus from an adult mouse. A) Intact coronal brain section at ~Bregma -1.35 stained with cresyl violet. B & C) dentate gyrus (DG) has been microdissected with laser. D) dentate gyrus has been isolated and captured for total RNA extraction and construction of SAGE libraries. Images were captured using a Sony DXC-390P 3-CCD color video camera attached to a Nikon Eclipse TE2000-S microscope (10× magnification). Scale bar = 100 μm. D: dorsal; V: ventral. Complexity of the adult mouse brain transcriptome and SAGE-based analysis of transcriptome similarity of brain regions Images were captured using a Sony DXC-390P 3-CCD color video camera attached to a Nikon Eclipse TE2000-S microscope (10× magnification). Scale bar = 100 μm. D: dorsal; V: ventral. ! !! !! ! " !! Transcript Figure 2 p y g p g g Transcriptome similarity among 17 brain tissues based on expression divergence at P value = 0.01. Tissues being compared are indicated on the Y-axis, and expression divergence (EDP) of clusters of tissues is plotted on the X-axis. At each node in the dendrogram, the number of genes shared between libraries in the tissue cluster is indicated. A threshold of 50% of maximum EDP was chosen for coloring of branch lines in the dendrogram. sion, microarray expression profile, radioactive/non-radi- oactive in situ hybridization) in publications, and confirmed with in situ hybridization data from the Allen Brain Atlas (see below). In addition to promoter-reporter fusion data from the literature, reporter expression data for BAC (Bacterial Artificial Chromosome) transgenic mice, when available from the GENSAT database [18], was also considered as complementary evidence of expres- sion [see Additional file 2]. Anatomic Search tool to identify additional genes whose expression patterns cluster within brain regions of inter- est. While this approach short-listed genes for major regions (Thalamus, Cerebral Cortex etc.) of the mouse brain listed under Anatomic Search, we also searched within these regions to identify expression in sub-regions of interest, e.g. within Pons for genes expressed in Locus Coeruleus. Recent introduction of the alternative ABA search tool, NeuroBlast, also proved to be useful. We used NeuroBlast to retrieve genes co-expressed with a seeded (query) gene in a region of interest. Identification of regionally enriched co-expressed genes in this manner is indispensable in subsequent identification of shared reg- ulatory elements for efficient mini-promoter design. Data mining genes showing regionally enriched expression from Allen Brain Atlas The entire Allen Brain Atlas (ABA) data set can be searched via a web-based application [13,14]. We used this feature to examine expression patterns of genes identified as regionally enriched by SAGE and/or the literature. This verification was particularly apt for SAGE because ABA in situ hybridization patterns were also derived from the same mouse strain C57BL/6J. We also employed the ABA Thus, SAGE analysis of the adult mouse brain transcrip- tome combined with meta-analysis using data mining resources described above identified 237 genes as show- ing regionally enriched expression (Table 4). A summary of the meta-analysis that supports regionally enriched expression is presented [see Additional file 2]; where available, this file includes examples of supporting ABA images downloaded from the ABA website (please see Availability & requirements for more information) Table 3: Boolean search terms to obtain Medline records with information about region-associated expression or promoter characterization Gene AND brain AND in situ [qualifiers: Mouse/Human] Gene AND brain region AND in situ Gene AND regulation Gene AND promoter Gene AND promoter AND brain Gene AND promoter AND brain region Gene AND promoter AND transgenic mice Gene AND promoter AND reporter (qualifiers: CAT/Luciferase/Gfp) Gene AND brain AND in situ [qualifiers: Mouse/Human] Gene AND brain region AND in situ Gene AND regulation Gene AND promoter Gene AND promoter AND brain Gene AND promoter AND brain region Gene AND promoter AND transgenic mice Gene AND promoter AND reporter (qualifiers: CAT/Luciferase/Gfp) Literature mining strategy to rapidly identify genes associated with brain regions of interest The candidature of literature-mined genes was verified by assessing available expression data (reporter gene expres- g We included in the present analysis several additional brain regions and cell-types, for example, Blood-Brain Page 4 of 13 (page number not for citation purposes) Page 4 of 13 (page number not for citation purposes) BMC Neuroscience 2008, 9:66 http://www.biomedcentral.com/1471-2202/9/66 Transcriptome similarity among 17 brain tissues based on expression divergence at P value = 0.01 Figure 2 Transcriptome similarity among 17 brain tissues based on expression divergence at P value = 0.01. Tissues being compared are indicated on the Y-axis, and expression divergence (EDP) of clusters of tissues is plotted on the X-axis. At each node in the dendrogram, the number of genes shared between libraries in the tissue cluster is indicated. A threshold of 50% of maximum EDP was chosen for coloring of branch lines in the dendrogram. & '! " % & ' (&) * ) + ! (' "' " &, &, (' "' & '! " " % & ' (&) &, &, (' "' ! (' "' Identification of over-represented GO terms among genes with region-enriched expression Signif- icant biological processes involved nervous system devel- opment, transmission of nerve impulse, cell-cell signaling, neurogenesis, behavior etc. Significant molecu- lar functions involved neuropeptide hormone activity, sequence-specific DNA binding, neurotransmitter recep- tor activity, steroid hormone receptor activity, neurotrans- mitter transporter activity etc. Products of some of these genes also tended to be localized in the extracellular region, plasma membrane, synapse, or within transcrip- tion factor complexes. Thus, it appears that many of the genes we identified have established neurological func- tions, which accounts for their regionally enriched expres- sion. It is noteworthy that we found 28 transcription factor encoding genes representing 16 of 30 regions/cell- types of interest (Table 5). This information combined with identification of regulatory sequences within pro- moters of selected genes will aid the design of mini-pro- moters specific for each brain region. Because our selection of the 237 genes was biased towards those with known functions, we also carried out GO analysis on among our set of genes from each of three ontology classes: Biological Process, Molecular Function and Cellu- lar Component (Methods). Of 237 genes in our selection, we found annotations for 216 genes in the whole mouse genome set of 18535 annotated genes (as of March 18, 2008). From this list, we determined the top 12 statisti- cally over-represented GO terms [see Additional file 3]. Annotations for the test selection of genes were compared with GO annotations of the whole mouse genome. Signif- icant biological processes involved nervous system devel- opment, transmission of nerve impulse, cell-cell signaling, neurogenesis, behavior etc. Significant molecu- lar functions involved neuropeptide hormone activity, sequence-specific DNA binding, neurotransmitter recep- tor activity, steroid hormone receptor activity, neurotrans- mitter transporter activity etc. Products of some of these genes also tended to be localized in the extracellular region, plasma membrane, synapse, or within transcrip- tion factor complexes. Thus, it appears that many of the genes we identified have established neurological func- tions, which accounts for their regionally enriched expres- sion. It is noteworthy that we found 28 transcription factor encoding genes representing 16 of 30 regions/cell- types of interest (Table 5). This information combined with identification of regulatory sequences within pro- moters of selected genes will aid the design of mini-pro- moters specific for each brain region. Because our selection of the 237 genes was biased towards those with known functions, we also carried out GO analysis on Identification of over-represented GO terms among genes with region-enriched expression The Gene Ontology (GO) resource [19] is a powerful tool to identify common functions shared by genes identified by high-throughput gene expression methods such as SAGE. We searched for over-representation of GO terms Page 5 of 13 (page number not for citation purposes) BMC Neuroscience 2008, 9:66 http://www.biomedcentral.com/1471-2202/9/66 Text mining data flow Figure 3 Text mining data flow. This shows the steps by which the medical sentence parser retrieves Medline records that contain expression information for a gene in a specific region of the brain. " ! -' -' -. -, - ' - # - . - / - , - . - . - % - , 0 % % % -' ! g g Text mining data flow. This shows the steps by which the medical sentence parser retrieves Medline records that contain expression information for a gene in a specific region of the brain. genes expressed in each of 18 SAGE libraries [see Addi- tional file 4]. Specific neurological functions were less apparent among over-represented GO terms for these larger sets than for the 237 genes presented in this study. among our set of genes from each of three ontology classes: Biological Process, Molecular Function and Cellu- lar Component (Methods). Of 237 genes in our selection, we found annotations for 216 genes in the whole mouse genome set of 18535 annotated genes (as of March 18, 2008). From this list, we determined the top 12 statisti- cally over-represented GO terms [see Additional file 3]. Annotations for the test selection of genes were compared with GO annotations of the whole mouse genome. Page 6 of 13 (page number not for citation purposes) Hunk, Klk8, Gpr161, Arfrp2, C630041L24Rik, Slc9a2, Neurod6, Pkp2, Fibcd1, Sstr4 Hunk, Klk8, Gpr161, Arfrp2, C630041L24Rik, Slc9a2, Neurod6, Pkp2, Fibcd1, Sstr4 Gabrd, Prox1, Dsp, C78409, Lct, Crlf1, Tdo2, A330019N05Rik, Lrrtm4, Htr4, Tspan18 Nr2e1, Dcx, Mki67, Vim, Dlx2, Nes, Dlx1, Dscam, Fabp7, Igfbpl1, Lrrn1, Rrm1, Sox2, Thbs4 Adora2a, Gpr88, Drd1a, Drd2, Gpr6, Rgs9, Adcy5, Crym, Foxp1, Lpl, Pde1b1, Pdyn, Rarb, Rasd2, Tgfa Tac1, Cyp26b1, Hap1, Cdh9, Ptprc, Gabra2, Hgf, Pdzrn3, Plxnd1, Wwox, Rasal1, Dock10, Prkcd Grp, Nov, Nr2f2 Atp6v1c2 Ramp3, Rgs16, Slitrk6, Tnnt1, 1110069I04Rik Amotl1, Rab37, Sh3d19, Grid2ip, Lef1, Plekhg1, Syt9, Tcf7l2, Gm804, Gja7, Socs6, Vangl1 Hcrt, Gpx3, Trh, Fezf1, Agrp, Calcr, Ghrh, Npy, Pmch, Pomc Gabra6, Cbln3 Hcrt, Gpx3, Trh, Fezf1, Agrp, Calcr, Ghrh, Npy, Pmch, Pomc Gabra6, Cbln3 Pcp2, Hbegf, Icmt, Atp2a3, Casq2, Gdf10, Grid2, Hes3, Lhx1, Ptprm, A930006D11Rik Gal, Ngfr, Tac2, Lhx8, Ecel1, Gbx1, Lancl3, Ntrk1 Ddc, Slc6a3, Ntsr1, Pitx3, Aldh1a1, Chrna6, Chrnb3, Th Fev, Gchfr, Slc6a4, Slc17a8, Tph2, Maob, Esr2 Dbh, Maoa, Slc6a2, Slc18a2 Gfap, S100b, Slc1a2, Plaur, Gcm1, Gcm2, Serpina3n Cd68, Aif1, P2rx7, Sulf2 Cx3cr1, Itgam Olig1, Ugt8a, Cnp, Gjb1, Klk6, Mag, Apod, Enpp2, Fa2h, Mal, Mbp, Mobp, Mog, Olig2, Pllp, Plp1, Sox10, Tmem63a Crh, Fgfr1 Slc6a5, Glra1, Pogz, Anxa4, Spp1, Esr1, Pou4f1, Slc4a2, Stac Egr1, Stmn1, Cckbr,Adcy1 Rspo1, Cyp39a1, Cartpt, Col5a1, Rorb, Loc433228, Gnb4 Lxn, Ntng2, Nr4a2, Fezf2, Ttc9b Avp, Oxt Pitx2, Lmx1b Abcb1a, Cldn5, Ednra, Fcgrt, Hspa12b, Lrp10, Lrp8, Rage, Slc2a1, Slc7a5, Slco1c1, Slc6a12, Slc28a2 Vip, Gpr88‡ also listed as a cortex-specific gene ‡also listed as a striatum-specific gene of humans and mice [1]. In general, transcripts that are regionally enriched in mice also appear to be regionally enriched in humans – further emphasizing conservation of mammalian brain gene expression. Nonetheless, we are exercising caution in assuming global conservation of expression across species as divergent as mouse and human, and will be testing multiple candidate genes for each region. Our study profiles region-enriched gene expression within 17 key areas of the adult mouse brain by LongSAGE anal- ysis. For the small number of brain regions for which we had no SAGE data we interrogated the literature and the ABA directly. We used several expression indicators including SAGE tag abundance and specificity, in situ hybridization, promoter-reporter fusion data etc. to assess candidacy of genes. Our data mining strategy was to start with SAGE-identified genes ranked on the basis of specif- Our study profiles region-enriched gene expression within 17 key areas of the adult mouse brain by LongSAGE anal- ysis. Discussion In another comparable study within the milieu of neurogenomics, it was demonstrated that there are signif- icant constraints on the evolution of gene expression and nucleotide sequence of region-specific genes in the brains Page 6 of 13 (page number not for citation purposes) Page 6 of 13 (page number not for citation purposes) http://www.biomedcentral.com/1471-2202/9/66 BMC Neuroscience 2008, 9:66 Table 4: List of regionally enriched genes in 30 brain regions and cell-types of therapeutic intere Brain Regions/Cell types Example Processes/Disease Associations Genes Cortex Alzheimer Disease, Amyotropic Lateral Sclerosis, Plasticity B3galt2, 3110035 Rbp4, Rtn4rl2, Stx Igfbp6, Ephb6, Mp Hippocampus Alzheimer Disease Adult Neurogenesis, Depression, Plasticity Htr1A, Tgfb2, Gria Hippocampus, Ammon's Horn Alzheimer Disease Adult Neurogenesis, Depression, Plasticity Hunk, Klk8, Gpr16 Pkp2, Fibcd1, Sstr4 Hippocampus, Dentate Gyrus Alzheimer Disease Adult Neurogenesis, Depression, Plasticity Gabrd, Prox1, Dsp, Lrrtm4, Htr4, Tspa Neurogenic Regions Adult Neurogenesis Nr2e1, Dcx, Mki6 Igfbpl1, Lrrn1, Rrm Striatum Huntington Disease, Parkinson Disease, Plasticity in Depression Adora2a, Gpr88, D Lpl, Pde1b1, Pdyn, Amygdala Huntington Disease, Depression, Plasticity Tac1, Cyp26b1, Ha Wwox, Rasal1, Do Amygdala, Basolateral Complex Huntington Disease, Depression, Plasticity Grp, Nov, Nr2f2 Amygdala, Central Nucleus Huntington Disease, Depression, Plasticity Atp6v1c2 Thalamus Huntington Disease Ramp3, Rgs16, Slit Sh3d19, Grid2ip, L Socs6, Vangl1 Hypothalamus Cancer Hcrt, Gpx3, Trh, Fe Cerebellum, Granule Cells Medulloblastoma, Ataxia, Cerebellar hypoplasia Gabra6, Cbln3 Cerebellum, Purkinje Cell Layer Spinocerebellar Ataxia, Autism, Plasticity Pcp2, Hbegf, Icmt, Ptprm, A930006D Basal Nucleus of Meynert Acetylcholine System, Alzheimer Disease Gal, Ngfr, Tac2, Lh Substantia Nigra Dopamine System, Parkinson Disease Ddc, Slc6a3, Ntsr1 Raphe Nuclei Norepinephrine System, Depression Fev, Gchfr, Slc6a4, Locus Coeruleus Serotonin System, Depression Dbh, Maoa, Slc6a2 Astroglia Alzheimer Disease Gfap, S100b, Slc1a Microglia (activated) Alzheimer Disease, Amyotropic Lateral Sclerosis Cd68, Aif1, P2rx7, Microglia (constitutive) Alzheimer Disease Cx3cr1, Itgam Oligodendroglia Alzheimer Disease, Multiple Sclerosis Olig1, Ugt8a, Cnp, Mbp, Mobp, Mog, Barrington's Nucleus Pain Crh, Fgfr1 Brainstem, Pons and Medulla Pain Slc6a5, Glra1, Pogz Cortex, Anterior Cingulate Pain Egr1, Stmn1, Cckb Cortex, Somatosensory Pain Rspo1, Cyp39a1, C Cortex, Insula Pain Lxn, Ntng2, Nr4a2 Hypothalamus, Paraventricular Nucleus Pain Avp, Oxt Subthalamic Nucleus Pain Pitx2, Lmx1b Blood Brain Barrier Drug therapy Abcb1a, Cldn5, Ed Slc2a1, Slc7a5, Slc GABAergic neurons Schizophrenia, Bipolar Disorder Vip, Gpr88‡ also listed as a cortex-specific gene ‡also listed as a striatum-specific gene of humans and mice [1]. Discussion Targeting gene therapy to specific regions of the brain requires the application of well-defined promoters that can drive expression in a region-specific manner. In this study our goal was to identify regionally enriched tran- scripts in sub-structures/cell-types of the mouse brain with a particular focus on those brain regions associated with diseases. We were encouraged by findings from the ABA project that above background level expression was found for ~80% of genes assayed – and approximately 70% of genes have been localized to fewer than 20% of all brain cells – suggesting that gene expression is clustered in small brain regions [14]. For a variety of reasons we believe that human orthologs of regionally enriched mouse genes would be good candidates to design promot- ers from. First, at the genomic level, approximately 99% of mouse genes have an ortholog in the human genome [20]. Second, it has been shown that 84% of human- mouse orthologous gene pairs show significantly lower expression divergence than that of random gene pairs [21]. Discussion In general, transcripts that are i ll i h d i i l t b i ll Our study profiles region-enriched gene expression within 17 k f th d lt b i b L SAGE l Table 4: List of regionally enriched genes in 30 brain regions and cell-types of therapeutic interest Brain Regions/Cell types Example Processes/Disease Associations Genes Cortex Alzheimer Disease, Amyotropic Lateral Sclerosis, Plasticity B3galt2, 3110035E14Rik, Ccl27, Ctgf, Emx1, Fhl2, Klf10, Myl4, Rbp4, Rtn4rl2, Stx1a, Tbr1, Vip, Ddit4l, Dkkl1, Rspo2, Ier5, Igfbp6, Ephb6, Mpped1, Pak7, Satb2, Cplx3, E430002G05Rik Hippocampus Alzheimer Disease Adult Neurogenesis, Depression, Plasticity Htr1A, Tgfb2, Gria1, Nr3c2 Hippocampus, Ammon's Horn Alzheimer Disease Adult Neurogenesis, Depression, Plasticity Hunk, Klk8, Gpr161, Arfrp2, C630041L24Rik, Slc9a2, Neurod6, Pkp2, Fibcd1, Sstr4 Hippocampus, Dentate Gyrus Alzheimer Disease Adult Neurogenesis, Depression, Plasticity Gabrd, Prox1, Dsp, C78409, Lct, Crlf1, Tdo2, A330019N05Rik, Lrrtm4, Htr4, Tspan18 Neurogenic Regions Adult Neurogenesis Nr2e1, Dcx, Mki67, Vim, Dlx2, Nes, Dlx1, Dscam, Fabp7, Igfbpl1, Lrrn1, Rrm1, Sox2, Thbs4 Striatum Huntington Disease, Parkinson Disease, Plasticity in Depression Adora2a, Gpr88, Drd1a, Drd2, Gpr6, Rgs9, Adcy5, Crym, Foxp1, Lpl, Pde1b1, Pdyn, Rarb, Rasd2, Tgfa Amygdala Huntington Disease, Depression, Plasticity Tac1, Cyp26b1, Hap1, Cdh9, Ptprc, Gabra2, Hgf, Pdzrn3, Plxnd1, Wwox, Rasal1, Dock10, Prkcd Amygdala, Basolateral Complex Huntington Disease, Depression, Plasticity Grp, Nov, Nr2f2 Amygdala, Central Nucleus Huntington Disease, Depression, Plasticity Atp6v1c2 Thalamus Huntington Disease Ramp3, Rgs16, Slitrk6, Tnnt1, 1110069I04Rik Amotl1, Rab37, Sh3d19, Grid2ip, Lef1, Plekhg1, Syt9, Tcf7l2, Gm804, Gja7, Socs6, Vangl1 Hypothalamus Cancer Hcrt, Gpx3, Trh, Fezf1, Agrp, Calcr, Ghrh, Npy, Pmch, Pomc Cerebellum, Granule Cells Medulloblastoma, Ataxia, Cerebellar hypoplasia Gabra6, Cbln3 Cerebellum, Purkinje Cell Layer Spinocerebellar Ataxia, Autism, Plasticity Pcp2, Hbegf, Icmt, Atp2a3, Casq2, Gdf10, Grid2, Hes3, Lhx1, Ptprm, A930006D11Rik Basal Nucleus of Meynert Acetylcholine System, Alzheimer Disease Gal, Ngfr, Tac2, Lhx8, Ecel1, Gbx1, Lancl3, Ntrk1 Substantia Nigra Dopamine System, Parkinson Disease Ddc, Slc6a3, Ntsr1, Pitx3, Aldh1a1, Chrna6, Chrnb3, Th Raphe Nuclei Norepinephrine System, Depression Fev, Gchfr, Slc6a4, Slc17a8, Tph2, Maob, Esr2 Locus Coeruleus Serotonin System, Depression Dbh, Maoa, Slc6a2, Slc18a2 Astroglia Alzheimer Disease Gfap, S100b, Slc1a2, Plaur, Gcm1, Gcm2, Serpina3n Microglia (activated) Alzheimer Disease, Amyotropic Lateral Sclerosis Cd68, Aif1, P2rx7, Sulf2 Microglia (constitutive) Alzheimer Disease Cx3cr1, Itgam Oligodendroglia Alzheimer Disease, Multiple Sclerosis Olig1, Ugt8a, Cnp, Gjb1, Klk6, Mag, Apod, Enpp2, Fa2h, Mal, Mbp, Mobp, Mog, Olig2, Pllp, Plp1, Sox10, Tmem63a Barrington's Nucleus Pain Crh, Fgfr1 Brainstem, Pons and Medulla Pain Slc6a5, Glra1, Pogz, Anxa4, Spp1, Esr1, Pou4f1, Slc4a2, Stac Cortex, Anterior Cingulate Pain Egr1, Stmn1, Cckbr,Adcy1 Cortex, Somatosensory Pain Rspo1, Cyp39a1, Cartpt, Col5a1, Rorb, Loc433228, Gnb4 Cortex, Insula Pain Lxn, Ntng2, Nr4a2, Fezf2, Ttc9b Hypothalamus, Paraventricular Nucleus Pain Avp, Oxt Subthalamic Nucleus Pain Pitx2, Lmx1b Blood Brain Barrier Drug therapy Abcb1a, Cldn5, Ednra, Fcgrt, Hspa12b, Lrp10, Lrp8, Rage, Slc2a1, Slc7a5, Slco1c1, Slc6a12, Slc28a2 GABAergic neurons Schizophrenia, Bipolar Disorder Vip, Gpr88‡ also listed as a cortex-specific gene ‡also listed as a striatum-specific gene Table 4: List of regionally enriched genes in 30 brain regions and cell-types of therapeutic interest B3galt2, 3110035E14Rik, Ccl27, Ctgf, Emx1, Fhl2, Klf10, Myl4, Rbp4, Rtn4rl2, Stx1a, Tbr1, Vip, Ddit4l, Dkkl1, Rspo2, Ier5, Igfbp6, Ephb6, Mpped1, Pak7, Satb2, Cplx3, E430002G05Rik Htr1A, Tgfb2, Gria1, Nr3c2 B3galt2, 3110035E14Rik, Ccl27, Ctgf, Emx1, Fhl2, Klf10, Myl4, Rbp4, Rtn4rl2, Stx1a, Tbr1, Vip, Ddit4l, Dkkl1, Rspo2, Ier5, Igfbp6, Ephb6, Mpped1, Pak7, Satb2, Cplx3, E430002G05Rik Htr1A, Tgfb2, Gria1, Nr3c2 Hunk, Klk8, Gpr161, Arfrp2, C630041L24Rik, Slc9a2, Neurod6, Pkp2, Fibcd1, Sstr4 Hunk, Klk8, Gpr161, Arfrp2, C630041L24Rik, Slc9a2, Neurod6, Pkp2, Fibcd1, Sstr4 Our data mining strategy was to start with SAGE-identified genes ranked on the basis of specif- Page 7 of 13 (page number not for citation purposes) Page 7 of 13 (page number not for citation purposes) http://www.biomedcentral.com/1471-2202/9/66 BMC Neuroscience 2008, 9:66 Table 5: Regionally enriched genes encoding transcription factors Gene Transcription Factor Description Nr2f2 Nuclear receptor subfamily 2, group F, member 2 Gbx1 Gastrulation brain homeobox 1 Lhx8 LIM homeobox protein 8 Esr1 Estrogen receptor 1 Pou4f1 POU domain, class 4, transcription factor 1 Lhx1 LIM homeobox protein 1 Emx1 Empty spiracles homeobox 1 Tbr1 T-box brain gene 1 Egr1 Early growth response 1/Zinc finger protein 225 Nr4a2 Nuclear receptor subfamily 4, group A, member 2 Nr3c2 Nuclear receptor subfamily 3, group C, member 2 Neurod6 Neurogenic differentiation 6; Basic HLH transcription factor Dlx1 Distal-less homeobox 1 Dlx2 Distal-less homeobox 2 Nr2e1 Nuclear receptor subfamily 2, group E, member 1 Sox2 SRY (sex determining region Y)-box 2 Esr2 Estrogen receptor 2 Foxp1 Forkhead box P1 Rarb Retinoic acid receptor, beta: Pitx3 Paired-like homeodomain transcription factor 3 Lmx1b LIM homeobox transcription factor 1, beta Pitx2 Paired-like homeodomain transcription factor 2 Lef1 Lymphoid enhancer binding factor 1 Tcf7l2 Transcription factor 7-like 2 (T-cell specific, HMG-box) Gcm1 Glial cells missing homolog 1 Gcm2 Glial cells missing homolog 2 Olig1 Oligodendrocyte transcription factor 1 Olig2 Oligodendrocyte transcription factor 2 Sox10 SRY (sex determining region Y)-box 10 Table 5: Regionally enriched genes encoding transcription factors Gene Transcription Factor Description Associated Brain Region Nr2f2 Nuclear receptor subfamily 2, group F, member 2 Amygdala, Basolateral Complex Gbx1 Gastrulation brain homeobox 1 Basal Nucleus of Meynert Lhx8 LIM homeobox protein 8 Basal Nucleus of Meynert Esr1 Estrogen receptor 1 Brainstem (Pons and Medulla) Pou4f1 POU domain, class 4, transcription factor 1 Brainstem (Pons and Medulla) Lhx1 LIM homeobox protein 1 Cerebellum, Purkinje Cell Layer Emx1 Empty spiracles homeobox 1 Cortex Tbr1 T-box brain gene 1 Cortex Egr1 Early growth response 1/Zinc finger protein 225 Cortex, Anterior Cingulate Nr4a2 Nuclear receptor subfamily 4, group A, member 2 Cortex, Insula Nr3c2 Nuclear receptor subfamily 3, group C, member 2 Hippocampus Neurod6 Neurogenic differentiation 6; Basic HLH transcription factor Hippocampus, Ammon's Horn Dlx1 Distal-less homeobox 1 Neurogenic Dlx2 Distal-less homeobox 2 Neurogenic Nr2e1 Nuclear receptor subfamily 2, group E, member 1 Neurogenic Sox2 SRY (sex determining region Y)-box 2 Neurogenic Esr2 Estrogen receptor 2 Raphe Nuclei Foxp1 Forkhead box P1 Striatum Rarb Retinoic acid receptor, beta: Striatum Pitx3 Paired-like homeodomain transcription factor 3 Substantia Nigra Lmx1b LIM homeobox transcription factor 1, beta Subthalamic Nucleus Pitx2 Paired-like homeodomain transcription factor 2 Subthalamic Nucleus Lef1 Lymphoid enhancer binding factor 1 Thalamus Tcf7l2 Transcription factor 7-like 2 (T-cell specific, HMG-box) Thalamus Gcm1 Glial cells missing homolog 1 White Matter – Glia, Astrocytes Gcm2 Glial cells missing homolog 2 White Matter – Glia, Astrocytes Olig1 Oligodendrocyte transcription factor 1 White Matter – Glia, Oligodendroglia Olig2 Oligodendrocyte transcription factor 2 White Matter – Glia, Oligodendroglia Sox10 SRY (sex determining region Y)-box 10 White Matter – Glia, Oligodendroglia Associated Brain Region icity and expression level, confirmed with supporting evi- dence from the literature, ABA or GENSAT. Hunk, Klk8, Gpr161, Arfrp2, C630041L24Rik, Slc9a2, Neurod6, Pkp2, Fibcd1, Sstr4 For the small number of brain regions for which we had no SAGE data we interrogated the literature and the ABA directly. We used several expression indicators including SAGE tag abundance and specificity, in situ hybridization, promoter-reporter fusion data etc. to assess candidacy of genes. Page 8 of 13 (page number not for citation purposes) http://www.biomedcentral.com/1471-2202/9/66 http://www.biomedcentral.com/1471-2202/9/66 elements, transcription factors and pathways influencing their interaction will stand in good stead for efficient mini-promoter design. domains based on such expression patterns [24]. Impor- tantly, it was revealed that embryological imprinting is still evident in the adult brain. Microarray analysis has similarly identified molecular markers for neuronal sub- types in the adult mouse forebrain [25], in brain regions in each of eight strains of inbred mice [26], as well as in the adult rat CNS [27,28]. Fang et al. have shown that the most regionally discriminative genes are associated with one of four specific factors: regional myelin/oligodendro- cyte levels, resident neuron types, neurotransmitter inner- vation profiles, and Ca+2-dependent signaling and second messenger systems [28]. We encountered challenges during in this study that are deserving of mention. In literature mining, curation was obfuscated by the existence of numerous synonyms for either mouse or human genes, references to a single pro- tein rather than two distinct isoforms, or different genes with the same synonym. Furthermore, where genes were not represented on either ABA or GENSAT it was not pos- sible to confirm expression, but nonetheless such genes were retained based on level and specificity of expression indicated by the literature or SAGE. Additionally, for a good number of genes there was low correlation between expression detected by SAGE and in situ hybridization. Despite the depth of sampling, expression of many genes was not detected by our SAGE procedure; for e.g Pde1b1, which has been shown to be strongly expressed in the striatum by in situ hybridization on ABA and in the litera- ture [35]. Also, Hcrt appeared to be Hypothalamus-spe- cific by SAGE but ABA indicated enrichment in the Hypothalamus with low level, widespread background expression. Although our SAGE procedure and ABA in situ hybridization profiled gene expression from the same mouse strain C57BL/6J, lack of correlation between the two could be due to inherent differences in the way RNA is processed and/or detected in these procedures. None- theless, Hcrt was retained in our study after considering significance of expression in SAGE analysis (P value = 0) and the description of minimal promoters in the literature [36,37]. By assessing over-representation of GO terms within our set of regionally expressed genes, we identified common- alities in molecular functions, cellular locations and involvement in key biological processes. Hunk, Klk8, Gpr161, Arfrp2, C630041L24Rik, Slc9a2, Neurod6, Pkp2, Fibcd1, Sstr4 Although we prioritized finding genes displaying absolute regional spe- cificity (no detectable background expression), for our data mining strategy to be practicable we did not limit ourselves to this level of stringency – especially for the brain nuclei e.g. Basal Nucleus of Meynert, Barrington's Nucleus etc. Therefore, we also selected genes that dis- played the highest level of regional enrichment with the idea that promoters of such genes can be manipulated to produce desired specificity of expression, as reported by Machon et al. for the mouse Dach1 gene [22]. Compared to ubiquitous expression of the native Dach1 gene, a trans- gene with 5.8 kb of Dach1 regulatory sequence restricts β- galactosidase reporter expression within the mouse brain to the neocortex. Deletion analysis of this 5.8 kb fragment further delimited cortex-specific activity to a minimal 2.5 kb promoter region. From a total of about 30,000 mouse genes [20], we have identified a set of 237 genes display- ing regional enrichment of expression. ters of non-neuronal tissues (Ependymal tissue and the highly myelinated Cerebellar White Matter tissue) and the neuronal outlier Cerebellar Purkinje Cell Layer. This pat- tern of tissue clustering appears to be borne out by unique tissue composition at the very least. Among neuronal tis- sues, tight clustering of the Ventral and Medial Thalamus regions is possibly a reflection of common diencephalic origin, although from a functional standpoint the two tis- sues can be considered to be different. The expression sig- nature of a tissue may either independently confer tissue uniqueness, or itself depend on unique tissue composi- tion, the surrounding cellular environment, or a combina- tion of factors. Other studies have also demonstrated the utility of gene expression patterns in assessing cytoarchitectural distinct- ness of rodent brain regions. During review of this manu- script another study was published that employed SAGE gene expression profiling to identify region expression in 11 regions of the adult mouse brain [23]. Interestingly, regional enrichment of some transcripts was found to be conserved in the human brain. http://www.biomedcentral.com/1471-2202/9/66 This offers the promise of a unique set of molecular markers for each region/cell-type, and could potentially provide a mecha- nistic explanation of unique functions performed by dis- crete brain regions. Because of the disease application of our work, we were assured by the over-representation of genes involved in neurotransmitter synthesis, reception and degradation. Importantly, we have also identified many regionally expressed transcription factor-encoding genes. This is consistent with previous findings of Suzuki et al. who have identified region-specific transcription fac- tors in 11 mouse brain regions by using medium-scale real-time RT-PCR [29]. They reported that 90% of known transcription factors display significant expression in at least one brain region. Additionally, it was found that 349 of over 1000 transcription factor and co-regulator genes, mapped by in situ hybridization in the brains of develop- ing mice, show restricted expression patterns adequate to describe the anatomical organization of the mouse brain [30]. Hunk, Klk8, Gpr161, Arfrp2, C630041L24Rik, Slc9a2, Neurod6, Pkp2, Fibcd1, Sstr4 Microarray analysis of gene expression patterns in 24 neural tissues in the mouse central nervous system has mapped discrete brain Analysis of SAGE data to delineate transcriptome similar- ity among 17 selected brain tissues revealed segregation of a large cluster of neuronal tissues from discrete single clus- Page 8 of 13 (page number not for citation purposes) Page 8 of 13 (page number not for citation purposes) BMC Neuroscience 2008, 9:66 SAGE library preparation y p p The LongSAGE-Lite method was used to construct the libraries as previously described [5]. In brief, first strand cDNA was synthesized with Powerscript Reverse Tran- scriptase (Clontech, BD Biosciences, Mississauga, Can- ada) and LITE1/LITE TS primer mix (Invitrogen, Carlsbad, CA) using 15–120 ng of DNase-treated total RNA, and amplified by a 20-cycle PCR according to the SAGE-Lite method [38]. SAGE-Lite biochemistry for the generation of full-length cDNA libraries is based upon the SMART (Switching Mechanism At the 5' end of RNA Transcripts) cDNA synthesis strategy (Clontech, BD Biosciences, Mis- sissauga, Canada). Following amplification, the cDNA were processed according to an adaptation of the standard LongSAGE protocol using the I-SAGE Long kit (Invitro- gen, Carlsbad, CA). The SAGE protocol includes steps of anchoring by NlaIII, tagging by MmeI, and generating 131 bp ditags by T4 DNA ligase. The 131 bp ditags were ampli- fied using the scale-up PCR varying from 23–27 cycles depending on the optimal scale up condition as described in the protocol, and were digested with NlaIII to remove adapter sequences. Purified 36-bp ditags were ligated to form concatemers that were cloned into SphI-digested pZErO-1 vector (Invitrogen, Carlsbad, CA), and transfor- mations were done using One Shot DH10B T1 electro- competent E. coli (Invitrogen, Carlsbad, CA). Conclusion We have successfully identified genes displaying region- enriched expression in the mouse brain by the application of SAGE and data mining from a variety of publicly avail- able sources. These genes represent useful molecular markers that could potentially aid in unraveling the func- tions of representative brain regions/cell-types. Impor- tantly, for the Pleiades Promoter Project, identification of these genes has brought us closer to our goal of designing well-defined human promoters for gene therapy. Indeed, we have further identified promoters of human orthologs of a subset of these mouse genes, and are now gearing up to test expression of reporter genes in transgenic mice (unpublished data). Ultimately, it will be of great interest to determine for how many of these promoters the mouse pattern of regional enrichment is recapitulated within the human brain, and which of these successfully remediate the disorders they may be designed for. The identification of brain region-specific transcription factors is a prelude to explaining expression patterns of similarly enriched genes regulated by these factors. Armed with this knowledge, we can now search for evidence of transcription factor co-regulation of genes by availing of existing repositories of regulatory sequence collections [31-33]. In particular, the PAZAR system [33] has been employed to integrate transcription factor data and anno- tated regulatory sequences from the Pleiades Promoter Project. Additionally, given that much is already known about pathways that activate transcription factors, it would now be possible to identify pathways with which genes regulated by these transcription factors are associ- ated. Indeed, a regulatory network comprising 15 impor- tant basic helix-loop-helix transcription factors and 153 target genes within the mouse brain has now been con- structed [34]. From the perspective of the Pleiades Pro- moter Project, the identification of DNA-binding Page 9 of 13 (page number not for citation purposes) BMC Neuroscience 2008, 9:66 http://www.biomedcentral.com/1471-2202/9/66 http://www.biomedcentral.com/1471-2202/9/66 Methods Mice tured for LCM. To identify the desired regions for process- ing by LCM, each slide was individually stained with a modified Nissl-substance stain using cresyl violet (CV) dye (Polysciences, Inc., Warrington, PA) as follows: Slide- mounted sections were air-dried for 2–3 min and the sur- rounding OCT medium was rinsed off with 1× PBS (made with DEPC water). Tissue was fixed for 30 sec with 75% ethanol, stained for 1 min with 0.5% CV, then sequen- tially rinsed for 5–10 sec with 75%, 95%, and 100% eth- anol. After air-drying for 2–3 min, sections were immediately dissected with the SL μCUT system (MMI AG; Glattbrugg, Switzerland) under the 10× objective of a Nikon Eclipse TE2000-S, at laser power < 70 mV, for no longer than 15 min. The cut regions were collected onto the adhesive cap of a 500-μl microfuge tube (MMI AG, Glattbrugg, Switzerland) designed for the SL μCUT sys- tem, digested with 30 μl lysis buffer RLT (RNeasy Micro Kit; Qiagen Inc., Missisauga, Canada), and transferred from the cap to the vial. The samples were vortexed, cen- trifuged for 5 sec, and then stored at -80°C until RNA extraction (as above). High-quality samples were pooled within groups for SAGE library generation. Mice used in our experiments were all adult male C57BL/ 6J mice (12-week old post-natal). All procedures used in these experiments were in accordance with the Canada Council on Animal Care and approved by the University of British Columbia Animal Care Committee (A05-1748). All experiments were conducted in accordance with Cana- dian and International standards for animal care. All efforts were made to minimize the number and suffering of any animals used in these experiments. Whole brain manual dissection and RNA extraction Whole brains were manually dissected at room tempera- ture from the intact bodies of mice. To minimize the effects of stress on gene expression, the mother, and the entire litter remained in the family cage until harvest. Mice were removed, one at a time and killed in a separate room, by cervical dislocation. Tissue was immediately flash fro- zen in liquid nitrogen and stored at -80°C until further processing. Frozen tissue was disrupted and homogenized for 30 seconds with a Polytron® PT 1200CL hand-held homogenizer (Kinematica AG, through Brinkmann™ Instruments Inc, Mississauga, Canada) at a setting of 3 (~13,000 RPM), which had been equipped with a 7-mm easy-care generator (PT-DA 1207/2EC). Total RNA was extracted using RNeasy Lipid Tissue Mini Kit (Qiagen Inc., Missisauga, Canada), following the manufacturer's proto- col with the modification of using 1.5-ml Phase Lock Gel™ Heavy Tube (Eppendorf Scientific, through Fisher Scien- tific, Ottawa, Canada) for more robust phase separation. Also, while on the column, samples underwent DNase I treatment during RNA extraction. Standard care was used to avoid RNA degradation: reagents were prepared with diethyl pyrocarbonate (DEPC)-treated water and all sur- faces and equipment were treated with an RNase decon- tamination solution (RNaseZap® and RNaseZap® Wipes; Ambion Inc., Austin, Texas, USA). The quality and quan- tity of the RNA samples were tested on an Agilent 2100 Bioanalyzer with the RNA 6000 Nano LabChip® Kit (Agi- lent Technologies Canada Inc., Mississauga, Canada). Harvesting adult brain regions by Laser Capture Microdissection (LCM) Brains (1–3 per region; exception: 7 per Ependymal and Subependymal Layers), recovered as above, were immedi- ately frozen on dry ice and mounted in OCT (Optimal Cutting Temperature) embedding medium. For the Visual Cortex (SM147), Cerebellar White Matter (SM182), Dor- sal Striatum (SM195), and Cerebellar Purkinje cells (SM196) sagittal sections were processed, while coronal sections were used for the remaining tissues. Cryosections (20 μm) of fresh-frozen tissues were mounted onto RNase-free membrane slides (Molecular Machines & Industries AG (MMI), Glattbrugg, Switzerland) manufac- After transformants had been screened by colony PCR, the fraction containing concatemers of sizes ranging from 900 bp-1300 bp was chosen for sequencing. Colonies were picked using a Q-Pix robot (Genetix, Beaverton, OR) and inoculated into 2xYT media with Zeocin (50 μg/ml) and Page 10 of 13 (page number not for citation purposes) Page 10 of 13 (page number not for citation purposes) BMC Neuroscience 2008, 9:66 http://www.biomedcentral.com/1471-2202/9/66 each target library (Table 2). Region-specific transcripts were obtained by selecting transcripts detected with 5 tags or more only in one target library. To identify region- enriched transcripts, those detected in one target library and one off-target library (PTL-OTL value < = 0.05) were selected. Transcripts detected in multiple libraries were ranked based on pre-defined P value limits of differential expression (PTL-TL, PTL-OTL), as well as additional criteria such as target and off-target library counts. Transcripts whose expression patterns did not fit these criteria were not ranked. glycerol (7.5%). After overnight culture, glycerol stocks were used to inoculate larger volume cultures for plasmid preparation, carried out using a standard alkaline-lysis procedure adapted for high-throughput processing with microtiter plates. DNA sequencing was performed with BigDye v3.1 dye terminator cycle sequencing reactions run on Tetrad thermal cyclers (MJ Research, Waltham, MA). Products from the sequencing reaction were purified by ethanol precipitation and then run on capillary DNA sequencers (Model 3730xl, Applied Biosystems, Foster City, CA). each target library (Table 2). Region-specific transcripts were obtained by selecting transcripts detected with 5 tags or more only in one target library. To identify region- enriched transcripts, those detected in one target library and one off-target library (PTL-OTL value < = 0.05) were selected. Transcripts detected in multiple libraries were ranked based on pre-defined P value limits of differential expression (PTL-TL, PTL-OTL), as well as additional criteria such as target and off-target library counts. Transcripts whose expression patterns did not fit these criteria were not ranked. SAGE data analysis To obtain high quality SAGE tags for this study, all raw SAGE tags underwent a three-step cluster modification process developed by Siddiqui et al. [8]. In the first step, we calculated for each tag a P value based on the Phred quality score [39] to identify single nucleotide variants likely to originate from sequencing error. In the second step, we used tag sequence clustering to group such vari- ants to combine tags likely to originate from a common transcript. Thus, some singletons were clustered and counted as a more abundant tag. The third step was to fil- ter out low quality tags and compare each P value to a meta-library P value calculated from all SAGE libraries. Tag-to-gene-mapping was then carried out using Discov- erySpace 4.0 application [40]. All cluster-modified tags were then mapped to transcripts in the NCBI Reference Sequence Collection [41]. The remaining unmapped tags were mapped to transcripts in the Mammalian Gene Col- lection [42], followed by the Ensembl database [43]. Only sense transcripts and unique mappings were considered, and tags that mapped to more than one transcript in any of the three transcript databases were discarded. The three mapping results were subsequently merged based on gene symbol. ED(p) = Ndiff(p)/N (Ndiff(p) = number of differentially expressed genes for a given P value, N = number of shared genes between two corresponding libraries). (Ndiff(p) = number of differentially expressed genes for a given P value, N = number of shared genes between two corresponding libraries). Harvesting adult brain regions by Laser Capture Microdissection (LCM) To analyze transcriptome similarity of tissues, a dendro- gram was generated using MATLAB 7 (The MathWorks, Natick, MA) based on hierarchical clustering using the Unweighted Pair Group Method with Arithmetic Mean (UPGMA). The input data is a list of objects (tissue SAGE libraries) with their pair-wise distances (expression diver- gence ED; see below), and the output is a dendrogram. Initially, each object is in its own cluster; then, at each step of the hierarchical clustering the nearest two clusters are combined into a higher-level cluster. The distance between any two clusters A and B is taken to be the aver- age of all distances between pairs of objects in A and B. Thus, we defined pair-wise distance or expression diver- gence (ED) between any two tissues as the fraction of dif- ferentially expressed genes in their corresponding SAGE libraries, using the formula: Following inspection of data quality from a first 384-well sequencing plate, each library was sequenced to a depth of > 100,000 raw tags. The resulting sequence data were col- lected automatically and processed by both trimming the reads for sequence quality and removing sequences from non-recombinant clones, vector DNA and linker-derived tags. Processed data can be found on the Mouse Atlas web- site (please see Availability & requirements for more infor- mation) Page 11 of 13 (page number not for citation purposes) Acknowledgements g We wish to acknowledge financial support from the BC Cancer Founda- tion, Genome British Columbia, Genome Canada, UBC Institute of Mental Health, Child and Family Research Institute, UBC Office of the Vice Presi- dent Research, BC Mental Health and Addiction Services, GlaxoSmithKline R & D Ltd., and Canada Research Chair in Genetics and Behaviour (to E.M.S). We are grateful to the SAGE Library Construction Group and the Sequencing Group at the Genome Sciences Centre for technical assistance. We would like to thank Charles De Leeuw for useful comments on this manuscript, and Tracey Weir and Russell Watkins for editorial assistance with the manuscript. S.J.M.J, R.A.H, W.W.W and M.A.M are Michael Smith Foundation for Health Research Scholars. WWW is also a CIHR New Investigator. Abbreviations SAGE: Serial Analysis of Gene Expression; LCM: Laser Capture Microdissection; OCT: Optimal Cutting Temper- ature; CV: Cresyl violet; DEPC: Diethyl Pyrocarbonate; ABA: Allen Brain Atlas; BAC: Bacterial Artificial Chromo- some; GENSAT: Gene Expression Nervous System Atlas; UPGMA: Unweighted Pair Group Method with Arithmetic Mean; ED: Expression Divergence; GO: Gene Ontology. The Pleiades Promoter Project: http://www.pleiades.org The Pleiades Promoter Project: http://www.pleiades.org ABA website: http://www.brain-map.org; Seattle (WA): Allen Institute for Brain Science © 2004–2007; in accord- ance with ABA Terms of Use and Citation Policy. Semi-automated Literature mining All synonyms for 28,000 mouse genes were obtained from Entrez (RefSeq release 14) combined with Ensembl (build 34) of the mouse genome. Synonyms for the human orthologs were obtained using Compara (Ensembl build 34) to identify similarities between human and mouse together with Homologene (version 47) for homolog detection. In each case, Ensembl and Entrez were used as cross-references for gene identifiers. From these search strings, all names found in the English dictionary were subtracted to remove obfuscating gene terms such as "Ice". Abstracts were parsed from Medline (extraction per- formed September 7, 2006) and the complete text of arti- cles were parsed from PubMed Central [45], and converted into individual sentences using the medical sentence parser [46]. Each sentence was searched for the co-occurrence of gene names with brain regions of inter- est. For each brain region, expanded search terms were applied referring to finer structures appropriate to the region as defined by the ontology available from the Allen For each gene, a P value was assigned to each target (TL; brain region of interest) and off-target (OTL; background region) library pair using the P value option in Discov- erySpace. The P value was computed based on Audic-Clav- erie algorithms [44] to assess confidence level of differential expression between two transcript libraries. A ranking system was implemented to facilitate selection of candidate genes with specific or enriched expression in Page 11 of 13 (page number not for citation purposes) Page 11 of 13 (page number not for citation purposes) http://www.biomedcentral.com/1471-2202/9/66 BMC Neuroscience 2008, 9:66 and coordination along with WWW, DG, MAM, RAH, and SJMJ. All authors read and approved the final manuscript. Additional material and coordination along with WWW, DG, MAM, RAH, and SJMJ. All authors read and approved the final manuscript. Brain Atlas website [13]. The number of sentences with gene names and brain regions obtained is greater than the number of sentences with only brain regions because of the plural nature of both search terms. We scrutinized retrieved publications for details indicating regionally enriched/specific expression in a brain region. Brain Atlas website [13]. The number of sentences with gene names and brain regions obtained is greater than the number of sentences with only brain regions because of the plural nature of both search terms. We scrutinized retrieved publications for details indicating regionally enriched/specific expression in a brain region. Authors' contributions CAD analyzed SAGE data, ABA in situ hybridization data and mined the PubMed database to identify region- enriched genes, carried out GO analysis, and drafted this manuscript. VC analysed SAGE data, ABA in situ hybridi- zation data, mined the PubMed database to identify region-enriched genes, and contributed to the compila- tion of gene expression summaries in Additional file 2. DG confirmed candidature of SAGE and literature mined genes by inspecting ABA images. RV and LLCL performed bioinformatics analysis of SAGE data. Y–YX and SB laser- microdissected tissues for construction of SAGE libraries. YZ participated in SAGE library construction. MB did the hierarchical clustering analysis of tissue transcriptomes utilizing the java script written by AH. EP–C participated in data mining and selection of region-enriched genes. EMS conceived of the study, and participated in its design Additional file 4 Top 10 over-represented GO terms in each ontology category among the genes in each of 18 SAGE libraries. Click here for file Top 10 over-represented GO terms in each ontology category among the genes in each of 18 SAGE libraries. Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2202-9-66-S4.xls] Gene Ontology over-representation analysis Gene Ontology [19] over-representation analysis was per- formed for the 237 genes using the BiNGO [47] plug-in for the Cytoscape [48] software package. Significance of over-representation of GO terms was calculated using the hypergeometric test, corrected for multiple testing with a Benjamini & Hochberg false discovery rate correction [49], and a cut-off of 0.05 was applied to the result. The test selection of 237 genes was compared to all GO anno- tated genes in the mouse genome (18535 genes, as of March 18, 2008). Additional material Additional file 1 Compilation of SAGE data for 237 regionally enriched genes. Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2202-9-66-S1.xls] Additional file 2 Summary of expression profiles of region-specific or enriched genes by sub- anatomical region. Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2202-9-66-S2.doc] Additional file 3 Top 12 over-represented GO terms in each ontology category among the 237 regionally enriched genes. Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2202-9-66-S3.xls] Additional file 4 Top 10 over-represented GO terms in each ontology category among the genes in each of 18 SAGE libraries. Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2202-9-66-S4.xls] Additional file 1 Compilation of SAGE data for 237 regionally enriched genes. Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2202-9-66-S1.xls] References 1. Strand AD, Aragaki AK, Baquet ZC, Hodges A, Cunningham P, Hol- mans P, Jones KR, Jones L, Kooperberg C, Olson JM: Conservation of regional gene expression in mouse and human brain. PLoS genetics 2007, 3(4):e59. g ( ) 2. 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https://openalex.org/W3161018481	https://bmcneurol.biomedcentral.com/track/pdf/10.1186/s12883-021-02461-9	English	null	Effect of long-term adjuvant temozolomide chemotherapy on primary glioblastoma patient survival	BMC neurology	2,021	cc-by	5,973	Huang et al. BMC Neurol (2021) 21:424 https://doi.org/10.1186/s12883-021-02461-9 Huang et al. BMC Neurol (2021) 21:424 https://doi.org/10.1186/s12883-021-02461-9 Abstract Objective: Glioblastoma multiforme (GBM) is the most common primary malignant central nervous system (CNS) tumor. The Stupp regimen is the standard treatment, although the optimal number of temozolomide (TMZ) treat- ment cycles remains controversial. We compared the effects of standard 6 cycles versus > 6 cycles of TMZ chemother- apy post-surgery with concurrent chemoradiotherapy on primary GBM patient survival. Patients and methods: We performed a single center retrospective study of GBM patients that underwent total resection, concurrent chemoradiotherapy, and at least 6 cycles of adjuvant TMZ chemotherapy from June 2011 to August 2018. Patients were divided into 2 groups based on adjuvant TMZ treatment plan: Group A(n = 27): stand- ard 6-cycle adjuvant TMZ therapy and Group B(n = 26): > 6 cycles of adjuvant TMZ therapy. Primary endpoints were progression-free survival (PFS) and overall survival (OS). Continuous variables were analyzed by ANOVA, and the Kaplan-Meier method was used to evaluate PFS and OS. Univariate and multivariate COX analyses determined cor- relation between survival rates and covariates. We used The Mini Mental State Examination (MMSE) and Karnofsky Performance Status (KPS) to assess patients’ neurocognitive function and quality of life. Results: After follow-up, median PFS was 15 months in in Group A (95%CI 9.5–20.5) and 20.1 months in Group B (95%CI 15.9–24.4). Group A median OS was 19.4 months (95%CI 15.5–23.2), compared to 25.6 months in Group B (95%CI 20.4–30.8). The 2-year survival rate of Groups A and B was 36% was 66%, respectively (P = 0.02). and 5-year survival was 7% in both. Multivariate COX regression analysis showed association between patient PFS and long- period adjuvant chemotherapy, but not OS. There were no significant difference in disability or quality of life during treatment with Stupp protocol, but differences in MMSE and KPS were in favour of the Groups B after year 1 of the treatment (P < 0.05). Conclusions: Long-term adjuvant TMZ chemotherapy was beneficial for PFS and 2-year survival rate in GBM patients, and improved their quality of life contemporarily. But OS was not significantly improved. Keywords: Glioblastoma, Long-term adjuvant chemotherapy;progression-freesurvival, Cancer survival Effect of long-term adjuvant temozolomide chemotherapy on primary glioblastoma patient survival Bin Huang, Zuan Yu and Risheng Liang* © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://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. Introduction Glioblastoma mulitforme (GBM) is the most common primary malignant tumor of the adult central nervous system (CNS), accounting for 45.2% of all CNS malig- nancies and an annual incidence of 3 out of 100,000 [1]. After the phase III clinical trial conducted by Stupp et al., the standard treatment for newly diagnosed GBM is Correspondence: doctorlr123@126.com Department of Neurosurgery, Fujian Medical University Union Hospital, No. 29 Xinquan Road, Gulou District, Fuzhou City, Fujian Province, China Correspondence: doctorlr123@126.com Department of Neurosurgery, Fujian Medical University Union Hospital, No. 29 Xinquan Road, Gulou District, Fuzhou City, Fujian Province, China *Correspondence: doctorlr123@126.com Department of Neurosurgery, Fujian Medical University Union Hospital, No. 29 Xinquan Road, Gulou District, Fuzhou City, Fujian Province, China . Open Access T d d © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://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. Huang et al. BMC Neurol (2021) 21:424 Page 2 of 8 Page 2 of 8 resection of the tumor in our Neurosurgery Depart- ment. Total resection is defined as complete tumor resection as determined via a T1-weighted MRI enhancement image of a postoperative brain [11]. All patients exhibited tumors located in non-critically functional regions of the cerebrum. The median time of radiation therapy (RT) was 4 weeks (3–6 weeks) surgery with adjuvant TMZ treatment. Later, TMZ was the first-line adjuvant chemotherapy for at least 6 treatment cycles. Patients in transition from low- grade glioma to GBM were excluded from the study. As the study objective was to evaluate potential adjuvant therapeutic benefits over 6 or more treatment cycles, patients that failed to complete the Stupp treatment program at our hospital were excluded, including those with tumor recurrence during 6-cycle adjuvant chemo- therapy and more than 6 weeks of post-surgical radio- therapy [12]. Additional eligibility criteria included a pre-operative minimum KPS score of 60 and follow-up MRI every 3 months. All patient data were reviewed by an experienced neurosurgeon, including clinical evalu- ations, pathology report, and all imaging results. post-surgical radiotherapy (RT) or biopsy and 75 mg/m2 daily adjuvant therapy with temozolomide (TMZ). TMZ is a common orally administered chemotherapeutic com- pound that acts via guanine methylation and subsequent inhibition of cellular proliferation. . Open Access T d d Six cycles of adjuvant TMZ therapy were administered performed follow- ing radiotherapy (28 days per cycle, with TMZ given the first 5 days per cycle). However, despite standard Stupp treatment, the prognosis of most GBM patients remains poor, with a median survival time of 14.6 months, a 26.5% 2-year survival rate, and < 5% five-year survival rate [2]. i Weller et al. indicated that favorable prognostic factors for GBM include age, preoperative KPS score, IDH1/2 mutation, and levels of methylguanine-DNA methyl- transferase (MGMT) promoter methylation [3]. The MGMT is recognized as a biomarker, as well as a pri- mary contributor to TMZ resistance in glioblastoma [4]. Long-term TMZ administration will minimize MGMT levels and weaken tumor cell resistance, thereby “autono- mously” enhancing anti-tumor effects of TMZ [5]. How- ever, it remains debatable what is considered the optimal number of adjuvant TMZ therapy cycles [6]. We collected all data from electronic medical records, which included age, gender, the number of cerebral lobes involving tumors, and molecular markers (IDH mutation, MGMT methylation). A record of treatment process and procedures was obtained, including radia- tion dose, range, and number of adjuvant TMZ therapy cycles. The date of diagnosis was defined as the date of GBM diagnosis through histology. Time to first relapse was determined by histological examination (includ- ing surgical resection and biopsy) or through follow- up imaging data evaluation. Overall survival (OS) was defined as the timeframe between initial diagnosis and date of death or last follow-up. We used the MMSE and KPS scoring scales to collect survival quality from preoperative(T1), 7 days after surgery (T2), at the com- pletion of the STUPP protocol (T3), and 1 year after surgery (T4), respectively. Primary adverse reactions of adjuvant TMZ chemo- therapy include thrombocytopenia and neutropenia, though studies suggest that long-term or high-dose adjuvant TMZ therapy does not increase the probabil- ity of neutropenia and thrombocytopenia compared to standard 6-cycle chemotherapy [7, 8]. Therefore, it is an appealing option for patients that have successfully com- pleted 6 cycles of adjuvant chemotherapy [9]. With regard to these points of consideration, the goal of the present study was to evaluate whether long-term adjuvant TMZ chemotherapy could confer clinical benefits. Patient information Th d The study was approved by Ethics Committee of Union Hospital of Fujian Medical University and performed according to the Declaration of Helsinki guidelines. Informed consent was obtained from the participants. In this study, included patients were from the Union Hospital of Fujian Medical University. Our hospital has conducted MGMT assessment since 2011 and TMZ has been a first-line medication for patients with GBM. Therefore, patients initially diagnosed with GBM (based on the WHO 2007 Central Nervous System Tumor Clas- sification) between 2011 and 2018 were included in our study [10].ii Results lasted 28 days, and all patients were given adjuvant chem- otherapy of 150 mg/m2 temozolomide for 5 consecu- tive days. If no treatment-related adverse reactions were observed in subsequent cycles, the dose was increased to a 200 mg/m2 adjuvant TMZ chemotherapy regimen. Our initial search of the medical database identified 319 patients with GBM as the primary diagnosis, of which 252 were excluded for failing to meet the Stupp ther- apy criteria or due to receipt of follow-up treatment at other institutions. Of the remaining 67 patients, 8 were excluded as they less than 18 years of age (3patients) older than 70 years of age (5 patients). Six were excluded due to diagnosis of secondary glioblastoma. The remain- ing 53 patients were grouped as described in Table 1. Included patients were divided into two groups: Group A and Group B. Group A (n = 27) completed the Stupp protocol, and Group B (n = 26) continued adjuvant TMZ chemotherapy following completion of the Stupp pro- tocol until tumor progression or patients refused con- tinuation of chemotherapy. All patients underwent total tumor resection and were examined by T1-enhanced weighted MRI within 1 week after surgery to confirm complete resection of the tumor. The median cycle of adjuvant TMZ chemotherapy in Group B patients was 10 (range = 7–41). The first cycle of adjuvant chemotherapy Upon completion of the follow-up on August 2019 (median follow-up of 26 weeks; range = 13–72 weeks), 42 progression events occurred (24 in 89% of Group A and 69% in Group B). Seven patients (26%) in Group A sur- vived to follow-up and 20 patients (74%) died from tumor progression. Ten patients (38%) in Group B survived to follow-up and 16 (62%) died from tumor progression. The 2-year survival rate for group A was 36%, compared to 66% in Group B (P = 0.02). Two patients (7%) in each group survived beyond 5 years.h The Kaplan-Meier method was used for comparing survival of standard and long-period adjuvant chemo- therapy. The median progression-free survival (PFS) in Group A was 15 months (95% CI 9.5–20.5), compared to 20.1 months in Group B (95% CI 15.9–24.4). The median overall survival (OS) in Group A was 19.4 months (95% CI 15.5–23.2) compared to 25.6 months in Group B (95% CI 20.4–30.8). The Log rank method was used to test differences in survival time distribution. Statistical analysis All d All data were recorded using Microsoft Excel (2007), and statistical analyses were performed using SPSS sta- tistical analysis software (version 21.0). Statistics were plotted using GraphPad Prism software (GraphPad, Inc.). The Kaplan-Meier method was used for univari- ate survival analysis to estimate patient PFS and OS probability distributions following treatment. Cox regression models were used to analyze relationships between survival and covariates. The effect of each covariate on GBM treatment was gradually determined by selecting and analyzing different covariates. Specific inclusion and exclusion criteria were defined as follows. Patients had to be adults (≥18 years old and ≤ 70 years old) with histologically diagnosed WHO grade IV GBM. All included patients underwent total Huang et al. BMC Neurol (2021) 21:424 Huang et al. BMC Neurol (2021) 21:424 Page 3 of 8 Page 3 of 8 Results Both patient group showed the highest KPS and MMSE at T3. After 1 year of treatment, the KPS score and MMSE score of patients in group B were higher group A, and the differ- ence between the two groups was statistically significant (Tables 4 and 5). a phase III clinical trial in 2005 (NCT00006353) on the standard treatment protocol of Glioblastoma mul- tiforme [2]. This trial established 6 cycles of adjuvant TMZ chemotherapy as the standard primary glioblas- toma treatment protocol post-surgery and concurrent chemoradiotherapy (ie, Stupp protocol). Ultimately, 36.5% (105/287) of patients completed the full adjuvant h th H th i ff ti Fig. 1 Progression-free survival curve for patients diagnosed with GBM after temozolomide adjuvant chemotherapy Fig. 2 Overall survival curve for patients diagnosed with GBM after temozolomide adjuvant chemotherapy Fig. 1 Progression-free survival curve for patients diagnosed with GBM after temozolomide adjuvant chemotherapy Fig. 1 Progression-free survival curve for patients diagnosed with GBM after temozolomide adjuvant chemotherapy Fig. 2 Overall survival curve for patients diagnosed with GBM after temozolomide adjuvant chemotherapy Fig. 2 Overall survival curve for patients diagnosed with GBM after temozolomide adjuvant chemotherapy a phase III clinical trial in 2005 (NCT00006353) on the standard treatment protocol of Glioblastoma mul- tiforme [2]. This trial established 6 cycles of adjuvant TMZ chemotherapy as the standard primary glioblas- toma treatment protocol post-surgery and concurrent chemoradiotherapy (ie, Stupp protocol). Ultimately, 36.5% (105/287) of patients completed the full adjuvant chemotherapy course. However, there is no effective supplementary treatment after completing the Stupp program. Although six adjuvant TMZ therapy cycles are used in the Stupp regimen, the optimal regimen for such a therapy is debated. In clinical practice, some a phase III clinical trial in 2005 (NCT00006353) on the standard treatment protocol of Glioblastoma mul- tiforme [2]. This trial established 6 cycles of adjuvant TMZ chemotherapy as the standard primary glioblas- toma treatment protocol post-surgery and concurrent chemoradiotherapy (ie, Stupp protocol). Ultimately, 36.5% (105/287) of patients completed the full adjuvant chemotherapy course. However, there is no effective supplementary treatment after completing the Stupp program. Although six adjuvant TMZ therapy cycles are used in the Stupp regimen, the optimal regimen for such a therapy is debated. In clinical practice, some were no significant difference at T1-T3 of MMSE scores and KPS scores between the two groups. Both patient group showed the highest KPS and MMSE at T3. Results After 1 year of treatment, the KPS score and MMSE score of patients in group B were higher group A, and the differ- ence between the two groups was statistically significant (Tables 4 and 5). Results Patients that received long-term adjuvant chemotherapy exhibited a statistically significant PFS (χ2 = 7.06, P = 0.008, Fig. 1), but no difference was observed for OS time distribution (χ2 = 2.04, P = 0.152, Fig. 2). Table 1 Summary of patient characteristics Group A Group B P (n = 27) (n = 26) Sex Male 16 (59%) 14 (53%) Female 11 (41%) 12 (47%) 0.74 Onset age < 45 16 (59%) 12 (46%) > 45 11 (41%) 14 (54%) 0.26 Pre-surgery KPS 60–80 5 (18%) 6 (23%) > 80 22 (82%) 20 (77%) 0.42 Brain lobes involved in tumor 1 14 (52%) 15 (57%) > 1 13 (48%) 11 (43%) 0.42 MGMT methylation Yes 12 (44%) 14 (54%) No 13 (48%) 10 (38%) Unknown 2 (8%) 2 (8%) 0.78 IDH mutation Mutated 5 (18.5%) 8 (31%) Wild type 17 (63%) 12 (46%) Unknown 5 (18.5%) 6 (23%) 0.14 TMZ therapeutic cycles 6 27 (100%) 0 7–9 10 (38%) 10–12 9 (34%) > 12 7 (28%) < 0.001 Total patient number 27 26 53 Table 1 Summary of patient characteristics Univariate analysis using the Cox proportional hazard model was performed (Table 2). Our results showed that the number of lobes involving the tumor, MGMT meth- ylation, IDH mutation, and adjuvant TMZ chemotherapy cycles (HR:0.454; P:0.01) are factors related to tumor pro- gression. Gender, age and pre-operative KPS score were not associated with tumor progression. OS was associ- ated with the methylation status of MGMT, IDH muta- tion, pre-surgical KPS score, but not with age, gender, adjuvant chemotherapy cycles (HR:0.628; P:0.159) or the number of lobes with tumor involvement. When adjusted to a multivariate COX risk model with known variables (age, MGMT methylation, IDH muta- tion, adjuvant chemotherapy cycle number, KPS score, and number of lobe involvement), our analysis showed that PFS was associated with MGMT methylation (HR:0.336, P:0.002), IDH mutation, number of adjuvant TMZ chemotherapy cycles (HR:0.224, P:< 0.01) and the number of tumor-involved lobes. OS was associated with the methylation status of MGMT, IDH mutation and pre- operative KPS score, but not with the number of TMZ chemotherapy cycles or the number of involved cerebral lobes (Table 3).h The median preoperative MMSE score of 21(range 12–29), a median preoperative KPS of 80/100. There Huang et al. BMC Neurol (2021) 21:424 Page 4 of 8 were no significant difference at T1-T3 of MMSE scores and KPS scores between the two groups. Discussion Th The European Organisation for the Research and Treat- ment of Cancer (EORTC) and the Canadian National Cancer Institute Clinical Trial Team (NCIC) conducted Huang et al. BMC Neurol (2021) 21:424 Page 5 of 8 Table 2 Univariate analysis with regard to tumor progression or death Table 2 Univariate analysis with regard to tumor progression or death Abbreviations: CI Confidence interval, KPS Kanovsky performance score, MGMT O-6-methylguanine-DNA-methyltransferase, IDH Isocitrate dehydrogenase, HR Hazard ratio Patient number PFS P值 OS P HR(95%CI) HR(95%CI) Age < 45 32 1 1 > 45 21 1.227 0.511 1.341 0.384 (0.667–2.258) (0.692–2.597) Sex Female 23 1 1 Male 30 1.062 0.843 1.19 0.586 (0.587–1.920) (0.629–2.269) Pre-surgery KPS < 80 11 1 1 > 80 42 0.701 0.33 0.387 0.013 (0.343–1.434) (0.183–0.817) Brain lobes involved in tumor Single lobe 34 1 1 Multiple lobes 19 0.521 0.04 0.783 0.365 (0.278–0.975) (0.382–1.424) MGMT methylation No 23 1 1 Yes 26 0.343 < 0.001 0.379 0.005 (0.178–0.663) (0.192–0.748) IDH Wild-Type 29 1 1 Mutation 10 0.222 0.01 0.112 < 0.001 (0.682–0.07) (0.027–0.56) TMZ therapeutic cycles 6 27 1 1 > 6 26 0.454 0.01 0.628 0.159 (0.244–0.842) (0.324–1.215) ce interval, KPS Kanovsky performance score, MGMT O-6-methylguanine-DNA-methyltransferase, IDH Isocitrate dehydrogenase, HR Hazar adverse reactions was greater in this group compared to the standard-dose group (52.5 and 34.1%). clinicians adopt a dose-intensive regimen or extend adjuvant treatment cycles beyond 6 weeks. Therefore, establishing a standardized adjuvant TMZ treatment plan is of high importance. Few large long-term adjuvant chemotherapy studies are present in the published literature. In several retro- spective studies [13–15], the median chemotherapy cycle number ranged from 14 to 16 cycles in long-term adju- vant chemotherapy groups. In these studies, patients undergoing long-term TMZ adjuvant chemotherapy treatment exhibited longer PFS and OS than those receiving standard chemotherapy regimens. Such stud- ies indicate that the number of adjuvant TMZ chemo- therapy cycles is an independent factor that benefits both PFS and OS; however, our findings suggest otherwise. Long-term adjuvant TMZ chemotherapy improved PFS (HR:0.454; P:0.01) not OS (HR:0.628; P:0.159) based on Cox regression and survival curve analyses in our study. Similar studies have been reported in the literature [16– 18]. Gramatzki, D. et al. evaluated 142 newly diagnosed Prolonged exposure to alkylating agents will deplete intracellular MGMT in peripheral blood mononu- clear cells, and low levels of MGMT will ensure opti- mal cytotoxicity of TMZ [4]. Discussion Th To verify whether lower MGMT levels were associated with improved survival, a phase III clinical trial divided patients with primary GBM into standard-dose and dose-dense TMZ treat- ment groups [8]. The results of this trial revealed a median OS of 16.6 and 14.9 months (HR 1.03; P = 0.63) and median PFS of 5.5 and 6.7 months (HR 0.87; P = 0.06), respectively, with no significant differences between the groups. Extended OS was not observed in the dose-dense group, although the incidence of Huang et al. BMC Neurol (2021) 21:424 Page 6 of 8 Table 3 Multivariate analysis with regard to tumor progression or death Abbreviations: CI Confidence interval, KPS Kanovsky performance score, MGMT O-6-methylguanine-DNA-methyltransferase, IDH Isocitrate dehydrogenase, HR Hazard ratio Patient number PFS P OS P HR HR (95%CI) (95%CI) MGMT methylation No 23 1 1 Yes 26 0.336 0.002 0.470 0.025 (0.167–0.674) (0.243–0.909) IDH Wild type 29 1 1 Mutation 10 0.094 0.077 (0.026–0.346) < 0.001 (0.016–0.384) 0.002 TMZ therapeutic cycles 6 27 1 – > 6 26 0.224 < 0.001 – (0.106–0.473) Brain lobes involved in tumor Single lobe 34 1 Multiple lobes 19 0.406 0.012 – (0.201–0.819) Pre-surgery KPS score < 80 11 – 1 > 80 42 – 0.192 < 0.001 (0.079–0.464) Table 3 Multivariate analysis with regard to tumor progression or death did not support and benefit to OS. Skardelly et al. stud- ied 107 recently diagnosed GBM patients from 2006 to 2014 [17]. In their study, long-period adjuvant chemo- therapy group exhibited a higher median survival time than the standard-period adjuvant chemotherapy group (28.6 months and 25.2 months). However, following mul- tivariate regression analysis, no significant differences between the two groups were determined (RR 0.77, P = 0.46). Table 4 Longitudinal comparison of KPS scores in the two groups KPS score Group A(n = 27) Mean ± SD Group B (n = 26) Mean ± SD P value T1 79.62 ± 8.07 80.38 ± 12.15 0.792 T2 73.70 ± 6.87 73.46 ± 6.89 0.899 T3 91.11 ± 8.00 93.46 ± 6.89 0.257 T4 71.11 ± 21.89 89.61 ± 13.10 0.001 GBM patients between 2004 and 2010 [16]. Discussion Th The study determined that long-term adjuvant chemotherapy was independently associated with PFS, but COX regression Table 4 Longitudinal comparison of KPS scores in the two groups KPS score Group A(n = 27) Mean ± SD Group B (n = 26) Mean ± SD P value T1 79.62 ± 8.07 80.38 ± 12.15 0.792 T2 73.70 ± 6.87 73.46 ± 6.89 0.899 T3 91.11 ± 8.00 93.46 ± 6.89 0.257 T4 71.11 ± 21.89 89.61 ± 13.10 0.001 Table 5 Longitudinal comparison of MMSE scores in the two groups MMSE score GroupA(n = 27) Mean ± SD Group B (n = 26) Mean ± SD P value T1 21.59 ± 4.19 21.65 ± 4.56 0.960 T2 19.96 ± 4.55 19.53 ± 4.36 0.730 T3 24.77 ± 2.76 24.84 ± 2.98 0.931 T4 20.37 ± 5.83 23.84 ± 4.49 0.019 Researchers have observed improvements in PFS from long-cycle chemotherapy on, as reported by multiple publications. Whether or not there is a statistically sig- nificant difference in OS, the long-cycle adjuvant TMZ chemotherapy group shows a higher 2-year survival rate [13–18]. In the present study, the 2-year survival rate of patients in the standard- and long-cycle adjuvant TMZ chemotherapy groups were 36 and 66%, respectively (P = 0.02). We believe that an increased 2-year survival rate in the long-term adjuvant chemotherapy group is associated with prolonged PFS from long-term chemo- therapy. In a retrospective analysis of phase II clinical tri- als, 437 GBM patients included were divided into 9-week, 18-week, and 26-week groups according to post-oper- ative PFS. The findings of this study show that patients Table 5 Longitudinal comparison of MMSE scores in the two groups Table 5 Longitudinal comparison of MMSE scores in the two groups MMSE score GroupA(n = 27) Mean ± SD Group B (n = 26) Mean ± SD P value T1 21.59 ± 4.19 21.65 ± 4.56 0.960 T2 19.96 ± 4.55 19.53 ± 4.36 0.730 T3 24.77 ± 2.76 24.84 ± 2.98 0.931 T4 20.37 ± 5.83 23.84 ± 4.49 0.019 GBM patients between 2004 and 2010 [16]. The study determined that long-term adjuvant chemotherapy was independently associated with PFS, but COX regression GBM patients between 2004 and 2010 [16]. The study determined that long-term adjuvant chemotherapy was independently associated with PFS, but COX regression Huang et al. Abbreviations GBM: Glioblalstoma; PFS: Progression-free Survival; CNS: Central Nervous Sys- tem; TMZ: Temozolomide; OS: Overa survive; MGMT: 0–6-Methyguanine DNA; IDH: Isocitrate dehydrogenase; KPS: Karnofsky; RT: Radiotherapy; OR: Odds ratio; RV: Residual volume. Funding N Funding No external funding was received for this research. g No external funding was received for this research. Ethics approval and consent to participate The study was approved by Ethics Committee of Union Hospital of Fujian Medical University and performed according to the Declaration of Helsinki guidelines. Informed consent was obtained from the participants. Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Acknowledgements We are grateful to all the medical staff on our interdisciplinary team fortreating the patients with GBM. We observed that patients with tumors involving only a single lobe of the brain exhibited longer PFS than patients with multi-lobe involvement. A possible expla- nation is that GBM aggressively invades surrounding tis- sues, and invisible tumors can be more easily removed in the patients with tumors involving a single lobe of the brain. Similar to our study, Filippini, G. et al. conducted a survival analysis of 676 GBM patients and found that the prognosis of patients with single lobe involvement was significantly better than those with multiple lobe involve- ment (HR: 0.78, 95% CI (0.65–0.94), P = 0.008) [22]. In a study by Kaisorn, L et al., RV was closely related to tumor recurrence. In addition to being negatively related to the degree of resection, RV was also affected by tumor loca- tion. When tumors involve multiple lobes and may not be fully removed by surgery, long-term adjuvant TMZ chemotherapy can be of benefit to these patients [23]. Authors’ contributions BH, ZY and LS designed the present study. BH, ZY contributed to the work equally. Material preparation, data collection and analysis were performed by BH, ZY. The first draft of the manuscript was written by BH, ZY. All authors read and approved the final manuscript. Competing interests The authors declare no conflicts of interest. Competing interests The authors declare no conflicts of interest. The authors declare no conflicts of interest. Received: 7 May 2021 Accepted: 22 October 2021 Received: 7 May 2021 Accepted: 22 October 2021 Discussion Th BMC Neurol (2021) 21:424 Page 7 of 8 with extended PFS also have higher survival rates after tumor recurrence [19]. A retrospective analysis of 831 GBM patients included in trial RTOG 0525 showed that the risk of death after GBM progression was 6.6 times higher than in the group that did not exhibit cancer pro- gression [20]. There is a close correlation between PFS and OS [21]. A longer PFS may improve the 2-year sur- vival rate by decreasing the risk of death. Consent for publication f bl i Despite our interesting results, our study has sev- eral limitations. First, the overall sample size of patients included in the study was small and they all came from the same clinical center. Second, this is a non-rand- omized retrospective study and that differences in treat- ment selection after tumor recurrence may affect OS. Therefore, a prospective multicenter clinical trial is nec- essary to evaluate the question of duration of TMZ ther- apy better. Written consent for publication was obtained from all patients and/or parents of the patients in this study. 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References py The Mini-Mental state examination (MMSE) is a sim- ple test that is able to briefly estimate the cognitive sta- tus of a patient affected by a cognitive impairment either induced by a tumour, in other studies, MMSE has proven to be very useful to describe the tumor-related cognitive impairment [24]. Post-hoc analysis of neurocognitive functioning in the first year. Patients had an improved postoperative MMSE scores and KPS scores in both group compared with preoperative patients, indicating that aggressive treatment helped improve quality-of-life. Neurocognitive function decreased in both groups at T4, but group B was significantly higher than group A, which was associated with the differences of tumor progression between the two groups (15/27 progressions in group A, 1/26 progressions in group B). 1. Ostrom QT, Gittleman H, Fulop J, Liu M, Blanda R, Kromer C, et al. 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Hegi ME, Diserens AC, Gorlia T, Hamou MF, de Tribolet N, Weller M, et al. MGMT gene silencing and benefit from temozolomide in glioblastoma. N Engl J Med. 2005;352(10):997–1003. https://doi.org/10.1056/NEJMoa0433 31. 6. Holdhoff M, Grossman SA. Controversies in the adjuvant therapy of high- grade gliomas. Oncologist. 2011;16(3):351–8. https://doi.org/10.1634/ theoncologist.2010-0335. In conclusion, long-term adjuvant TMZ chemotherapy was beneficial for PFS and 2-year survival rate in GBM patients, and improved their quality of life contemporar- ily. But OS was not significantly improved. g 7. Hau P, Koch D, Hundsberger T, Marg E, Bauer B, Rudolph R, et al. Safety and feasibility of long-term temozolomide treatment in patients with 7. Hau P, Koch D, Hundsberger T, Marg E, Bauer B, Rudolph R, et al. Safety and feasibility of long-term temozolomide treatment in patients with Huang et al. BMC Neurol (2021) 21:424 Huang et al. 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https://openalex.org/W3152989031	https://aclanthology.org/2021.eacl-main.315.pdf	English	null	“Killing Me” Is Not a Spoiler: Spoiler Detection Model using Graph Neural Networks with Dependency Relation-Aware Attention Mechanism	null	2,021	cc-by	3,311	Abstract Several machine learning-based spoiler detec- tion models have been proposed recently to protect users from spoilers on review websites. Although dependency relations between con- text words are important for detecting spoilers, current attention-based spoiler detection mod- els are insufﬁcient for utilizing dependency re- lations. To address this problem, we propose a new spoiler detection model called SDGNN that is based on syntax-aware graph neural networks. In the experiments on two real- world benchmark datasets, we show that our SDGNN outperforms the existing spoiler de- tection models. Figure 1: Attention-based models focused on the word “killing” because the word is frequently used in spoiler sentences, which results in incorrect predictions. review documents achieve state-of-the-art perfor- mance on the SD task (Chang et al., 2018; Wan et al., 2019). However, the attention-based SD models have a lack of using dependency relations between con- text words. Dependency relations are useful for capturing the semantics of given sentences and de- tecting spoilers. As shown in Figure 1, although the phrase “killing me” is not a spoiler because the phrase is a metaphor, the attention-based SD models often focus on the word “killing” and clas- sify sentences that contain the phrase “killing me” as spoilers. By providing the information that the word “me” is used as the direct object of the verb “killing,” SD models can understand that the phrase is a metaphor. Proceedings of the 16th Conference of the European Chapter of the Association for Computational Linguistics, pages 3613–3617 April 19 - 23, 2021. ©2021 Association for Computational Linguistics †This work was done while the author was afﬁliated with Korea University. Buru Chang† Inggeol Lee‡ Hyunjae Kim‡ Jaewoo Kang‡∗ Hyperconnect† Korea University‡ buru@hpcnt.com {ingulbull,hyunjae-kim,kangj}@korea.ac.kr Theresa killing Thomas plans on it killing me is but I think I will forgive Label O X Predicted O O pcomp dobj nsubj dobj aux Figure 1: Attention-based models focused on the word “killing” because the word is frequently used in spoiler sentences, which results in incorrect predictions. ∗Corresponding author. † 1 Introduction Spoilers on review websites, which reveal criti- cal details of the original works, can ruin an ap- preciation for the works. Review websites, such as Rotten Tomato, IMDb, and Metacritic, provide self-reporting systems that tag spoiler information to warn users of spoilers. However, since self- reporting systems depend solely on the active par- ticipation of users, they cannot handle the fast- growing volume of newly generated reviews. Dur- ing the past decade, several machine learning-based spoiler detection (SD) models have been proposed to solve the inefﬁciency of self-reporting systems. Guo and Ramakrishnan (2010) proposed an auto- matic SD model that measures the similarity be- tween reviews and synopses of movies. Support vector machine (SVM)-based SD models using handcrafted features have been proposed (Boyd- Graber et al., 2013; Jeon et al., 2016). Recently, attention-based SD models that utilize metadata of In this paper, we propose SDGNN, which is a new Spoiler Detection model based on syntax- aware Graph Neural Networks (GNNs) (Marcheg- giani and Titov, 2017) for leveraging dependency relations between context words in sentences to fully capture the semantics. With the success of GNNs We also propose a dependency relation- aware attention mechanism, which is a modiﬁ- cation of the gating mechanism used by syntax- aware GNNs, to be suitable for the spoiler detection task. In SD, considering the relative importance of dependency relations. However, existing syntax- aware GNN-based models compute the importance of each dependency relation individually in sen- tences without considering the context of the given 3613 where W(k) ∈Rd×d and b(k) ∈Rd are the weight matrix and bias term, respectively, for the k-th layer. f is a non-linear function. h(0) is the outputs of the bi-directional LSTMs in the previous stage. sentence. Our proposed dependency relation-aware attention mechanism considers the relative impor- tance of dependency relations. Also, we adopt a previously proposed genre-aware pooling method (Chang et al., 2018) to utilize the genre of works efﬁciently. In the experiments, we demonstrate the effectiveness of SDGNN on two real-world bench- mark datasets in both quantitative and qualitative ways. There are two main differences between our pro- posed dependency-aware attention mechanism and the gating mechanism used by syntax-aware GNNS. First, the dependency-aware attention mechanism employs the softmax function to capture the rel- ative importance of dependency relations, while the gating mechanism computes the scalar weights by the inner-product of latent features of words and dependency relations. 2 Our Approach SDGNN classiﬁes whether a given sentence x = (w1, w2, · · · , wn) is a spoiler sentence. SDGNN consists of three stages: contextualized word rep- resentation, dependency relation-aware attention mechanism, and genre-aware pooling. Contextualized Word Representation Each word w in the given sentence x is represented with the pretrained word embedding vector (Pennington et al., 2014). We then utilize bi-directional LSTMs (Hochreiter and Schmidhuber, 1997) to encode con- textualized word representations h(0) ∈Rd. Dependency Relation-Aware Attention Mecha- nism While the gating mechanism in syntax- aware GNNs (Marcheggiani and Titov, 2017; Nguyen and Grishman, 2018) computes the scalar weight of each dependency relation, it does not consider the relative importance of dependency relations, which varies depending on the context of the given sentence. We present a dependency relation-aware attention mechanism that considers the relative importance of dependency relations in the given sentence. The relation-aware attention weights are computed as follows: Genre-Aware Pooling Genre information is use- ful for detecting spoilers. To leverage genre infor- mation, we employ a genre-aware pooling method following Chang et al. (2018). The genre-aware pooling computes the attention weights between the latent features of words and a genre feature captured from genre information of works. We then obtain a latent feature vector x for the given sentence x. Optimization We compute the spoiler probabil- ity ˆy of the given sentence x with the following the linear transformation: a(k) L(u,v) = g h(k) u W (k)e(k) L(u,v) + b (k) L(u,v) , (1) ˆa(k) L(u,v) = exp a(k) L(u,v) P v′∈N (u) exp a(k) L(u,v′) , (2) (1) ˆy = σ(wx + b), (4) (2) ˆy = σ(wx + b), (4) (2) (4) where w and b are trainable parameters, and σ is a sigmoid function. We use the weighted binary cross entropy (Wan et al., 2019) as the loss function. where ˆak L(u,v) is a scalar attention weight of the dependency relation label L(u, v) of the edge be- tween word nodes u, v. g is the non-linear function and exp(·) is an exponential function. W (k) ∈ Rd×d is the attention weight matrix for the k-th layer. e(k) L(u,v) ∈Rd and b k L(u,v) ∈R are latent features of the dependency relation L(u, v). 1 Introduction Second, the gating mechanism utilizes only three dependency rela- tions (forward, backward, and self) because of the over-parameterization issue. On the other hand, our proposed dependency relation-aware at- tention mechanism utilizes all the 82 types of de- pendency relations without suffering from the over- parameterization issue since the weight matrix in Equation 3 does not depend on the number of re- lations. The number of trainable parameters of SDGNN is proportionate to d2 while that of syntax- aware GNNs is proportionate to \|L\| · d2, where \|L\| is the number of relations. 1We obtained the datasets from Wan et al. (2019) and Boyd-Graber et al. (2013), respectively. 3.1 Experimental Setup Datasets We evaluated our proposed model on the two public spoiler datasets: Goodreads (Wan et al., 2019) and TVTropes (Boyd-Graber et al., 2013) 1. The Goodreads dataset consists of spoiler sentences on book reviews, and only 3.22% of en- tire sentences are labeled as spoiler sentences. The TVTropes dataset consists of descriptions of 884 TV programs from the TVTropes site, and 52.7% of the descriptions are labeled as spoilers. The statistics of the datasets are summarized in Table 1. Table 3: Evaluation results on the Goodreads dataset. size of 1024 was used for training TextCNN, HAN, and DNSD, and a batch size of 512 was used for training SpoilerNet and SDGNN. To prevent over- ﬁtting, we applied L2-normalization with λ = 1e-5 and a dropout rate of 0.5. For TextCNN, we used 50 ﬁlters with kernel sizes of 3, 4, and 5. For efﬁcient training on deep learning libraries, SDGNN set the maximum number of words to 50. For SDGNN, we used Leaky ReLU for the non-linear function g, and ReLU for f. We set k = 2 for SyntacticGCN, C-GCN, and SDGNN. We use η = 0.05 for the Goodreads dataset, which is unbalanced. Baseline Models We compared our proposed model with the following state-of-the-art SD mod- els: SVM (Boyd-Graber et al., 2013; Jeon et al., 2016), CNN (Kim, 2014), HAN (Yang et al., 2016), SpoilerNet (Wan et al., 2019) and DNSD (Chang et al., 2018). Note that the implementation de- tails about our experiments are described in the Appendix due to space limitations. 2 Our Approach L = −1 \|D\| X xi∈D (yilog(ˆyi) + η · (1 −yi)log(1 −ˆyi)), (5) (5) where y id the ground truth of spoiler information and D indicates the dataset. η is a hyperpameter used to balance the number of spoiler and non- spoiler labels in the training data. All the trainable parameters of SDGNN are updated by minimizing the loss function with gradient descent. Finally, we aggregate the latent feature for each node u as follows: h(k) u = f X v∈N (u) ˆa(k) L(u,v)W(k)h(k−1) v + b(k) , (3) (3) 3614 Statistics Goodreads TVTropes # of Training Sentences 14,007,593 11,970 # of Validation Sentences 128,718 2,808 # of Test Sentences 3,536,341 1,477 # of Edge Types 82 82 # of Genre 542 30 Avg. # of Nodes per Sentence 17.7 21.03 Avg. # of Edges per Sentence 33.4 40.06 Avg. # of Genre per Sentence 4.95 2.40 Table 1: Statistics of the datasets. Models Goodreads TVTropes AUROC F1 AUROC F1 SVM 0.880 0.162 0.735 0.698 TextCNN 0.904 0.188 0.779 0.738 HAN 0.915 0.190 0.785 0.750 SpoilerNet 0.924 0.194 0.808 0.768 DNSD 0.928 0.199 0.818 0.788 SDGNN 0.938 0.210 0.828 0.801 Table 2: Evaluation results on two benchmark datasets. The best results are highlighted in bold. Table 2: Evaluation results on two benchmark datasets. The best results are highlighted in bold. Table 1: Statistics of the datasets. Table 1: Statistics of the datasets. Models Goodreads AUROC F1 SytacticGCN 0.933 0.204 C-GCN 0.923 0.193 SDGNN 0.938 0.210 Table 3: Evaluation results on the Goodreads dataset. 3.2 Results The experimental results are summarized in Table 2. Evaluation results show that our proposed SDGNN outperforms all the baseline models including attention-based models. This result demonstrates that our proposed dependency relation-aware at- tention mechanism contributes to improving SD performance. Metrics We use Area Under the Receiver Oper- ating Characteristics curve (AUROC) used in Wan et al. (2019) as an evaluation metric. We also use an F1 score following Chang et al. (2018). Implemention Details We trained and evaluated the models on two TITAN X (Pascal) GPUs. We implemented SDGNN using PyTorch v1.1. We used Stanford CoreNLP (Manning et al., 2014) to generate dependency parse trees. We employed GloVe (Pennington et al., 2014) to represent word vectors in neural network-based models including SDGNN. Using the validation set and grid search, we searched optimal hyper-parameters for each SD model. All the neural network-based models were trained with the learning rate of 0.001 and the Adam optimizer (Kingma and Ba, 2014). A batch 4.1 Analysis of Relative Importance To further demonstrate the usefulness of the relative importance of dependency relations, we conducted quantitative and qualitative analysis. Quantitative We compared SDGNN with the more syntax-aware GNN-based models, Syntac- ticGCN (Marcheggiani and Titov, 2017) and C- GCN (Zhang et al., 2018). We trained and eval- uated the models on the Goodreads dataset. We 3615 Models Genres Sentences Prediction Label DNSD Romance it ’s killing me but i think i ’ll forgive him no matter Positive Negative what he did or did n’t do . Fantasy the villains are decidedly vicious and in some cases insane . Positive Negative SDGNN Romance it ’s killing me but i think i ’ll forgive him no matter Negative Negative what he did or did n’t do . Fantasy the villains are decidedly vicious and in some cases insane . Negative Negative Table 4: Visualization of attention scores from DNSD, SpoilerNet, and SDGNN on test data. Models Genres Sentences Prediction Label DNSD Romance it ’s killing me but i think i ’ll forgive him no matter Positive Negative what he did or did n’t do . Fantasy the villains are decidedly vicious and in some cases insane . Positive Negative SDGNN Romance it ’s killing me but i think i ’ll forgive him no matter Negative Negative what he did or did n’t do . Fantasy the villains are decidedly vicious and in some cases insane . Negative Negative Table 4: Visualization of attention scores from DNSD, SpoilerNet, and SDGNN on test data. Table 4: Visualization of attention scores from DNSD, SpoilerNet, and SDGNN on test data. will Mara eventually Noah kill reunited ending guards viciously mother kill Figure 2: Partial graphs of dependency parse trees with dependency relation-aware attention weights. is a metaphor. In this case, DNSD failed to cor- rectly classify the sentence since DNSD cannot fully capture the semantics of the sentence. On the other hand, SDGNN focused on not only the word “killing” but also on the word “me” and classiﬁed the sentence correctly since SDGNN employs the dependency relation (dobj) between the word “me” and the word “killing”. reunited ending Figure 2: Partial graphs of dependency parse trees with dependency relation-aware attention weights. utilize contextualized word representations and the genre-aware pooling method to SyntacticGCN and C-GCN. The evaluation results are summarized in Table 3. Our proposed SDGNN outperformed Syn- tacticGCN and C-GCN. 4.3 Discussion Qualitative In Figure 2, the attention weights of the adverbial modiﬁer (advmod) linked to the words “eventually” and “viciously” are high, which indicates that adverbial modiﬁers frequently can be important hints for detecting spoilers. In the right partial graph, the attention weight of the (dobj) is relatively higher than that in the left partial graph. Since the word “mother” is not typically used as the object of the word “kill” in the original works, the phrase “kill mother” is a critical hint in detect- ing spoilers, and SDGNN effectively captures the phrase. Dependency Parsing on User-Generated Texts The spoiler datasets are user-generated texts, which are intrinsically noisy. To examine the inﬂuence of noises on dependency parsing results and the per- formance of SDGNN, we sampled 100 sentences from Goodreads. We manually classiﬁed whether the sentences are noise or not, and 28 of 100 sen- tences were classiﬁed as noisy sentences. Depen- dency parsing results on well-structured sentences seem good, but dependency parsing results on noisy sentences are poor. However, there is no signiﬁ- cant gap in performance. SDGNN achieved 85.7% accuracy on noisy sentences and 87.5% accuracy on well-structured sentences. Since our proposed dependency relation-aware attention mechanism of SDGNN ﬁlters noisy information, SDGNN could detect spoilers even on noisy sentences. 4.1 Analysis of Relative Importance This result demonstrates that our proposed attention mechanism is effective by considering the relative importance of depen- dency relations. Although SDGNN signiﬁcantly re- duced the number of parameters, SDGNN achieved better results compared to SyntacticGCN and C- GCN. The second sentence is a non-spoiler because it is obvious that villains are vicious in most original works. DNSD classiﬁed the sentence as a spoiler because the model solely focused on individual words such as “villains”, “vicious”, and “insane”, rather than the understanding of the overall seman- tics of the sentence. On the other hand, SDGNN classiﬁed the sentence correctly as the word “are” is used to describe characters in many cases, and SDGNN understands the semantics of the sentence. 4.2 Case Study We sample several sentences from the test set of the Goodreads dataset to explore how the models detect spoilers. Table 4 shows the visualization of attention scores in the pooling layer obtained by DNSD and SDGNN, respectively. The ﬁrst sen- tence contains the verb “killing,” but it is not a spoiler sentence because the phrase “killing me” Subjectivity in Judging Spoilers Since judging a sentence as a spoiler is a subjective task, label inconsistency occurs in spoiler datasets crawled 3616 linguistic cues. In Proceedings of the 23rd Inter- national Conference on Computational Linguistics, pages 412–420. Association for Computational Lin- guistics. from self-reporting systems. Guo and Ramakrish- nan (2010) found that 23% of the labels of their manually labeled data is different from the original labels of IMDb reviews. One of the ways to miti- gate label inconsistency is to solidify the deﬁnition of a spoiler. Although the TV Tropes site deﬁnes spoilers, efforts should be made for a more rigorous and linguistic deﬁnition in future studies. Another possible way is to employ reviewers’ information in detecting spoilers. Reviewer biases of SpoilerNet can alleviate label inconsistency between users. Sepp Hochreiter and J¨urgen Schmidhuber. 1997. Long short-term memory. Neural computation, 9(8):1735–1780. Sungho Jeon, Sungchul Kim, and Hwanjo Yu. 2016. Spoiler detection in tv program tweets. Information Sciences, 329:220–235. Yoon Kim. 2014. Convolutional neural net- works for sentence classiﬁcation. arXiv preprint arXiv:1408.5882. Acknowledgments This research was supported by National Research Foundation of Korea (NRF-2020R1A2C3010638). This research was also supported by the MSIT (Ministry of Science and ICT), Korea, under the ICT Creative Consilience program (IITP-2020-0- 01819) supervised by the IITP (Institute for Infor- mation & communications Technology Planning & Evaluation). Jeffrey Pennington, Richard Socher, and Christopher Manning. 2014. Glove: Global vectors for word rep- resentation. In Proceedings of the 2014 conference on empirical methods in natural language process- ing (EMNLP), pages 1532–1543. Mengting Wan, Rishabh Misra, Ndapa Nakashole, and Julian McAuley. 2019. Fine-grained spoiler detec- tion from large-scale review corpora. arXiv preprint arXiv:1905.13416. 5 Conclusion Diederik P Kingma and Jimmy Ba. 2014. Adam: A method for stochastic optimization. arXiv preprint arXiv:1412.6980. In this paper, we proposed a novel spoiler detection model called SDGNN which is based on syntax- aware GNNs that utilize dependency relations be- tween context words. We also proposed a depen- dency relation-aware attention mechanism for con- sidering the relative importance of dependency re- lations. In the experiments, our proposed SDGNN model achieved the state-of-the-art performance on two spoiler datasets. Our experimental results demonstrate the effectiveness of dependency rela- tions in the spoiler detection task and our depen- dency relation-aware attention mechanism. Christopher Manning, Mihai Surdeanu, John Bauer, Jenny Finkel, Steven Bethard, and David McClosky. 2014. The stanford corenlp natural language pro- cessing toolkit. In Proceedings of 52nd annual meet- ing of the association for computational linguistics: system demonstrations, pages 55–60. Diego Marcheggiani and Ivan Titov. 2017. En- coding sentences with graph convolutional net- works for semantic role labeling. arXiv preprint arXiv:1703.04826. Thien Huu Nguyen and Ralph Grishman. 2018. Graph convolutional networks with argument-aware pool- ing for event detection. In Thirty-Second AAAI Con- ference on Artiﬁcial Intelligence. References Zichao Yang, Diyi Yang, Chris Dyer, Xiaodong He, Alex Smola, and Eduard Hovy. 2016. Hierarchi- cal attention networks for document classiﬁcation. In Proceedings of the 2016 conference of the North American chapter of the association for computa- tional linguistics: human language technologies, pages 1480–1489. Jordan Boyd-Graber, Kimberly Glasgow, and Jackie Sauter Zajac. 2013. Spoiler alert: Ma- chine learning approaches to detect social media posts with revelatory information. In Proceedings of the 76th ASIS&T Annual Meeting: Beyond the Cloud: Rethinking Information Boundaries, page 45. American Society for Information Science. Yuhao Zhang, Peng Qi, and Christopher D Manning. 2018. Graph convolution over pruned dependency trees improves relation extraction. arXiv preprint arXiv:1809.10185. Buru Chang, Hyunjae Kim, Raehyun Kim, Deahan Kim, and Jaewoo Kang. 2018. A deep neural spoiler detection model using a genre-aware atten- tion mechanism. In Paciﬁc-Asia Conference on Knowledge Discovery and Data Mining, pages 183– 195. Springer. Sheng Guo and Naren Ramakrishnan. 2010. Find- ing the storyteller: automatic spoiler tagging using 3617
https://openalex.org/W2806457395	https://www.nature.com/articles/s41598-018-25651-x.pdf	English	null	Tissue distribution and cell tropism of Brucella canis in naturally infected canine foetuses and neonates	Scientific reports	2,018	cc-by	8,274	Tissue distribution and cell tropism of Brucella canis in naturally infected canine foetuses and neonates canis infection occurred in owners of dogs with no history of abortion, and these patients presented unspecific symptoms when compared to those of patients infected with other Brucella spedies, which may lead to the lack of clinical suspicion and diagnostic failure4–8. Although these case reports have not been characterized by a labour-related transmis- sion, human infection with B. canis is also considered an occupational underdiagnosed disease9. B. canis differs from other Brucella species in its pathogenicity and preferential host, affecting mainly dogs, and, occasionally, humans and wild canids2,3. Antigenically, B. canis is classified as a rough Brucella due to its outer membrane lipopolysaccharide that characterizes a rough surface on colonies grown for more than 48 hours10. Rough lipopolysaccharide, also naturally expressed in B. ovis, is associated with impaired intracellular survival in vitro, and induction of lower levels of cytokine expression in human monocytes11,12. Furthermore, B. canis does not cross-react in traditional serological test antigens from smooth species, namely B. melitensis, B. abortus, 1Departamento de Clínica e Cirurgia Veterinárias, Escola de Veterinária, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil. 2Curso de Medicina Veterinária, Universidade Vila Velha, Vila Velha, ES, Brazil. 3Departamento de Patologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil. Correspondence and requests for materials should be addressed to R.L.S. (email: rsantos@vet.ufmg.br) Brucella canis is a zoonotic coccobacillus described for the first time as a cause of canine abortion in 19661. Since then, B. canis has been recognized as the etiologic agent of reproductive disorders and, less frequently, of dis- cospondylitis and ocular infection in dogs2. Although B. canis is recognized as an important bacterial zoonosis, human brucellosis is mainly related to B. melitensis, B. abortus, and B. suis, while brucellosis due to B. canis infec- tion in humans has historically been considered less relevant3. Zoonotic infection may occur mainly in humans in close contact with bitches that aborted. However, several cases of human brucellosis due to B. canis infection occurred in owners of dogs with no history of abortion, and these patients presented unspecific symptoms when compared to those of patients infected with other Brucella spedies, which may lead to the lack of clinical suspicion and diagnostic failure4–8. Although these case reports have not been characterized by a labour-related transmis- sion, human infection with B. canis is also considered an occupational underdiagnosed disease9.ff B. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Tissue distribution and cell tropism of Brucella canis in naturally infected canine foetuses and neonates Received: 19 January 2018 Accepted: 24 April 2018 Published: xx xx xxxx Tayse Domingues de Souza1,2, Tatiane Furtado de Carvalho1, Juliana Pinto da Silva Mol1, João Vítor Menezes Lopes2, Monique Ferreira Silva3, Tatiane Alves da Paixão3 & Renato Lima Santos 1 Brucella canis infection is an underdiagnosed zoonotic disease. Knowledge about perinatal brucellosis in dogs is extremely limited, although foetuses and neonates are under risk of infection due to vertical transmission. In this study, immunohistochemistry was used to determine tissue distribution and cell tropism of B. canis in canine foetuses and neonates. Diagnosis of B. canis in tissues of naturally infected pups was based on PCR and sequencing of amplicons, bacterial isolation, and immunohistochemistry, whose specificity was confirmed by laser capture microdissection. PCR positivity among 200 puppies was 21%, and nine isolates of B. canis were obtained. Tissues from 13 PCR-positive puppies (4 stillborn and 9 neonates) presented widespread immunolabeling. Stomach, intestines, kidney, nervous system, and umbilicus were positive in all animals tested. Other frequently infected organs included the liver (92%), lungs (85%), lymph nodes (69%), and spleen (62%). Immunolabeled coccobacilli occurred mostly in macrophages, but they were also observed in erythrocytes, epithelial cells of gastrointestinal mucosa, renal tubules, epidermis, adipocytes, choroid plexus, ependyma, neuroblasts, blood vessels endothelium, muscle cells, and in the intestinal lumen. These results largely expand our knowledge about perinatal brucellosis in the dog, clearly demonstrating a pantropic distribution of B. canis in naturally infected foetuses and neonates. 1 SCIEntIFIC ReporTS \| (2018) 8:7203 \| DOI:10.1038/s41598-018-25651-x mucosa, renal tubules, epidermis, adipocytes, choroid plexus, ependyma, neuroblasts, blood vessels endothelium, muscle cells, and in the intestinal lumen. These results largely expand our knowledge about perinatal brucellosis in the dog, clearly demonstrating a pantropic distribution of B. canis in naturally infected foetuses and neonates. Brucella canis is a zoonotic coccobacillus described for the first time as a cause of canine abortion in 19661. Since then, B. canis has been recognized as the etiologic agent of reproductive disorders and, less frequently, of dis- cospondylitis and ocular infection in dogs2. Although B. canis is recognized as an important bacterial zoonosis, human brucellosis is mainly related to B. melitensis, B. abortus, and B. suis, while brucellosis due to B. canis infec- tion in humans has historically been considered less relevant3. Zoonotic infection may occur mainly in humans in close contact with bitches that aborted. However, several cases of human brucellosis due to B. Tissue distribution and cell tropism of Brucella canis in naturally infected canine foetuses and neonates Brucella-positive puppies, their breed, kennel, age, bacterial isolates origin, and positivity by immunohistochemistry in organs and tissues. Abbreviations: GIT: Gastrointestinal tract; LN: Lymph node; CNS: Central nervous system; NE: Negative; NA: Not available to be tested. + = only one positive mark in ten fields (400X) or more; ++ = at least one positive mark in every two fields; +++ = more than two positive marks in more than ten fields. Table 1. Brucella-positive puppies, their breed, kennel, age, bacterial isolates origin, and positivity by immunohistochemistry in organs and tissues. Abbreviations: GIT: Gastrointestinal tract; LN: Lymph node; CNS: Central nervous system; NE: Negative; NA: Not available to be tested. + = only one positive mark in ten fields (400X) or more; ++ = at least one positive mark in every two fields; +++ = more than two positive marks in more than ten fields. and B. suis13. The lack of cross-reactivity between smooth and rough Brucella antigens in serology contributes to diagnostic failure and under diagnosis of B. canis human infections5,9.l B. canis induces less inflammation and more insidious lesions when compared to other Brucella spp.14, but its ability to persist in the host, disseminate and perpetuate in dog population, is demonstrated by its prevalence in many countries15,16, and the difficulty for clearing the pathogen from infected dogs17 and eradicating it in ken- nels18–20. Diagnosis of B. canis infection remains a challenge due to frequent false negative results in direct and indirect diagnostic methods employed to detect the infection in adult dogs and humans6,21,22.f g p y g Despite the relevance of canine brucellosis as a reproductive disease that is vertically transmitted to the off- spring2,19,23, and the high canine perinatal mortality rates of unknown cause in breeding kennels24,25, canine per- inatal brucellosis remains neglected worldwide. One of the reasons for this situation is that B. canis infection is suspected mostly in cases of abortion, but not in cases of perinatal mortality1,10,15. p y p y Cellular and tissue tropism of B. canis in its natural preferential host remains poorly understood. It has been assessed based on isolation and histopathology10,26, but not through in situ localization of the bacteria. In canine foetal brucellosis, bronchopneumonia, myocarditis, and renal haemorrhage have been described in an exper- imental study26, while neonatal canine brucellosis remains to be described. Tissue distribution and cell tropism of Brucella canis in naturally infected canine foetuses and neonates canis differs from other Brucella species in its pathogenicity and preferential host, affecting mainly dogs, and, occasionally, humans and wild canids2,3. Antigenically, B. canis is classified as a rough Brucella due to its outer membrane lipopolysaccharide that characterizes a rough surface on colonies grown for more than 48 hours10. Rough lipopolysaccharide, also naturally expressed in B. ovis, is associated with impaired intracellular survival in vitro, and induction of lower levels of cytokine expression in human monocytes11,12. Furthermore, B. canis does not cross-react in traditional serological test antigens from smooth species, namely B. melitensis, B. abortus, 1Departamento de Clínica e Cirurgia Veterinárias, Escola de Veterinária, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil. 2Curso de Medicina Veterinária, Universidade Vila Velha, Vila Velha, ES, Brazil. 3Departamento de Patologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil. Correspondence and requests for materials should be addressed to R.L.S. (email: rsantos@vet.ufmg.br) SCIEntIFIC ReporTS \| (2018) 8:7203 \| DOI:10.1038/s41598-018-25651-x 1 www.nature.com/scientificreports/ Puppy Breed Kennel Puppy age at death Isol. Tissue distribution and cell tropism of Brucella canis in naturally infected canine foetuses and neonates Therefore, canine brucellosis is a disease that has been neglected both in man and in the dog, possibly due to its stealthy behaviour, difficulty for diagnosis, and limited knowledge about B. canis infection, treatment, and eradication. A better understanding of canine brucellosis in foetuses and neonates will contribute for improving diagnosis and prevention of this disease in dogs and humans. The aim of this study was to describe cell and tissue tropism of B. canis in naturally infected stillborn and neonatal dogs, which generated a novel perspective on B. canis pathogenesis, with implications regarding the persistence of the pathogen and the risk of zoonotic transmission. Tissue distribution and cell tropism of Brucella canis in naturally infected canine foetuses and neonates GIT Heart Lung Spleen Liver Kidney Urinary bladder LN Adipose tissue Blood Genital Umbilical Cord CNS Eye 1 (127) German Spitz A Stillborn NE +++ ++ ++ + +++ + + − − ++ + +++ NA NA 2 (143) German Spitz B Stillborn Placenta +++ ++ +++ + + + ++ + + ++ ++ +++ NA NA 3 (30) Border Collie C Stillborn NE ++ +++ − +++ ++ ++ + − ++ +++ − + ++ NA 4 (33) Brazilian Mastiff D Stillborn Placenta Kidney + +++ − − + +++ + − + − ++ +++ NA ++ 5 (53) Miniature Pinscher E 1 day Lung Heart ++ + ++ ++ + + + + + − + +++ NA NA 6 (51) Miniature Pinscher E 2 days Kidney Spleen ++ − ++ + +++ ++ + + − − ++ +++ ++ NA 7 (16) Miniature Poodle E 2 days NE +++ + ++ − + + − − + ++ − + ++ − 8 (156) Rottweiler F 2 days NE +++ + + + ++ + + + + + NA ++ NA NA 9 (185) English Bulldog G 2 days NE +++ + +++ − + ++ ++ + − − NA ++ NA NA 10 (90) Miniature Pinscher E 3 days NE +++ + +++ +++ +++ ++ +++ +++ ++ ++ +++ +++ ++ ++ 11 (50) German Spitz H 4 days NE + + +++ − ++ ++ + ++ + ++ + NA NA NA 12 (111) Yorkshire Terrier I 10 days Spleen ++ ++ ++ + − ++ + + ++ − − NA NA NA 13 (112) German Spitz A 13 days Heart +++ − +++ − + ++ NA + ++ − ++ NA NA NA % Positive 100 85 85 62 92 100 92 69 77 54 73 100 100 67 Table 1. Brucella-positive puppies, their breed, kennel, age, bacterial isolates origin, and positivity by immunohistochemistry in organs and tissues. Abbreviations: GIT: Gastrointestinal tract; LN: Lymph node; CNS: Central nervous system; NE: Negative; NA: Not available to be tested. + = only one positive mark in ten fields (400X) or more; ++ = at least one positive mark in every two fields; +++ = more than two positive marks in more than ten fields. Table 1. Results PCR PCR, sequencing, and Brucella sp. isolation. A total of 42 puppies out of the 200 (21%) that were tested, from 16 kennels, were PCR-positive. Amplicon sequences had 100% identity with Brucella spp. (GenBank access number: M20404.1). PCR, sequencing, and Brucella sp. isolation. A total of 42 puppies out of the 200 (21%) that were tested, from 16 kennels, were PCR-positive. Amplicon sequences had 100% identity with Brucella spp. (GenBank access number: M20404.1). B. canis was isolated from tissues of six out the 10 puppies subjected to bacterial isolation, totalling 42 tissue samples. From these six puppies, nine isolates were obtained from placenta (n = 2), spleen (n = 2), kidney (n = 2), heart (n = 2), and lung (n = 1). Isolates and their origins are listed in Table 1. Pantropic distribution of Brucella canis in foetal and neonatal canine tissues. Immunolabeled coccobacilli were widely distributed in several organs and tissues from all 13 Brucella PCR-positive puppies (Table 1). Organs with the highest frequency of positivity (100%) included the stomach, intestine, kidney, and umbilicus, which contained numerous intracytoplasmic immunolabeled coccobacilli located mostly within mac- rophages (Fig. 1). Macrophages were also positive in the hepatic parenchyma as well as in portal spaces and around central lobular veins (12/13), in lungs (11/13), and heart (11/13). Reproductive organs, including the gonads, uterus and prostate, although pre-pubertal and in a very young age, were frequently positive (8/11), with immunolabeling mostly in macrophages, in the albuginea (Fig. 1), prostate, myometrium, uterine broad liga- ment. Spleen and lymph nodes, positive in 62–69% of the puppies, most of the time presented low intensity (−or SCIEntIFIC ReporTS \| (2018) 8:7203 \| DOI:10.1038/s41598-018-25651-x 2 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 1. Immunohistochemistry for detection of Brucella sp. in naturally infected canine stillborn and neonates. (a) Stillborn. Duodenum, with several diffusely distributed immunolabeled macrophages in the lamina propria and submucosa (arrow). Bar = 100 μm. (b) Stillborn. Cecum, macrophages containing intracytoplasmic immunolabeled coccobacilli in lamina propria and muscle layer (arrow). Bar = 100 μm. (c) Neonate, 10-day-old. Lung, with several interstitial immunostained macrophages around an arteriole and bronchioles (arrow). Bar = 50 μm. (d) Neonate, 13-day-old. Testicle with several immunostained macrophages in the albuginea (arrow). Bar = 100 μm. Figure 1. Immunohistochemistry for detection of Brucella sp. in naturally infected canine stillborn and neonates. (a) Stillborn. Duodenum, with several diffusely distributed immunolabeled macrophages in the lamina propria and submucosa (arrow). Results PCR Bar = 100 μm. (b) Stillborn. Cecum, macrophages containing intracytoplasmic immunolabeled coccobacilli in lamina propria and muscle layer (arrow). Bar = 100 μm. (c) Neonate, 10-day-old. Lung, with several interstitial immunostained macrophages around an arteriole and bronchioles (arrow). Bar = 50 μm. (d) Neonate, 13-day-old. Testicle with several immunostained macrophages in the albuginea (arrow). Bar = 100 μm. +, 10/13; − or +11/13), respectively. In these tissues, positive macrophages often had perivascular localization Fig. 1), and infiltrated the wall of arteries (pulmonary, umbilical, coronary, and hepatic).hf i There was also positive coccoid staining in intravascular erythrocytes (7/13) in different organs, including the lungs, genital organs, kidneys, liver, heart chambers, and cerebrum (Fig. 2). In addition to macrophages and erythrocytes, positivity was also observed in other cell types in different organs, as in superficial epithelium of gastric mucosa, in enterocytes, and in goblet cells in the small and large intestines (Fig. 3). Meconium was positive in 75% of stillborn puppies while only 33% of neonates had immunolabeled coccobacilli in the intestinal luminal content, with a lower intensity of immunolabeling (+, 3/9). The gastrointestinal mucosa was intensely positive in all puppies, although immunolabeled coccobacilli were observed mostly intracytoplasmically in macrophages diffusely distributed in the lamina propria, submucosa, muscular layer, mesentery and gastro-splenic ligament, in stillborn and neonates. Only two puppies had low intensity of immunolabeling in stomach and intestine: one premature stillborn and one four-day-old premature neonate who did not nurse in the bitch and was fed artificial milk formula by orogastric tube due to palatoschisis. In five puppies, immature adipocytes in the mesenteric fat contained immunolabeled coccobacilli in the cytoplasm (Fig. 3).i y p g In five puppies, three stillborn and two neonates, Brucella was observed intracytoplasmically in the epithelial cells of proximal and distal convoluted tubules, Henle loops, and collecting tubules (Fig. 3). Immunolabeled coc- cobacilli occurred also intracytoplasmically in adipocytes in perirenal fat (++, 2/13). Occasionally, intracytoplasmic immunolabeled coccobacilli were present in smooth muscle cells in umbili- cal, pulmonary and coronary arteries, where few or no macrophages and neutrophils were present (Fig. 4). The same finding was observed in myocardium cells, with variable number of coccobacilli intracytoplasmically in the absence of inflammation or associated with rare interstitial macrophages and neutrophils (Fig. 3). l In the umbilical cord, immunostained coccobacilli occurred on the epidermis in the stratum corneum (Fig. SCIEntIFIC ReporTS \| (2018) 8:7203 \| DOI:10.1038/s41598-018-25651-x was no evidence of inflammatory reaction in haematoxylin and eosin-stained sections (Fig. 5), and those cells were interpreted as resident macrophages of the placenta. In the nervous tissue, there was positive staining intracytoplasmically in the choroid plexus (Fig. 6) (++ to +++, 3/4), ependyma (+ to ++, 3/4), neuroblasts (+ to +++, 2/4), and in macrophages in the meninges (+, Results PCR 4) in stillborn and neonates (+ to +++), and also intracytoplasmically in the apocrine gland epithelial cells (++) in only one sample, and in adipocytes in subcutaneous fat (+, 3/10), besides the positivity in macrophages and in arterial smooth muscle cells already mentioned above.h y There were only two placentas available for this study, and they were both positive by immunohistochemistry, with intracellularly immunolabeled B. canis predominantly in macrophages located in the foetal mesenchyme, and occasionally within trophoblasts (Fig. 5). Although, immunolabeling was observed in macrophages, there 3 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 2. Immunohistochemistry for detection of Brucella sp. in stillborn dogs. Erythrocytes in a blood vessel in the kidney with granular immunostaining compatible with intraerythrocytic B. canis (arrow). Inset: Blood in a cardiac chamber with moderate number of immunostained erythrocytes. Bar = 20 μm. Figure 2. Immunohistochemistry for detection of Brucella sp. in stillborn dogs. Erythrocytes in a blood vessel in the kidney with granular immunostaining compatible with intraerythrocytic B. canis (arrow). Inset: Blood in a cardiac chamber with moderate number of immunostained erythrocytes. Bar = 20 μm. Figure 3. Immunohistochemistry for detection of Brucella sp. in canine foetuses and neonates. (a) Neonate, 2-day-old. Ileum with numerous intracellular coccobacilli in goblet cells of the villus epithelium. Bar = 50 μm. (b) Stillborn. Immunostained adipocytes in the gastro-splenic ligament, with a granular pattern adjacent to lipid vacuoles. Bar = 20 μm. (c) Neonate, 2-day-old. Kidney with granular immunostaining in tubular epithelial cells and in tubular lumen. Bar = 50 μm. (d) Stillborn. Myocardium with intracellular granular immunostaining in myocardial cells. Bar = 20 μm. Figure 3. Immunohistochemistry for detection of Brucella sp. in canine foetuses and neonates. (a) Neonate, 2-day-old. Ileum with numerous intracellular coccobacilli in goblet cells of the villus epithelium. Bar = 50 μm. (b) Stillborn. Immunostained adipocytes in the gastro-splenic ligament, with a granular pattern adjacent to lipid vacuoles. Bar = 20 μm. (c) Neonate, 2-day-old. Kidney with granular immunostaining in tubular epithelial cells and in tubular lumen. Bar = 50 μm. (d) Stillborn. Myocardium with intracellular granular immunostaining in myocardial cells. Bar = 20 μm. was no evidence of inflammatory reaction in haematoxylin and eosin-stained sections (Fig. 5), and those cells were interpreted as resident macrophages of the placenta. In the nervous tissue, there was positive staining intracytoplasmically in the choroid plexus (Fig. Discussionh This study largely expanded our knowledge regarding the tissue and cell tropism of B. canis in canine foetuses and neonates, clearly demonstrating a previously unknown pantropic distribution of this pathogen in several organs and tissues. This is the first thorough description of B. canis tissue distribution and cell tropism in its preferential host, and therefore this study contributed to a better understanding of canine brucellosis. y g Abundant immunohistochemistry positivity in the gastrointestinal mucosa and in intestinal contents sug- gested that the oral route of infection may play an important role in canine perinatal brucellosis, possibly through ingestion of bacteria in suspension in the amniotic fluid. Carmichael and Kenney26 hypothesized that foetal infec- tion could occur through the oral route, based on their findings of high bacterial loads in the amniotic fluid and mild inflammation observed in the digestive system of canine foetuses. Furthermore, high positivity in the gastrointestinal mucosa was also observed in this study in one neonate at 12 days after birth, which may be due to transmammary transmission. Importantly, B. abortus endocytosis by M cells in the Peyer’s patches had been described after oral experimental infection of calves27 and in mice28, but the pathogen has not been observed within enterocytes and goblet cells. While the gastrointestinal mucosa and mesentery were highly positive in all puppies in this study, mesenteric lymph nodes were negative in most of the puppies, which is possibly due to the early stages of infection in these cases.h y g The ability of B. canis to infect renal tubular epithelial cells strongly suggested a previously unsuspected route of excretion of the organism. Transmission of B. canis has been attributed to prolonged contact with urine of an infected dog, but it was assumed that the organism originated from prostatic secretions of adult males29. Although the prostate gland in puppies was also positive, immunolabeled coccobacilli were located in interstitial macrophages, while in renal tubules they were in epithelial cells and in the lumen, indicating a source for urine contamination in the kidney itself. Zoonotic transmission to humans in close contact with young puppies has been documented5,8, and based on our findings, it may have occurred due to shedding of the organism in the urine, since in these cases abortion or vaginal secretions that are considered the conventional means of zoonotic transmission were absent23.hi This study has demonstrated for first time that B. Results PCR 6) (++ to +++, 3/4), ependyma (+ to ++, 3/4), neuroblasts (+ to +++, 2/4), and in macrophages in the meninges (+, SCIEntIFIC ReporTS \| (2018) 8:7203 \| DOI:10.1038/s41598-018-25651-x 4 www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 4. Immunohistochemistry for detection of Brucella sp. in the umbilical cord of a stillborn dog. (a) Skin adjacent to the umbilicus with immunolabeled coccobacilli in stratum corneum (arrow) and in macrophages in the dermis (arrow heads). Bar = 20 μm. (b) Arterial wall in the umbilical cord with intracellular immunostained coccobacilli in smooth muscle cells. Bar = 50 μm. Figure 4. Immunohistochemistry for detection of Brucella sp. in the umbilical cord of a stillborn dog. (a) Skin adjacent to the umbilicus with immunolabeled coccobacilli in stratum corneum (arrow) and in macrophages in the dermis (arrow heads). Bar = 20 μm. (b) Arterial wall in the umbilical cord with intracellular immunostained coccobacilli in smooth muscle cells. Bar = 50 μm. Figure 5. Immunohistochemistry for detection of Brucella sp. in the placenta. Section of the foetal placenta infected with Brucella canis, with absence of inflammatory reaction. Haematoxylin and eosin. Bar = 50 μm. Inset: intracellular immunostaining in a cell located in the mesenchyme with morphology compatible with a macrophage. Figure 5. Immunohistochemistry for detection of Brucella sp. in the placenta. Section of the foetal placenta infected with Brucella canis, with absence of inflammatory reaction. Haematoxylin and eosin. Bar = 50 μm. Inset: intracellular immunostaining in a cell located in the mesenchyme with morphology compatible with a macrophage. SCIEntIFIC ReporTS \| (2018) 8:7203 \| DOI:10.1038/s41598-018-25651-x 5 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 6. Immunohistochemistry for detection of Brucella sp. in the choroid plexus of a stillborn dog. Choroid plexus with intracytoplasmic immunostaining with a granular pattern in cells of the choroid plexus. Bar = 50 μm. Figure 6. Immunohistochemistry for detection of Brucella sp. in the choroid plexus of a stillborn dog. Choroid plexus with intracytoplasmic immunostaining with a granular pattern in cells of the choroid plexus. Bar = 50 μm. Figure 6. Immunohistochemistry for detection of Brucella sp. in the choroid plexus of a stillborn dog. Choroid plexus with intracytoplasmic immunostaining with a granular pattern in cells of the choroid plexus. Bar = 50 μm. 1/3). In the eye, there was a mild immunostaining in the cornea and retina (+, 2/3), while in the dermal surface of the eyelids there were numerous macrophages containing positive coccobacilli (++, 2/3). 1/3). Results PCR In the eye, there was a mild immunostaining in the cornea and retina (+, 2/3), while in the dermal surface of the eyelids there were numerous macrophages containing positive coccobacilli (++, 2/3). y p g g p Extracellular immunolabeled coccobacilli occurred rarely, in small numbers, in the lumen of renal tubules ntestinal content and epicellular on choroid plexus cells.i p p Considering the originality of these findings, immunolabeled epithelial cells of renal tubules were microdis- sected by using a laser capture microdissection apparatus. DNA extracted from these immunolabeled microdis- sected immunolabeled renal tubular epithelia was PCR-positive for Brucella sp., further confirming the specificity of immunolabeling. SCIEntIFIC ReporTS \| (2018) 8:7203 \| DOI:10.1038/s41598-018-25651-x Discussionh canis is associated with erythrocytes in naturally infected canine foetuses and neonates. Canine brucellosis is recognized as a disease with intermittent cell-associated bac- teremia, presumably intracytoplasmically in leucocytes2,23,26. B. melitensis is capable of infecting erythrocytes of experimentally infected mice, in which bacterial cells were demonstrated either epi- or intracellularly in erythro- cytes30. However, association of Brucella spp. with erythrocytes has not been previously demonstrated in naturally infected animals. Distribution in tissues and frequency of positivity in erythrocytes was variable, a finding that is consistent with the intermittent detection of B. canis in the blood of infected dogs21. g B. canis infection in myocardial cells and arterial smooth muscle cells had not been documented before. It may explain the frequent finding of myocarditis and arteritis associated with this disease26. In humans, arterial aneurysm has been diagnosed in a teenager infected with B. canis4, what may have occurred due to the direct infection of the smooth muscle cells. SCIEntIFIC ReporTS \| (2018) 8:7203 \| DOI:10.1038/s41598-018-25651-x 6 www.nature.com/scientificreports/ This study also demonstrated that B. canis are often associated with cells of the choroid plexus. Importantly, neurobrucellosis is a highly relevant clinical presentation in different host species31. B. abortus has been described infecting neuroblasts and glial cells32, but, in dogs, encephalic neurological signs are usually not attributed to B. canis infection. Based on our findings, research on neurobrucellosis in dogs should be encouraged, and perhaps this agent should be included among the differentials in canine neurological patients. g gf g p Interestingly, immunostaining was observed in genital organs of canine foetuses and neonates. B. canis tro- pism to the genital tract in males has been well documented in adult dogs33, but it has not been documented in prepubertal animals. The early infection in epididymis and testicles may compromise irreversibly the reproduc- tive potential of the dogs. Interestingly, in the uterus, positivity was observed in myometrium, and seldom in endometrium. Brucella spp. are known to have tropism for the placenta34, although there are clearly differences in terms of clinical and pathological manifestations according to combination of Brucella and host species35. Interestingly, the two placentas available for this study had B. canis infection as demonstrated by bacterial isola- tion and immunohistochemistry, but no inflammatory reaction was observed histologically. This contrasts with the severe necrotizing and neutrophilic placentitis observed in other species, particularly in B. abortus-infected pregnant cows34. Discussionh However, these observations require additional studies since only two samples are not sufficient for a definitive conclusion.h i Ocular lesions have been reported in adult dogs infected with B. canis17. Therefore, immunolabeling in the cornea and retina in neonates is not unexpected, but it indicated that infection and ocular disease may occur in perinatal brucellosis, even before the opening of the eyelid. p p g y B. canis infection of adipocytes has not been previously described, although adipocytes are permissive cells to bacterial infection. Mycobacterium tuberculosis in human adipocytes has been demonstrated by IHQ and in situ PCR, especially in adipose tissue adjacent to kidneys, lymph nodes, stomach, heart and skin36, the same distri- bution observed for B. canis in this study. Adipose tissue may represent a site for persistence of Brucella infection in dogs, a localization that is suitable to evade treatment and immune response, as is the case for M. tuberculosis in humans37. In adipocytes, the endoplasmic reticulum is localized adjacent to the lipid vacuole38, the same loca- tion where the immunohistochemistry positive coccobacilli were visualized in this study. Furthermore, adipo- cytes in canine foetuses and neonates are immature and undergoing differentiation. In a previous in vitro study, pre-adipocytes expressed unfolded proteins during the differentiation process39 a response also observed during Brucella intracellular replication40, which suggests that differentiating adipocytes may represent a viable niche for Brucella replication. The presence of B. canis in mature adipocytes remains to be investigated.l h Apart from the infected macrophages, immunolabeling was not associated with other inflammatory cells or significant inflammation in tissues, what seems to be a feature of B. canis, since naturally infected dogs do not develop signs of sepsis26, and experimentally infected animals develop only mild inflammation14. The low inflam- matory tissue reaction, observed in this study, may contribute to the prolonged unapparent infection and diffi- culty to eradicate the organism, which is typical of canine brucellosis2,18,20.h y g yp The data generated in this study provided a vast body of novel information that expands our comprehension of canine brucellosis, and provided the basis for new hypothesis regarding transmission of infection to humans or other dogs, through urine, faeces and skin of canine neonates. These hypotheses should be further investigated since it may be extremely valuable to establish new preventive measures when handling neonatal dogs. y y p g g In conclusion, B. Discussionh canis is widespread in macrophages in foetal and neonatal tissues, and cell tropism is diverse, including enterocytes, renal tubular epithelia, adipocytes, myocardium, and choroid plexus cells. Gastrointestinal infection occurs in congenital brucellosis in dogs. Bacteria may be excreted in the faeces and urine of young puppies. Methods E i Data was analysed as described by Altschul et al.43 with Phred Software (http://asparagin.cenargen. embrapa.br/phph/), Sequence Scanner Software (Applied Biosystems) (https://products.appliedbiosystems.com/ ab/en/US/adirect/ab?cmd = catNavigate2&catID = 600583&tab = DetailInfo) and Blast (https://blast.ncbi.nlm. nih.gov/Blast.cgi). Immunohistochemistry. Paraffin embedded tissues from Brucella-positive puppies were selected for immunohistochemistry based on the absence of autolysis. The 13 puppies were included in this analysis: four stillborn and nine neonates, from eight different breeds, and nine breeding kennels. gf g Brucella sp. immunolabeling was performed in the following organs: lung, heart, liver, spleen, stomach, intes- tines, kidney, bladder, prostate, uterus, gonads, umbilical cord, encephalon, and eye. Paraffin embedded tissues were sectioned (4 μm thick) and processed for immunohistochemistry as described by Xavier et al.34, with mod- ifications. Tissue sections were dewaxed, hydrated, and incubated three times in hydrogen peroxide 10% for 20 minutes, after each bath tissue sections were washed three times in PBS. To block unspecific reactions, tissues sections were incubated in 2.5% powdered milk suspension for one hour. The primary antibody was applied over the slides at the dilution of 1:1000, incubated in humidified chamber overnight and washed in PBS. Secondary biotinylated universal antibody with streptavidin-biotin complex (LSAB2 System-HRP, DAKO, USA) was added and slides were incubated for 1 hour and 40 minutes at room temperature. Reaction was revealed by using the chromogen AEC (DAKO, USA). Meyer’s haematoxylin was used for counterstaining. Th l l l b d l d h b l d b d44 l d d d g y y g The primary polyclonal antibody employed has been previously described44. Reactions included positive and neg- ative controls. Positive controls consisted of Brucella infected mouse tissues. Negative controls consisted of infected mouse and puppy tissues incubated overnight with PBS with suppression of the primary antibody in the reaction. Tissue sections were analysed under light microscopy and the frequency of the immunostaining was estimated under high magnification field (400x) as follows: + = one positive mark in ten fields or more; ++ = one positive mark in every two fields; +++ = more than two positive marks in more than ten fields. Laser capture microdissection. In order to confirm that immunohistochemically Brucella sp.-positive cells indeed contained Brucella sp., we performed laser capture microdissection using the MMI CellCut® System. A sample of renal tissue of one stillborn, positive by immunohistochemistry, was used. Methods E i Experimental design. Canine foetuses and neonates of 19 breeds that died spontaneously in 32 breeding kennels were submitted for necropsy by the owners. Kennels were located in the State of Espirito Santo, Brazil. Puppies were collected from November 2014 until June 2016. Necropsy, sample collection and analysis were performed according to a protocol approved by the Ethics Committee of the Federal University of Minas Gerais (CEUA/UFMG), under protocol number 197/2014. All methods were performed in accordance with the relevant guidelines and regulations. Brucella diagnosis in puppies - DNA Extraction, PCR and sequencing. For diagnosis of Brucella-positive puppies, approximately 50 mg of lung, kidney, spleen, liver, and myocardium, from 200 canine foetuses and neo- nates were macerated individually with a scalpel blade in 100 μL of TE buffer, and processed for DNA extraction according to the method described by Pitcher et al.41. After spectrophotometry, DNA samples from the five organs of each puppy were mixed to a final concentration of 250 ng/µL. PCR was directed to the bcsp31 gene with primers 5′-TGGCTCGGTTGCCAATATCAA-3′ and 5′-CGCGCTTGCCTTTCAAGGTCTG-3′42. PCR reaction was per- formed with 2.5 μL of sample DNA, 18.5 µL of Supermix PCR (22 mM Tris-HCl [pH 8.4], 55 mM KCl, 1.65 mM Magnesium Chloride, 220 μM dGTP, 220 μM dATP, 220 μM dTTP, 220 μM dCTP, 22 U/mL recombinant Taq DNA Polymerase - Invitrogen, USA), 1.0 µL of each primer (10 µM) in total volume of 25 μL, with expected product of 223 base pairs (bp). Amplification parameters were: 94 °C for 3 minutes; 40 cycles (94 °C for 30 seconds, 60 °C for 30 seconds, 72 °C for 30 seconds) and 72 °C for 10 minutes. The amplified products were subjected to 1.5% agarose gel electrophoresis stained with SYBR®Safe DNA Gel Stain (Invitrogen, USA) and examined in an ultraviolet light transilluminator.iff Amplified PCR products of bcsp31 gene from three different puppies (from different kennels) were sequenced in order to confirm specificity of Brucella sp. amplification. Amplicons were extracted from agarose gel and puri- fied using QIAEXII Gel Extraction Kit (Qiagen, USA) and sequenced by capillary electrophoresis in ABI3130 SCIEntIFIC ReporTS \| (2018) 8:7203 \| DOI:10.1038/s41598-018-25651-x 7 www.nature.com/scientificreports/ device. Data was analysed as described by Altschul et al.43 with Phred Software (http://asparagin.cenargen. embrapa.br/phph/), Sequence Scanner Software (Applied Biosystems) (https://products.appliedbiosystems.com/ ab/en/US/adirect/ab?cmd = catNavigate2&catID = 600583&tab = DetailInfo) and Blast (https://blast.ncbi.nlm. nih.gov/Blast.cgi). device. Methods E i Paraffin-embedded tissue was mounted on the MMI slide membrane, processed for immunohistochemistry as described, and immunos- tained tubular epithelial cells were microdissected and subsequently the DNA was extracted using the Dneasy blood & tissue kit (Qiagen, Germany) according to the manufacturer’s instructions. To investigate the integ- rity of the extracted DNA, polymerase chain reaction (PCR) was performed for the β-actin gene45 with 22 μL SuperMix PCR (22 mM Tris-HCl (pH 8.4), 55 mM KCl, 1.65 mM Magnesium Chloride, 220 μM dGTP, 220 μM dATP, 220 μM dTTP, 220 μM dCTP, 22 U/mL recombinant Taq DNA Polymerase (Invitrogen, USA), 0.5 μL of each primer (10 μM), 5′-GGCATCCTGACCCTGAAGTA-3′ and 5′-CGCAGCTCGTTGTAGAAGGT-3′, and 2.5 μL of DNA sample. Amplification was performed at 95 °C for 10 minutes, 95 °C for 30 seconds, 60 °C for 30 sec- onds, and 72 °C for 30 seconds with 35 consecutive cycles, followed by final extension at 72 °C for 10 minutes, with expected product of 98 bp. The amplified products were subjected to 1.5% agarose gel electrophoresis stained with SYBR®Safe DNA Gel Stain (Invitrogen, USA) and examined in an ultraviolet light transilluminator. For Brucella sp. detection, PCR for the bcsp31 gene was performed as described. Isolation and identification of Brucella canis. Frozen samples of heart, kidney, spleen and lung, from ten PCR and immunohistochemistry Brucella-positive puppies, from eight different kennels, were submitted in duplicates to bacterial isolation46. Samples of placenta were available from two puppies, and were included in this study. Tissue samples stored at −80 °C were thawed and homogenized with a scalpel blade in 500 µL of PBS. Then, 100 µL of the homogenate were inoculated in selective Tryptose agar (BD Difco, USA) with and without antibiotics (2,500 UI of polymyxin B; 12,500 UI bacitracin; 50,000 UI of nystatin; 50 mg of cycloheximide; 2.5 mg of nalidixic acid; and 10 mg of vancomycin - Sigma Aldrich, USA) and tryptose broth (BD Difco, USA). Plates were incubated at 37 °C and 5% CO2, checked every 48 hours for colony growth. After seven days of incubation, broth was plated on tryptose soy agar without antibiotics and these plates incubated in the same conditions for a maximum of 21 days, when, if no growth was detected, they were considered negative and discarded.il y g y g B. canis isolates were identified based on colony morphology, acriflavine test47, PCR for the bcsp31 gene42, and B. ovis-specific PCR34. Methods E i For molecular analyses, bacterial colonies were collected from agar plates into 200 µL PBS and heat-killed (100 °C/1 hour) and DNA was extracted as described by Pitcher et al.41. For the acriflavine agglutination test, 30 μL of a 0.001% aqueous solution of acriflavine was mixed with bacterial colony. PCR for the bcsp31 gene was performed as described. For B. ovis-specific PCR 22 μL of SuperMix PCR (22 mM Tris-HCl [pH 8.4], 55 mM KCl, 1.65 mM Magnesium Chloride, 220 μM dGTP, 220 μM dATP, 220 μM dTTP, 220 μM dCTP, and 22 U/mL of recombinant Taq DNA Polymerase) were used (Invitrogen, USA), 0.5 μL of each primer (25 μM), 5′-GCCTACGCTGAAACTTGCTTTTG-3′ and 5′-ATCCCCCCATCACCATAACCGAAG-3′, and 2.0 μL of DNA sample. Amplification was performed at 95 °C for 5 minutes, 95 °C for 1 minute, 57 °C for 1 minute and 72 °C for 1 minute for 35 consecutive cycles, followed by final extension at 72 °C for 5 minutes, with an expected product of 228 bp, which was analysed in 1.5% agarose gel stained with SYBR®Safe DNA Gel Stain (Invitrogen, USA).i g Isolates with positive amplification of the bcsp31 Brucella gene, negative in PCR for B. ovis, and agglutination n the acriflavine test were identified as B. canis. Data availability. The datasets generated during and/or analysed during the current study are available from the corresponding author on request to anyone who wishes to repeat our analyses or collaborate with us. 8 SCIEntIFIC ReporTS \| (2018) 8:7203 \| DOI:10.1038/s41598-018-25651-x www.nature.com/scientificreports/ References Brucella canis is an intracellular pathogen that induces a lower proinflammatory response than smooth zoonotic counterparts. Infect. 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E. & Santos, R. L. Pathogenesis and pathobiology of brucellosis in livestock. Rev. Sci. Tech. 32, 105–115 33. Moore, J. A. & Kakuk, T. J. Male dogs naturally infected with Brucella canis. J. Am. Vet. Med. Assoc. 155, 1352 1358 (1969). 34. Xavier, M. N., Paixão, T. A., Poester, F. P., Lage, A. P. & Santos, R. L. Pathological, immunohistochemical and bacteriological study of tissues and milk of cows and fetuses experimentally infected with Brucella abortus. J, Comp. Pathol. 140, 149–157 (2009). 4. Xavier, M. N., Paixão, T. A., Poester, F. P., Lage, A. P. & Santos, R. L. Pathological, immunohistochemical and bacteriological study of tissues and milk of cows and fetuses experimentally infected with Brucella abortus. J, Comp. Pathol. 140, 149–157 (2009). 5. Poester, F. P., Samartino, L. E. & Santos, R. Author Contributions T.D.S., T.A.P. and R.L.S. contributed to study creation and design. T.D.S., T.F.C., J.P.S.M., J.V.M.L., M.F.S. and T.A.P. participated in data acquisition. T.D.S., J.P.S.M., T.A.P. and R.L.S. contributed to the analysis and interpretation of data, All the authors contributed to drafting and critically reviewing the manuscript. Acknowledgements g Work in RLS lab is supported by CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil), FAPEMIG (Fundação de Amparo a Pesquisa do Estado de Minas Gerais, Brazil), and CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brazil). RLS and TAP have fellowships from CNPq (Brazil). References Biotyping and genotyping (MLVA16) of Brucella abortus isolated from cattle in Brazil, 1977 to 2008. PLoS One 8, e81152 (2013). 47. Alton, G. G., Jones, L. M. & Pietz, D. E. Laboratory Techniques in Brucellosis. WHO Monograph Series no. 55. (WHO, 1975). 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https://openalex.org/W3133516572	https://qmro.qmul.ac.uk/xmlui/bitstream/123456789/70303/2/Majid%20Quantum%20gravity%20and%202021%20Accepted.pdf	English	null	Quantum gravity and Riemannian geometry on the fuzzy sphere	letters in mathematical physics/Letters in mathematical physics	2,021	cc-by	10,099	Ver. 2. The ﬁrst author was partially supported by CONACyT and Fundaci´on Alberto y Dolores Andrade (M´exico). QUANTUM GRAVITY AND RIEMANNIAN GEOMETRY ON THE FUZZY SPHERE EVELYN LIRA-TORRES AND SHAHN MAJID Abstract. We study the quantum geometry of the fuzzy sphere deﬁned as the angular momentum algebra [xi, xj] = 2ıλpϵijkxk modulo setting ∑i x2 i to a constant, using a recently introduced 3D rotationally invariant diﬀerential structure. Metrics are given by symmetric 3 × 3 matrices g and we show that for each metric there is a unique quantum Levi-Civita connection with constant coeﬃcients, with scalar curvature 1 2 (Tr(g2)−1 2 Tr(g)2)/ det(g). As an application, we construct Euclidean quantum gravity on the fuzzy unit sphere. We also calculate the charge 1 monopole for the 3D diﬀerential structure. Key words and phrases. Quantum gravity, fuzzy sphere, quantum geometry, fuzzy monopole, noncommutative geometry, angular momentum algebra, coadjoint quantisation. EVELYN LIRA-TORRES AND SHAHN MAJID 2 EVELYN LIRA-TORRES AND SHAHN MAJID Indeed, one might expect such quantum Riemannian geometry to be more in- teresting for the corresponding ‘fuzzy sphere’ quotient, but it turns out that the diﬀerential structure proposed in [4] does not descend to the fuzzy sphere. To ad- dress this problem, [9, Example 1.46] recently proposed a diﬀerent 3D diﬀerential structure on the fuzzy sphere and in the present paper we explore its quantum Rie- mannian geometry with this calculus. We ﬁnd that it is indeed curved for general metrics, including its natural rotationally invariant ‘round metric’. We will denote the unit fuzzy sphere here by Cλ[S2]. The term ‘fuzzy sphere’ is also used in the literature, e.g. [16], for matrix algebras Mn(C) viewed in our terms as further quotients of Cλ[S2] for certain values of λ (those values for which the irreducible n-dimensional representations of spin n/2 descend). We use the constructive ‘quantum groups’ approach to quantum Riemannian geometry as in the recent text [9]. This was established in recent years e.g. [5, 6, 8, 19, 21, 22, 24, 2] using particularly (but not only) the notion of a bimodule connection[11, 27]. The formalism is recalled brieﬂy in Section 2 along with the new diﬀerential structure on Cλ[S2] proposed in [9]. Section 3 contains the ﬁrst new results, namely uniqueness and construction of a quantum-Levi Civita connection for each metric. Metrics here can be chosen freely as symmetric 3×3 matrices in the natural basis of the 3D calculus. In Section 3.2 we look at the curvature as a function of the metric and use this in Section 4 to explore Euclidean quantum gravity on the fuzzy sphere. Conventionally, the Euclidean case, although not usual quantum gravity itself, is nevertheless of interest on any compact Riemannian manifold with boundary[14]. Note also that our approach to quantum Riemannian geometry is very diﬀerent from that of Connes[10] based on spectral triples as abstract ‘Dirac’ operators, but the two approaches can sometimes be related [7]. A ﬁrst step for the fuzzy sphere would be to compute the Grassmann or monopole connection, which we do in the short Section 5. It remains to explain why the cotangent bundle on the fuzzy sphere in this paper is 3D not 2D. Indeed, it often happens in quantum geometry that there is an obstruction to having the same dimension connected diﬀerential calculus as classically and preserving symmetries. 1. Introduction The angular momentum algebra U(su2) has been viewed since the 1970s as the quantisation of R3 viewed as su∗ 2 with its Kirillov-Kostant bracket as part of a general theory for any Lie algebra. As such, setting the quadratic Casimir to a constant quantises the coadjoint orbits, again in a standard way. The angular momentum algebra was also proposed as ‘position coordinates’ for Euclideanised 2+1 quantum gravity by ’t Hooft[15]. We denote it Cλ[R3] or ‘fuzzy R3 with generators xi and relations [xi,xj] = 2λϵijkxk as in [4, 9, 18, 13] to indicate that we consider it a deformation of ﬂat spacetime. Its covariance under the quantum double D(U(su2)) = U(su2)▷<C[SU2] as ‘Poincare group’ was found in [4] along with a 4D quantum-Poincar´e invariant calculus, and further studied in [18][13] among other places. This is by now a well-established picture of a deformed R3 in Euclideanised 2+1 quantum gravity with point sources and without cosmological constant, see e.g. [12] at the group algebra level. Moreover, it deforms naturally to the quantum enveloping algebra Uq(su2) with quantum Poincar´e group D(Uq(su2)) ≅Cq[SO1,3] in 2+1 quantum gravity with cosmological constant, see [26] for an overview and the relationship to the bicrossproduct model Majid-Ruegg quantum spacetime[25]. In physical terms, λ = ıλp where λp is a real deformation parameter which, in the above context (but not necessarily), should be of order the Planck scale. The general idea that spacetime geometry is ‘quantum’ or noncommutative was spec- ulated since the 1920s but in modern times was proposed in [17] coming out of ideas for quantum gravity of quantum Born reciprocity or observable-state/Hopf algebra duality. See also subsequent works by many authors, including [1]. Not surprisingly, however, the quantum spacetime Cλ[R3] with its natural rotationally invariant quantum metric is ﬂat and admits only the zero Levi-Civita connection in the standard coordinates [9, Example 8.15]. The same is true for the correspond- ing bicrossproduct model spacetime with quantum Poincar´e covariant calculus[9, Prop. 9.20], the two models being related by twisting[23]. For quantum gravity eﬀects with background gravity present we should look at curved quantum space- times. 1 2. Preliminaries Here we give a very short introduction to the general formalism, with more details to be found in [9] and references there in. This provides the framework whereby our constructions for the fuzzy sphere should not be seen as ad-hoc but natural within this context. To this end, let A be a unital algebra, possibly noncommutative, over a ﬁeld K (we will be mainly interested in C). By a ﬁrst order diﬀerential calculus (Ω1,d) we mean that: (1) Ω1 is an A-bimodule; ( ) (2) A linear map d ∶A →Ω1 such d(ab) = (da)b + adb for all a,b ∈A; 1 (2) A linear map d ∶A →Ω1 such d(ab) = (da)b + adb for all a,b ∈A; 1 (2) A linear map d ∶A →Ω1 su 1 (3) Ω1 = span{adb∣a,b ∈A}; (4) (Optional) ker(d) = K.1A Here d is called the exterior derivative, the condition (3) is the surjectivity condition and (4) is the connectedeness property, which is not an axiom but is desirable. We also require Ω1 to extend to an exterior algebra Ω= ⊕iΩi of forms of diﬀerent degree, generated by A = Ω0 and Ω1 with d extending by the graded Leibniz rule and d2 = 0. The product of Ωis denoted ∧. For quantum Riemannian geometry we only need up to Ω2. See [9, Chap. 1]. [ ] In this context, we deﬁne a quantum metric as g ∈Ω1 ⊗A Ω1 such that there exists an inverse ( , ) ∶Ω1 ⊗A Ω1 →A which is a bimodule map. Inverse here means in the usual sense, explicitly (ω,g1)g2 = ω = g1(g2,ω) for all ω in a notation where g = g1 ⊗g2 (sum of terms and tensor product over A understood). This implies[9] that g is central, since ag = g1′(g2′,ag1)⊗g2 = g1′ ⊗(g2′a,g1)g2 = ga for all a, where g = g1′ ⊗g2′ is another copy of g. We usually (but not always) also require g to be quantum symmetric in the sense ∧(g) = 0. If this does not hold, we speak of an asymmetric or ‘generalised’ metric. Next, a left connection on Ω1 for us means a linear map ∇∶Ω1 →Ω1 ⊗A Ω1 obeying the left Leibniz rule ∇(a.ω) = da⊗ω + a.∇ω for all a ∈A and ω ∈Ω1. QUANTUM GRAVITY AND RIEMANNIAN GEOMETRY ON THE FUZZY SPHERE 3 EVELYN LIRA-TORRES AND SHAHN MAJID This is because most highly noncommutative geometries are inner in the sense of a 1-form θ such that the exterior derivative is d = [θ, } but this equation has no meaning in classical geometry (the right hand side would be zero) and indeed it is a purely quantum phenomenon. Hence quantum calculi often contain an extra dimension not visible classically, which we could think of as an internally generated ‘time’ direction θ = dt, since quite often the partial derivative in the θ direction turns out to be the natural wave operator or Laplacian. This was explained in [18] in the context of the 4D calculus on Cλ[R3], where this external time is not part of spacetime but may be more related to geodesic ﬂow proper time according to recent ideas in [8] or to renormalisation group ﬂow according to ideas in [13]. In our context, for the new calculus on Cλ[S2] in [9] which we use, there is a more direct geometric interpretation of this extra dimension: there is in fact a (nonconnected) 3D calculus on Cλ[R3] with no extra dimension needed there, but when this descends to the sphere quotient, the normal direction does not decouple as it would classically and instead one has θ = (2ıλp)−2xidxi. So one should think of the fuzzy sphere with this calculus as embedded in a fuzzy R3 and retaining the normal vector as an additional direction. This then makes its calculus 3D and not 2D. 2. Preliminaries If X ∶Ω1 →A is a right vector ﬁeld in the sense that it commutes with the action of A from the right, then we may deﬁne ∇X = (X ⊗id)∇∶Ω1 →Ω1 as an associated covariant derivative-like map. It obeys ∇X(aω) = (X(da))ω + a∇Xω as expected if X also commutes with the action of A from the left. In the classical case with local coordinates xi, say, we would set ∇dxi = −Γijkdxj ⊗dxk in terms of Christoﬀel symbols. One can apply a similar deﬁnition for any vector bundle in the sense of a left A-module E (typically required to be projective). In our case E = Ω1 is a bimodule and we demand ∇(ηa) = (∇η)a + σ(η ⊗da), ∇(ηa) = (∇η)a + σ(η ⊗da), for a bimodule map σ ∶Ω1 ⊗A Ω1 →Ω1 ⊗A Ω1, called the generalised braiding. If this exists, it is uniquely determined by this formula, so this is not additional data, just a property of some left connections. We say that ∇is then a bimodule connection[11, 27]. This case is nice because bimodule connections can be tensor producted. Relevant to us is that Ω1 ⊗A Ω1 gets a bimodule connection ∇(ω ⊗η) = ∇ω ⊗η + (σ(ω ⊗())⊗id)∇η for ω,η ∈Ω1. In this case ∇g = 0 makes sense and we say when that holds that g is metric compatible[6]. Explicitly, we need ∇g ∶= (∇⊗id)g + (σ ⊗id)(id ⊗∇)g = 0. See [9, Chap 8] for more details. 4 EVELYN LIRA-TORRES AND SHAHN MAJID Also, for any left connection on Ω1 and a choice of Ω2, we have a canonical notion of torsion, which is the standard notion but written in terms of diﬀerential forms as T∇= ∧∇−d ∶Ω1 →Ω2 Given a generalised metric we also have a notion of ‘cotorsion’ deﬁne by coT∇= dg1 ⊗g2 −g1 ∧∇g2 ∈Ω2 ⊗A Ω1, which classically is a skew-symmetrized version of metric compatibility. We say that a connection is a quantum Levi-Civita connection (QLC) for a metric g if it is torsion free and metric compatible. We say that it is a weak QLC (WQLC) if it is cotorsion free and torsion free. One can show that a QLC is necessarily a WQLC so it can be useful to impose the WQLC condition ﬁrst, being linear in ∇compared to the QLC condition which is quadratic (due to the σ, which is linear in ∇) and hence much harder to solve. Finally, over C, we need everything to be ‘unitary’ or ‘real’ in a suitable sense. It means that A is a ∗-algebra in the usual sense, and that ∗extends to Ωin a way that commutes with d and is a graded-order reversing involution (it means there is an extra minus sign on a product of odd degree forms). We require the metric and connection to be ‘real’ in the sense ﬂip(∗⊗∗)g = g, σ ○ﬂip(∗⊗∗)∇= ∇○∗. This means we impose [si,xj] = 0, and we deﬁne a diﬀerential dxi = ϵijkxjsk QUANTUM GRAVITY AND RIEMANNIAN GEOMETRY ON THE FUZZY SPHERE 5 which one can check is translation and rotation invariant calculus, but not con- nected. Indeed, d∑i x2 i = 0 so there are diﬀerent connected components according to any constant value of ∑i x2 i . The calculus is inner with θ = 1 2ıλp xisi = 1 (2ıλp)2 xidxi = − 1 (2ıλp)2 (dxi)xi. Finally, for the exterior algebra we take si to be the Grassmann algebra, with Finally, for the exterior algebra we take si to be the Grassmann algebra, with si ∧sj + sj ∧si = 0, dsi = −1 2ϵijksj ∧sk and Ωfree over this (so in each degree it has a basis over A given by a basis over C of the Grassmann algebra). The reader should be warned, however, that this is no longer inner by θ in higher degree. That in turn means it is not the maximal prolongation of the ﬁrst order calculus, but is a natural quotient. We then take the same form of calculus and d for A = Cλ[S2], where we add the unit sphere relation. This is compatible with d for reasons already given and this time we obtain a connected calculus. These facts are all covered in [9, Example 1.46] and one could do the same for a sphere of any ﬁxed radius. Lemma 2.1. In Ω(Cλ[S2]), one has sl = 1 (1−λ2p) (2ıλpxlθ + ϵlim(dxi)xm) = 1 (1−λ2p) ( 1 2ıλp xlxidxi + ϵlim(dxi)xm). Proof. First observe from the form of dxi that ϵlmidxi = ϵlmiϵijkxjsk = xlsm −xmsl. Then ϵlmi(dxi)xm = (xlsm −xmsl)xm = xlxmsm −xmxmsl = 2iλpxlθ −xmxmsl Lemma 2.1. In Ω(Cλ[S2]), one has sl = 1 (1−λ2p) (2ıλpxlθ + ϵlim(dxi)xm) = 1 (1−λ2p) ( 1 2ıλp xlxidxi + ϵlim(dxi)xm). Proof. First observe from the form of dxi that ϵlmidxi = ϵlmiϵijkxjsk = xlsm −xmsl. Th Lemma 2.1. In Ω(Cλ[S2]), one has Lemma 2.1. In Ω(Cλ[S2]), one has emma 2.1. In Ω(Cλ[S2]), one has sl = 1 (1−λ2p) (2ıλpxlθ + ϵlim(dxi)xm) = 1 (1−λ2p) ( 1 2ıλp xlxidxi + ϵlim(dxi)xm). sl = 1 (1−λ2p) (2ıλpxlθ + ϵlim(dxi)xm) = 1 (1−λ2p) ( 1 2ıλp xlxidxi + ϵlim(dxi)xm). Proof. ﬂip(∗⊗∗)g = g, σ ○ﬂip(∗⊗∗)∇= ∇○∗. ﬂip(∗⊗∗)g = g, σ ○ﬂip(∗⊗∗)∇= ∇○∗. The key here is the map † = ﬂip(∗⊗∗) ∶Ω1 ⊗A Ω1 →Ω1 ⊗A Ω1 deﬁned by (ω ⊗η)† = η∗⊗ω∗, which one can check is well-deﬁned on the tensor product over A under- stood here, and obeys (aω ⊗ηb)† = b∗(ω ⊗η)†a∗. It is shown in [9] that (σ ○†)2 = id then holds. Just imposing the latter is a weaker notion of ∗-compatibility as in [5]. In the classical case with self-adjoint local coordinates, the displayed ‘reality’ conditions would ensure that the metric and connection coeﬃcients are real. These are a well-studied set of axioms for which many interesting examples are known, e.g., [6, 21, 22, 24]. Also note that if the reality property for ∇holds on ω then σ((∇(aω))†) = σ(ω∗⊗da∗+(∇ω)†a∗) = σ(ω∗⊗da∗)+(∇ω∗)a∗= ∇(ω∗a∗) = ∇((aω)∗) using both Leibniz rules, so it also holds on aω. It follows that it suﬃces to prove it on a generating set of 1-forms. σ((∇(aω))†) = σ(ω∗⊗da∗+(∇ω)†a∗) = σ(ω∗⊗da∗)+(∇ω∗)a∗= ∇(ω∗a∗) = ∇((aω)∗) using both Leibniz rules, so it also holds on aω. It follows that it suﬃces to prove it on a generating set of 1-forms. 2.1. Fuzzy sphere and its 3D diﬀerential calculus. We work over C and start with the enveloping algebra U(su2) of the angular momentum Lie algebra, with basis xi normalised so that [xi,xj] = 2ıλpϵijkxk for a parameter λp. We call this Cλ[R3] as a quantisation of functions on R3. We take it as a ∗algebra with x∗ i = xi and λp real. Note that this has ﬁnite-dimensional irreducible representations ρj labelled by a non-negative half-integer j of dimension n = 2j + 1 and in which ∑i x2 i = (n2 −1)λ2 p in our normalisation. We deﬁne the unit fuzzy sphere A = Cλ[S2] as the quotient U(su2) modulo the relation ∑ i x2 i = 1 −λ2 p which we see descends to the spin j representation precisely when λp = 1/n. We keep λp as a free parameter, however. Note that in all cases A = Cλ[S2] is inﬁnite- dimensional and therefore never a matrix algebra. Next we deﬁne Ω(Cλ[R3]) as a free 3D calculus with central basis si, i = 1,2,3. ﬂip(∗⊗∗)g = g, σ ○ﬂip(∗⊗∗)∇= ∇○∗. First observe from the form of dxi that ϵlmidxi = ϵlmiϵijkxjsk = xlsm −xms Then ϵlmi(dxi)xm = (xlsm −xmsl)xm = xlxmsm −xmxmsl = 2iλpxlθ −xmxmsl = 2iλpxl( 1 (2iλp)2 xidxi) −xmxmsl = 1 2iλp xlxidxi −xmxmsl using the two forms of θ. We then use the sphere relation and rearrange as stated. □ From this we see that sl does not necessarily have a classical analogue when λp →0, due to the 1 2ıλp xidxi in the second expression. However, if we take this limit with θ ﬁxed then xidxi is order λ2 p and this ‘purely quantum’ term in sl then vanishes in the limit. In this sense, we think of sl as a deformation of the Killing forms sl class = ϵlim(dxi)xm for the action of the rotation group on the classical sphere with its standard round metric. These are the standard orbital angular momentum vector ﬁelds on the sphere when viewed via the metric as 1-forms[9, Cor 9.10]. 3. Moduli of QLCs on the fuzzy sphere Once we have ﬁxed the calculus it is clear, since Ω1(Cλ[S2]) has a central basis, a general metric, as already observed in [9, Example 1.46], takes the form g = gijsi ⊗sj where, since g has to be central, we need the coeﬃcients gij to be central. Since the centre of U(su2) is generated by the quadratic Casimir, it follows that Cλ[S2] has trivial centre, so gij ∈C. For quantum symmetry, we need 0 = gijsi ∧sj = ∑i<j(gijsi∧sj +gjisj ∧si) = ∑i<j(gij −gji)si∧sj, i.e. gij symmetric. We assume such symmetry throughout, for simplicity. For the reality property, we note that si∗= si so we need gij to be hermitian which, given the symmetry, means gij ∈R. Finally, we need gij to be an invertible matrix with inverse gij, say. Then (si,sj) = gij. The new question, which we address, is what are the QLCs and WQLCs. Part of this was answered in [30], although not our main result Proposition 3.4. where, since g has to be central, we need the coeﬃcients gij to be central. Since the centre of U(su2) is generated by the quadratic Casimir, it follows that Cλ[S2] has trivial centre, so gij ∈C. For quantum symmetry, we need 0 = gijsi ∧sj = ∑i<j(gijsi∧sj +gjisj ∧si) = ∑i<j(gij −gji)si∧sj, i.e. gij symmetric. We assume such symmetry throughout, for simplicity. For the reality property, we note that si∗= si so we need gij to be hermitian which, given the symmetry, means gij ∈R. Finally, we need gij to be an invertible matrix with inverse gij, say. Then (si,sj) = gij. The new question, which we address, is what are the QLCs and WQLCs. Part of this was answered in [30], although not our main result Proposition 3.4. 6 EVELYN LIRA-TORRES AND SHAHN MAJID EVELYN LIRA-TORRES AND SHAHN MAJID 6 3.1. Quantum Levi-Civita connection. First, we consider an arbitrary connec- tion equivalently in the form 3.1. Quantum Levi-Civita connection. First, we consider an arbitrary connec- tion equivalently in the form ∇si = −1 2Γi jksj ⊗sk, where Γi jk ∈A. As si form a basis, this extends by the Leibniz rule to a left connection. Proposition 3.1. Let Γijk ∶= gimΓmjk. Proposition 3.1. Let Γijk ∶= gimΓmjk. (1) ∇is torsion free if and only if Γijk −Γikj = 2gimϵmjk. (1) ∇is torsion free if and only if Γijk −Γikj = 2gimϵmjk. (1) ∇is torsion free if and only if Γijk −Γikj = 2gimϵmjk. j j j (2) ∇is cotorsion free if and only if Γijk −Γjik = 2gkmϵmij. (2) ∇is cotorsion free if and only if Γijk −Γjik = 2gkmϵmij. Proof. (1) is immediate from T∇si = ∧∇si −dsi = ∧(−1 2Γi klsk ⊗sl) + 1 2ϵiklsk ∧sl that torsion freeness needs Γi kl −Γi lk + 2ϵilk = 0, which we write to match (2) in terms of the lowered index version. that torsion freeness needs Γi kl −Γi lk + 2ϵilk = 0, which we write to match (2) in terms of the lowered index version. (2) We calculate coT∇= d(gijsi) ⊗sj −gijsi ∧(−1 2Γj klsk ⊗sl) = gij(−1 2ϵimnsm ∧sn) ⊗sj + 1 2gijsi ∧(Γj klsk ⊗sl) = −1 2(gmlϵmik −gijΓj kl)(si ∧sk) ⊗sl. So vanishing of cotorsion requires that gmlϵmik −gijΓj kl is symmetric in i,k by a similar argument as for symmetry of the metric, which we have written as stated. □ Therefore, the moduli of WQLCs (where torsion and cotorsion vanish) are given by the two conditions (1) and (2) simultaneously. Note that the two conditions have a very similar form, which is part of the symmetric role of torsion and cotorsion in the WQLC theory. Finally, for the full QLC theory, we need to know when ∇is a bimodule connection, i.e. when there exists a suitable generalised braiding σ and what it looks like. Lemma 3.2. If ∇is a bimodule connection on Ω1(Cλ[S2]) then σ(si ⊗sj) = sj ⊗si + 1 2(1−λ2p)( 1 2iλp xjxn[Γi lk,xn] + ϵjmn[Γi lk,xm]xn)sl ⊗sk σ(si ⊗sj) = sj ⊗si + 1 2(1−λ2p)( 1 2iλp xjxn[Γi lk,xn] + ϵjmn[Γi lk,xm]xn)sl ⊗sk Proof. The generalised braiding if it exists, given that [si,xj] = 0, is characterised b Proof. The generalised braiding if it exists, given that [si,xj] = 0, is characterised by σ(si ⊗dxj) = dxj ⊗si −[∇si,xj] = ϵjmnxmsn ⊗si + 1 2[Γi mn,xj]sm ⊗sn, h th t i th l b A N b tit ti n f L 2 1 σ(si ⊗dxj) = dxj ⊗si −[∇si,xj] = ϵjmnxmsn ⊗si + 1 2[Γi mn,xj]sm ⊗sn, where the tensor is over the algebra A. Proposition 3.1. Let Γijk ∶= gimΓmjk. Now substituting sn from Lemma 2.1, σ(si ⊗dxj) = dxj ⊗si −[∇si,xj] = ϵjmnxmsn ⊗si + 1 2[Γi mn,xj]sm ⊗sn, where the tensor is over the algebra A. Now substituting sn from Lemma 2.1, σ(si ⊗sn) = σ(si ⊗ 1 2iλp(1−λ2p)xnxjdxj + 1 1−λ2p ϵnmj(dxm)xj) = 1 2iλp(1−λ2p)xnxjσ(si ⊗dxj) + 1 1−λ2p ϵnmjσ(si ⊗dxm)xj = 1 2iλp(1−λ2p)xnxj(ϵjmkxmsk ⊗si + 1 2[Γi mk,xj]sm ⊗sk) + 1 1−λ2p ϵnmj(ϵmlkxlsk ⊗si + 1 2[Γi lk,xm]sl ⊗sk)xj = 1 1−λ2p ( 1 2iλp ϵjmkxnxjxm + ϵnmjϵmlkxlxj)sk ⊗si+ + 1 2(1−λ2p)( 1 2iλp xnxj[Γi lk,xj] + ϵnmj[Γi lk,xm]xj)sl ⊗sk = sn ⊗si + 1 2(1−λ2p)( 1 2iλp xnxj[Γi lk,xj] + ϵnmj[Γi lk,xm]xj)sl ⊗sk σ(si ⊗sn) = σ(si ⊗ 1 2iλp(1−λ2p)xnxjdxj + 1 1−λ2p ϵnmj(dxm)xj) = 1 2iλp(1−λ2p)xnxjσ(si ⊗dxj) + 1 1−λ2p ϵnmjσ(si ⊗dxm)xj = 1 2iλp(1−λ2p)xnxj(ϵjmkxmsk ⊗si + 1 2[Γi mk,xj]sm ⊗sk) + 1 1−λ2p ϵnmj(ϵmlkxlsk ⊗si + 1 2[Γi lk,xm]sl ⊗sk)xj = 1 1−λ2p ( 1 2iλp ϵjmkxnxjxm + ϵnmjϵmlkxlxj)sk ⊗si+ + 1 2(1−λ2p)( 1 2iλp xnxj[Γi lk,xj] + ϵnmj[Γi lk,xm]xj)sl ⊗sk = sn ⊗si + 1 2(1−λ2p)( 1 2iλp xnxj[Γi lk,xj] + ϵnmj[Γi lk,xm]xj)sl ⊗sk □ as stated, using the commutation relations in A. as stated, using the commutation relations in A. □ as stated, using the commutation relations in A. UANTUM GRAVITY AND RIEMANNIAN GEOMETRY ON THE FUZZY SPHERE QUANTUM GRAVITY AND RIEMANNIAN GEOMETRY ON THE FUZZY SPHERE 7 If follows that σ has to the ﬂip on the si basic forms if the Γijk ∈A are central, i.e. Γijk ∈C as our algebra has trivial centre and g already has constant coeﬃcients. In this constant coeﬃcients case, σ(si ⊗sj) = sj ⊗si has a well-deﬁned extension as a bimodule map since the si are a central basis of Ω1, and ∇is indeed then a bimodule connection. Also in this case, the ‘reality’ property with respect to ∗ reduces to Γijk ∈R. We are now ready to consider the condition for full metric compatibility under this assumption. Lemma 3.3. [30] The metric compatibility condition assuming Γ are constant co- eﬃcients is Γlik + Γkil = 0. Proof. ∇g = (∇⊗id)(gijsi ⊗sj) + (σ ⊗id)(gijsi ⊗∇(sj)) = −gij 1 2Γi mnsm ⊗sn ⊗sj −(σ ⊗id)(gijsi ⊗1 2Γj mnsm ⊗sn) = −gij 1 2Γi mnsm ⊗sn ⊗sj −1 2gijΓj mnσ(si ⊗sm) ⊗sn = (−1 2(Γnlk + Γkln) − 1 4(1−λ2p)Γimn( xmxp 2ıλp [Γi lk,xp] + ϵmpq[Γi lk,xp]xq))sl ⊗sk ⊗sn. In the natural case of constant coeﬃcients of Γ, we can drop the second term. □ It is a nice check that torsion free and metric compatible (in our constant Γ case) implies cotorsion free, as it must. Indeed, we can write torsion freeness as Γlki −Γlik = 2glmϵmki by Proposition 3.1. Given metric compatibility in the form just found, this is equivalent to −Γikl + Γkil = 2glmϵmki, which is the cotorsion free condition in Proposition 3.1. It remains to solve for the moduli of constant coeﬃcient QLC solutions for a given a metric gijsi ⊗sj. Proposition 3.4. For any metric gij, there is a unique QLC among those with constant coeﬃcients, namely Γijk = 2ϵikmgmj + Tr(g)ϵijk. There are real, hence the connection is ∗-preserving. Proof. We have to solve the joint system Γikl −Γilk = 2gimϵmkl, Γlik + Γkil = 0, the second of which is solved by setting Γijk = ϵikmγmj for some matrix γ. Letting Li be the matrices (Li)mn = ϵimn, the ﬁrst equation is then the second of which is solved by setting Γijk = ϵikmγmj for some matrix γ. Letting Li be the matrices (Li)mn = ϵimn, the ﬁrst equation is then Liγ −(Liγ)t + 2gimLm = 0 as matrices. This is a linear system for γ with a unique solution as matrices. QUANTUM GRAVITY AND RIEMANNIAN GEOMETRY ON THE FUZZY SPHERE This is a linear system for γ with a unique solution γ = 2g −Tr(g)id which translates into the solution stated. (Note that we do not have to solve the cotorsion equation Γkil −Γikl + 2glmϵmki = 0 as this is implied, as mentioned.) □ For example, when gij = δij (the rotationally invariant or ‘round’) metric, we have a unique solution Γijk = ϵijk. The classical limit of this metric with θ ﬁxed is gclass = dxi ⊗dxi, which is the classical round metric on the unit sphere in this description[9, Sec. 9.3.1]. EVELYN LIRA-TORRES AND SHAHN MAJID 8 8 3.2. Ricci curvature. Now that we understand the moduli of QLCs, we explore their curvature on the fuzzy sphere for general metrics. The curvature in quantum Riemannian geometry is 3.2. Ricci curvature. Now that we understand the moduli of QLCs, we explore their curvature on the fuzzy sphere for general metrics. The curvature in quantum Riemannian geometry is R∇∶Ω1 →Ω2 ⊗ A Ω1, R∇= (d ⊗id −id ∧∇)∇ which one can check is well-deﬁned and a left A-module map[9, Lemma 8.1]. In our case, we can necessarily write it in the form which one can check is well-deﬁned and a left A-module map[9, Lemma 8.1]. In our case, we can necessarily write it in the form R∇(si) = ρi jkϵjmnsm ∧sn ⊗sk for some curvature coeﬃcients ρijk ∈A. We are also interested in taking a ‘trace’ for the Ricci tensor and the Ricci scalar, which in the current framework [9] means with respect to a further, but in our case canonical, bimodule ‘lift’ map i ∶Ω2 →Ω1 ⊗ A Ω1, i(si ∧sk) = 1 2(si ⊗sk −sk ⊗si). For this choice of i and form of R∇, we have Ricci = (( , )⊗id)(id⊗i⊗id)(id⊗R∇)(g) Ricci = (( , )⊗id)(id⊗i⊗id)(id⊗R∇)(g) = 1 2gpiρi jkϵjmn((sp, )⊗id)(sm ⊗sn ⊗sk −sn ⊗sm ⊗sk) = 1 2ρi jkϵjinsn ⊗sk −1 2ρi jkϵjmism ⊗sk = ρi jnϵjimsm ⊗sn. Hence the Ricci tensor deﬁned by Ricci = Rmnsm ⊗sn and Ricci scalar S = ( , )Ricci are Rmn = ρi jnϵjim, S = ρi jnϵjimgmn. Rmn = ρi jnϵjim, S = ρi jn Finally, we adopt the convention that indices of ϵ can be raised with the inverse metric ( , ) = g−1 with matrix entries gij. Proposition 3.5. QUANTUM GRAVITY AND RIEMANNIAN GEOMETRY ON THE FUZZY SPHERE For Cλ[S2], the scalar curvature for the QLC in Proposition 3.4 is is S = 1 2(Tr(g2) −1 2Tr(g)2)/det(g). Proof. We ﬁrst compute from its deﬁnition as given above that R∇(si) = −1 2(Γi kldsk + (dΓi kl)sk) ⊗sl + 1 2Γi klsk ∧∇sl = 1 4Γi klϵkmnsm ∧sn ⊗sl −1 2dΓi kl ∧sk ⊗sl −1 4Γi klΓl mnsk ∧sm ⊗ which corresponds to which corresponds to ρi jk = 1 4Γi jk −1 4ϵjmn∂mΓi nk −1 8ϵjmnΓi mlΓl nk (1) where df = (∂if)si deﬁnes the partial derivatives. One can check that this repro- duces the same R∇. This applies for any left connection on Ω1. where df = (∂if)si deﬁnes the partial derivatives. One can check that this repro- duces the same R∇. This applies for any left connection on Ω1. We now specialise to the QLC in Proposition 3.4. Then ρi jk = 1 2(ϵi kmgmj −ϵi lpϵjmnϵl kqgpmgqn) + 1 4Tr(g)(ϵi jk −ϵj ik + ϵij pgpk) −1 8Tr(g)2ϵij k which we contract to obtain Ricci as Rst = 1 2ϵi tmgmjϵjis+ 1 2Tr(g−1)gst−1 2δst+ 1 2Tr(g)(gst−Tr(g−1)δst−1 2ϵij pϵijsgpt)+ 1 8Tr(g)2ϵij tϵijs. This then contracts further to S = Tr(g−1)+ 1 2ϵij m(gmk−1 2Tr(g)δmk+ 1 4Tr(g)2gmk)ϵijk+ 1 2Tr(g)(Tr(g−2)−Tr(g−1)2). S = Tr(g−1)+ 1 2ϵij m(gmk−1 2Tr(g)δmk+ 1 4Tr(g)2gmk)ϵijk+ 1 2Tr(g)(Tr(g−2)−Tr(g−1)2). S = Tr(g−1)+ 1 2ϵij m(gmk−1 2Tr(g)δmk+ 1 4Tr(g)2gmk)ϵijk+ 1 2Tr(g)(Tr(g−2)−Tr(g−1)2) Finally, we identify the middle ϵ...ϵ expression in terms of detg−1. This is most easily seen assuming that g = diag(λ1,λ2,λ3), say, but then holds generally. Here ϵijkgmkϵijk = ϵ2 ijkλ−1 i λ−1 j λ−1 k = 6detg−1 (summing over i,j,k) is well-known, but a similar method gives ϵij kϵijk = ϵ2 ijkλ−1 i λ−1 j ϵ2 ijkλ−1 i λ−1 j λ−1 k λk = 2detg−1Tr(g) ϵij mgmkϵijk = ϵ2 ijkλ−1 i λ−1 j λk = ϵ2 ijkλ−1 i λ−1 j λ−1 k λ2 k = 2detg−1Tr(g2). QUANTUM GRAVITY AND RIEMANNIAN GEOMETRY ON THE FUZZY SPHERE 9 In this way, we obtain In this way, we obtain S = Tr(g−1) + (Tr(g2) + 1 4Tr(g)2)detg−1 + 1 2Tr(g)(Tr(g−2) −Tr(g−1)2) which then simpliﬁes further to the form stated, as one can again check in the diagonal case. 4. Euclideanised quantum gravity on the fuzzy sphere The scalar curvature found above is the main, but not the only, ingredient for quantum gravity in a functional integral approach. Here we brieﬂy consider the other elements and formulate the theory, although it will remain too hard to com- pute explicitly. Moreover, this will not be physical gravity since our spherical form of coordinates where ∑i x2 i = 1−λ2 p are suitable for a Euclidean signature (the actual ‘shape’ depends on the metric but the choice of algebra plays the role of the mani- fold in some sense) but this is still of interest in certain contexts. In the Euclidean case, the partition function for the functional integral should take the form Z = ∫Dg e−2 G ∫S[g] (and similarly with operators inserted for expectation values), where G is some real positive coupling constant and (and similarly with operators inserted for expectation values), where G is some real positive coupling constant and QUANTUM GRAVITY AND RIEMANNIAN GEOMETRY ON THE FUZZY SPHERE In terms of the λi, this is (2) S = λ2 1 + λ2 2 + λ2 3 −2(λ1λ2 + λ1λ3 + λ2λ3) 4λ1λ2λ3 , (2) which one can also regard as a function on the space of metrics modulo conjugation if we think of the λi as the (not necessarily distinct) eigenvalues of g. □ Note that the classical limit of S in the noncommutative geometry conventions here is −1 2 of the classical value. Hence a classical unit sphere with its usual round metric would in our conventions have S = −1. In the fuzzy case, for the rotationally invariant ‘round’ metric gij = δij on Cλ[S2] and the unique QLC Γijk = ϵijk in Proposition 3.4, we have Rmn = −1 4δmn, S = −3 4. This is not quite the same as the classical value, presumably as a consequence of the extra cotangent direction. If we perturb around this metric by setting g = id+ϵ then we can write This is not quite the same as the classical value, presumably as a consequence of the extra cotangent direction. If we perturb around this metric by setting g = id+ϵ then we can write S = −3 4 + 1 4Trϵ −1 12(Trϵ)2 + 1 4(ϵ2 12 + ϵ2 13 + ϵ2 23) + 1 24 ((ϵ11 −ϵ22)2 + (ϵ11 −ϵ33)2 + (ϵ22 −ϵ33)2) + O(ϵ3), showing an unbounded mode for the average of the diagonal entries plus a positive deﬁnite part for the ‘ﬂuctuations’ oﬀdiagonal or between the diagonal entries. (3) S[g] = g2 11 + g2 22 + g2 33 −2(g11g22 + g11g33 + g22g33) + 4(g2 12 + g2 13 + g2 23) 4detg (3) S[g] = g2 11 + g2 22 + g2 33 −2(g11g22 + g11g33 + g22g33) + 4(g2 12 + g2 13 + g2 23) 4detg (3) S[g] = g2 11 + g2 22 + g2 33 −2(g11g22 + g11g33 + g22g33) + 4(g2 12 + g2 13 + g2 23) 4detg is the scalar curvature in Proposition 3.5. We also need for the action a map ∫∶A →C which classically would be the Lebesgue measure in local spacetime coordinates times √ ∣det(g)∣. One could at- tempt to characterise this map in the quantum case by requiring that it is a positive linear functional (so ∫a∗a ≥0 for all a in the algebra) compatible with the Rie- mannian metric in some way (classically this would be so as to vanish on a total divergence). This is an interesting question but is not needed for our present pur- poses as S[g] ∈R is a multiple of the constant function, so the only thing we need is ∫1. The natural choice here similar to integration on a Riemannian manifold would be power of ∣detg∣(but not necessarily its square root as classically, given EVELYN LIRA-TORRES AND SHAHN MAJID 10 that our tangent space and metric have the wrong dimension compared to classical sphere). Bearing in mind the det(g) in the denominator of S[g], the most natural choice here is to cancel this by setting ∫1 = ∣det(g)∣. Finally, we need a measure Dg for integration over the space of metrics. In our case, this is the 6-dimensional space P3 of 3×3 positive-deﬁnite symmetric matrices, which has the natural structure of a Riemannian manifold as the noncompact sym- metric space GL3(R)/O3(R), with an invariant metric gP 3 given in line element terms as ds2 = Tr((g−1dg)2). Integration over P3 therefore has a canonical Riemannian manifold measure Dg deﬁned relative to Lebesgue measure in local coordinates by √ ∣det(gP 3)∣. The latter works out at metric g as a factor ∣det(g)∣−2, see [31, Sec 4.1.3]. Putting these ingredients together, we thus deﬁne Euclideanised quantum gravity on the fuzzy sphere by partition function latter works out at metric g as a factor ∣det(g)∣ , see [31, Sec 4.1.3]. QUANTUM GRAVITY AND RIEMANNIAN GEOMETRY ON THE FUZZY SPHERE 11 QUANTUM GRAVITY AND RIEMANNIAN GEOMETRY ON THE FUZZY SPHERE 11 11 35 40 45 50 100 200 300 400 ⟨λi⟩ ⟨λi⟩2 ⟨λiλj⟩ L 200 400 600 800 1000 0.99994 0.99996 0.99998 1.00000 1.00002 1.00004 1.00006 L ⟨λiλj⟩ 16 3 ⟨λi⟩2 ⟨λiλj⟩ 3 16 L2 Figure 1. Expectation values for Euclidean quantum gravity on the fuzzy sphere as a function of the cutoﬀL. The right graph shows ⟨λiλj⟩converging to 16 3 ⟨λi⟩2 and 3 16L2. 200 400 600 800 1000 0.99994 0.99996 0.99998 1.00000 1.00002 1.00004 1.00006 L ⟨λiλj⟩ 16 3 ⟨λi⟩2 ⟨λiλj⟩ 3 16 L2 35 40 45 50 100 200 300 400 ⟨λi⟩ ⟨λi⟩2 ⟨λiλj⟩ L Figure 1. Expectation values for Euclidean quantum gravity on the fuzzy sphere as a function of the cutoﬀL. The right graph shows ⟨λiλj⟩converging to 16 3 ⟨λi⟩2 and 3 16L2. we can do the integration over a dense subset of SO3 to give a constant, which we ignore. Hence (4) becomes eﬀectively (6) Z = ∫ L ϵ ∏ i dλi ∣(λ1 −λ2)(λ1 −λ3)(λ2 −λ3)∣ λ2 1λ2 2λ2 3 e−1 2G (λ2 1+λ2 2+λ2 3−2(λ1λ2+λ1λ3+λ2λ3)), where we have introduced cut-oﬀs L >> ϵ > 0 to regulate divergences at both ends. The story here turns out to be very similar to [21] in that the divergence as ϵ →0 does not show up when we look as vacuum expectation values as these are ratios (both top and bottom diverge as ϵ →0 but the ratio is well deﬁned in this limit). Moreover, where we have introduced cut-oﬀs L >> ϵ > 0 to regulate divergences at both ends. The story here turns out to be very similar to [21] in that the divergence as ϵ →0 does not show up when we look as vacuum expectation values as these are ratios (both top and bottom diverge as ϵ →0 but the ratio is well deﬁned in this limit). Moreover, ⟨λi1⋯λin⟩∼3 16Ln for large L, independently of which λi are involved (this was checked numerically to several orders of λ), as plotted for n = 1,2 in Figure 1. (3) S[g] = g2 11 + g2 22 + g2 33 −2(g11g22 + g11g33 + g22g33) + 4(g2 12 + g2 13 + g2 23) 4detg Putting these ingredients together, we thus deﬁne Euclideanised quantum gravity on the fuzzy sphere by partition function (4) Z = ∫P 3 ∏ i≤j dgij ∣det(g)∣−2 e−1 G (Tr(g2)−1 2 Tr(g)2), (4) where G is a real coupling constant. Here expectation values are the ratio of the same expression with operators inserted divided by Z, both parts of which can be expected to diverge given the above initial remarks and the noncompact nature of P3. One can also write det(g)−2 = (2π)−3 2 ∫R3 d3⃗x e−1 2 ⃗xtg2⃗x so that up to a discarded constant and assuming we can swap the order of integra- tion, (5) Z = ∫R3 ∏ i dxi ∫P 3 ∏ i≤j dgij e−1 G (Tr(g2)−1 2 Tr(g)2)−1 2 ⃗xtg2⃗x, (5) giving an idea of the formal content of the theory. Moreover, if we ignored the restriction to P3, we could then do the dg integration as a Gaussian to give the inverse determinant of a quadratic form built from the xi. We can also write (detg)−2 = e−2Tr ln g in (4) as a non-quadratic ‘interaction’ term. We now look more closely at the theory in a reduced form where we look only at SO3-invariant expressions. Our ﬁrst step it to parameterize positive symmetric matrices according to the spectral decomposition g = Ctdiag(λ1,λ2,λ3)C for some ⃗λ ∈R3 >0 and some C ∈SO3. The latter is not unique but the multiplicity is discrete and generically we can convert dg to these new coordinates with an appropriate Jacobean. To do this explicitly, we let E(θ,φ,ψ) be the Euler rotation matrix for angles θ,φ,ψ and let g = E(θ,φ,ψ)tdiag(λ1,λ2,λ3)E(θ,φ,ψ). This change of variables is locally invertible for sinφ ≠0 and distinct λi, indeed the Jacobean can be computed and we ﬁnd ∏ i≤j dgij = dθ dφdψ ∣sin(φ)∣∏ i dλi ∣(λ1 −λ2)(λ1 −λ3)(λ2 −λ3)∣ On the other hand, the action itself does not depend on the SO3 conjugation (having the value in (2) but without the λ1λ2λ3 denominator there), so for the partition function and for any insertions that depend only on the λi and not on the angles, 5. Fuzzy monpole We have focussed on the quantum Riemannian geometry of the fuzzy sphere with its new calculus from [9]. However, we could also ask about the monopole connection for this calculus. Classically, this arises naturally on the tautological line bundle over the sphere which in algebraic terms is the Grassmann connection for the rank 1 projector associated with that. We recall that if A is a unital algebra, a vector bundle can be viewed as, say, a left ﬁnitely generated projective module S. This property is characterised by existence of a ﬁnite number n of generators eα ∈S and corresponding left module maps eβ ∶S →A such that s = eα(s)eα for all s ∈S. There is an associated projection Pαβ = eβ(eα) obeying P 2 = P as an element of Mn(A) and expressing relations eα = Pαβeβ in S. Equivalently, given a projection P we can realise S concretely as generated by the rows eα of P and set eβ(eα) = Pαβ extended as left-module maps. The associated Grassmann connection and its curvature in the conventions of [9, Prop. 3.24] are ∇Seα = (dPαβ)Pβγ ⊗eγ, RS(eα) = −dPαβ ∧(dPβγ)Pγδ ⊗eδ. The construction is best-known in the case of the universal calculus, where it un- derlies the Chern-Connes pairing between K-theory and cyclic cohomology[10]. The construction is best-known in the case of the universal calculus, where it un- derlies the Chern-Connes pairing between K-theory and cyclic cohomology[10]. [ ] It was already shown in [20] that the fuzzy sphere (as well as the standard q-sphere and the 2-parameter q-fuzzy sphere) has such a projection matrix Pαβ associated to a tautological rank 1 projective module S. Here P ∈M2(A) and [9, Example 3.27] covers the Grassmann connection but depending on the choice of calculus; here we carry this through for our choice of Ω(Cλ[S2]). In terms of natural ‘coordinates’ and relations x = 1 2(x3+1+λp), z = 1 2(x1+ıx2); [x,z] = λpz, [z,z∗] = 2λpx−λp(1+λp), z∗z = x(1−x), the projection matrix is [20] x = 1 2(x3+1+λp), z = 1 2(x1+ıx2); [x,z] = λpz, [z,z∗] = 2λpx−λp(1+λp), z∗z = x(1−x), the projection matrix is [20] P = (1 + λp −x z z∗ x) = 1 2 (1 + λp −x3 x1 + ıx2 x1 −ıx2 1 + λp + x3) which one can check obeys P 2 = P. QUANTUM GRAVITY AND RIEMANNIAN GEOMETRY ON THE FUZZY SPHERE 11 It follows that we have well-deﬁned ratios ⟨λi1⋯λin⟩ ⟨λi⟩n = ( 16 3 )n−1 in the limit L →∞, and in particular that there is a uniform relative uncertainty in the λi, in the limit L →∞, and in particular that there is a uniform relative uncertainty in the λi, ∆λi ⟨λi⟩∶= √ ⟨λ2 i ⟩−⟨λi⟩2 ⟨λi⟩ = √ 13 3 similarly to quantum gravity on a square in [21]. Note, however, that these are formal interpretations given that this is a Euclidean theory. We can also follow the pattern of [21] and look at a partial theory where we regard the average of the λi as a background metric with respect to which we are quantising only the diﬀerences. Thus, we let λ1 = u −2v, λ2 = u + v −w, λ3 = u + v + w with inverse u = 1 3(λ1 + λ2 + λ3), v = 1 6(λ2 + λ3 −2λ1), w = 1 2(λ3 −λ2), which diagonizes the quadratic form in the action to which diagonizes the quadratic form in the action to λ2 1 + λ2 2 + λ2 3 −2(λ1λ2 + λ1λ3 + λ2λ3) = −3u2 + 12v2 + 4w2. In the partial theory, we leave out the du integral and regard u > 0 as a parameter. Putting in the region of u,v,w corresponding to λi > 0, we have the eﬀective theory EVELYN LIRA-TORRES AND SHAHN MAJID 12 for the ‘ﬂuctuation’ variables v,w, Zu = 2∫ u 2 −u dv ∫ u+v −(u+v) dw ∣(9v2 −w2)w∣ (u −2v)2((u + v)2 −w2)2 e−2 G (3v2+w2) = 4∫ u 2 −u dv ∫ u+v 0 dw ∣9v2 −w2∣w (u −2v)2((u + v)2 −w2)2 e−2 G (3v2+w2). This is still divergent at the boundaries corresponding previously to λi = 0, but has the merit that the inner integral can now be done analytically. The previous partition function Z = ∫ ∞ 0 du e 3 2G u2Zu still contains the other divergence at λi = ∞, requiring a cut-oﬀ. One can make this change for the diagonal entries of any metric and have the oﬀ-diagonals as three further Gaussian variables according to (3), but the restriction on the variables for a positive metric is then much harder to describe. 5. Fuzzy monpole Starting with P, one can realise S as spanned by the rows of P with one relation, e1 = (1,0).P = (1 + λp −x,z), e2 = (0,1).P = (z∗,x); (x −λp)e1 = ze2 and forming dual bases with eβ deﬁned by eβ(eα) = Pαβ as explained. Here, we will stick with the xi generators of A in order to match the rest of the paper. will stick with the xi generators of A in order to match the rest of the paper. QUANTUM GRAVITY AND RIEMANNIAN GEOMETRY ON THE FUZZY SPHERE 13 Lemma 5.1. The monopole Grassmann connection on S for our projection P and calculus on Cλ[S2] is Lemma 5.1. The monopole Grassmann connection on S for our projection P and calculus on Cλ[S2] is ∇eα = (1 + λp 2 dPαβ + λpPαβθ + ı1 −λ2 p 4 Qαβ −λp(1 −λp) 2 δαβθ) ⊗eβ, here Q = ( −s3 s1 + ıs2 s1 −ıs2 s3 ). ∇eα = (1 + λp 2 dPαβ + λpPαβθ + ı1 −λ2 p 4 Qαβ −λp(1 −λp) 2 δαβθ) ⊗eβ, Proof. Here ∇S ∶S →Ω1 ⊗A S obeys similar axioms to those for a left connection on Ω1 in Section 2. The calculation of the stated formula from the projector is a straightforward from dP = 1 2 ( x2s1 −x1s2 x2s3 −x3s2 + ıx3s1 −ıx1s3 x2s3 −x3s2 −ıx3s1 + ıx1s3 x1s2 −x2s1 ) and the commutation relations in the algebra. For example, ((dP)P)11 = 1 4 ((x2s1 −x1s2)(1 + λp −x3) + (x2s3 −x3s2 −ıx3s1 + ıx1s3)(x1 −ıx2)) = 1 4 ((1 + λp)(x2s1 −x1s2) + ıx3(x1s1 + x2s2 + x3s3) −ı(1 −λ2 p)s3+ +[x3,x2]s1 + [x1,x3]s2 + [x2,x1]s3) ((dP)P)11 = 1 4 ((x2s1 −x1s2)(1 + λp −x3) + (x2s3 −x3s2 −ıx3s1 + ıx1s3)(x1 −ıx2)) = 1 4 ((1 + λp)(x2s1 −x1s2) + ıx3(x1s1 + x2s2 + x3s3) −ı(1 −λ2 p)s3+ +[x3,x2]s1 + [x1,x3]s2 + [x2,x1]s3) = 1 4 ((1 + λp)(x2s1 −x1s2) −2x3λpθ + ı(λ2 p −1)s3 −2ıλp(x1s1 + x2s2 + x3s3)) = 1 4 ((1 + λp)d(−x3) + ı(λ2 p −1)s3 −2λpx3θ + 4λ2 pθ) = 1 2(1 + λp)dP11 + λpP11θ −1 2(1 −λp)λpθ −ı 4(1 −λ2 p)s3. 6. Concluding remarks Fuzzy-R3 in the form of the angular momentum algebra U(su2) has a long history as a ‘quantisation’ as well as clear applications such as at the heart of 3D Euclideanised quantum gravity without cosmological constant. Its quotient the fuzzy sphere is likewise well known as the quantisation of a coadjoint orbit. Although less relevant perhaps to 3D quantum gravity, it is nevertheless related to Penrose’ spin network geometry[28], and could also have a role for the geometry of angular momentum in actual quantum systems. A diﬀerential calculus that works with it was, however, only recently proposed[9, Example 1.46] and it is signiﬁcant that, using this, we have now solved its quantum Riemannian geometry (found a natural quantum Levi-Civita connection) for any quantum metric, including the canonical rotationally invariant ‘round’ metric given by δij. y g y ij After understanding this moduli of quantum Riemannian geometries on the fuzzy sphere, we went on and constructed Euclidean quantum gravity on it, where we integrate over all quantum geometries. Even though the fuzzy sphere is an inﬁnite- dimensional algebra which becomes functions (in some form) on the usual sphere in the classical limit, its quantum geometry turned out to be much more rigid and to admit only quantum metrics of the form of a single 3 × 3 real symmetric matrix gij transported over the whole algebra. It turned out that the quantum Levi-Civita connection, hence the whole moduli of quantum Riemannian geometries could simi- larly be developed with constant coeﬃcients, hence the quantum geometry behaves eﬀectively like just one point. This was not put in by hand, but forced by the rigidity of the axioms of noncommutative geometry and the noncommutativity of our particular algebra. Moreover, our results were strikingly similar to quantum gravity on a quadrilateral in [21] even though the details are completely diﬀerent not to mention that that model was Lorentzian with ı in action, whereas ours is Euclidean. In both cases, the functional integral over all metrics of the natural ac- tion built from the quantum Ricci scalar has UV and IR divergences, appearing in our case at λi = 0,∞. In both cases, the divergence at metric zero modes cancels in the ratio of functional integrals when we look at expectation values. 5. Fuzzy monpole = 1 4 ((1 + λp)(x2s1 −x1s2) −2x3λpθ + ı(λ2 p −1)s3 −2ıλp(x1s1 + x2s2 + x3s3)) = 1 4 ((1 + λp)d(−x3) + ı(λ2 p −1)s3 −2λpx3θ + 4λ2 pθ) = 1 2(1 + λp)dP11 + λpP11θ −1 2(1 −λp)λpθ −ı 4(1 −λ2 p)s3. = 1 2(1 + λp)dP11 + λpP11θ −1 2(1 −λp)λpθ −ı 4(1 −λ2 p)s3. Similary for the other entries of dP.P, the proof of which we omit. Similary for the other entries of dP.P, the proof of which we omit. The curvature of ∇was also recalled in general as −dP ∧(dP)P acting as a 2-form valued operation on our eα. Proposition 5.2. The curvature of the monopole connection is given by −dP∧(dP)P = (1−λp) 2ı ((x1 λp λp x1)s2 ∧s3 + ( x2 ıλp −ıλp x2 )s3 ∧s1 + (x3 −λp 0 0 x3 + λp)s1 ∧s2)P. Proof. We similarly compute the matrix-valued coeﬃcients of s1 ∧s2,s2 ∧s3,s3 ∧s1 of dP ∧(dP)P, using the commutation relations in the diﬀerential algebra. For example, Proof. We similarly compute the matrix-valued coeﬃcients of s1 ∧s2,s2 ∧s3,s3 ∧s1 of dP ∧(dP)P, using the commutation relations in the diﬀerential algebra. For example, 8(dP ∧(dP)P)11 = 8(dP11 ∧dP11 + dP12 ∧dP21)P11 + 8(dP11 ∧dP12 + dP12 ∧dP22)P21 = ((x2s1 −x1s2)(x2s1 −x1s2) + (x2s3 −x3s2 + ıx3s1 −ıx1s3)(x2s3 −x3s2 + ıx1s3 −ıx3s2)) (1 + λp −x3) + ((x2s1 −x1s2)(x2s3 −x3s2 + ıx3s1 −ıx1s3) + (x2s3 −x3s2 + ıx3s1 −ıx1s3)(x1s2 −x2s1)) (x1 −ıx2) and from this we pick out the coeﬃcient of s1 ∧s2 as ([x1,x2] −2ıx2 3)(1 + λp −x3) + (−[x2,x3]x2 −2x3x2 + ı[x1,x3] + 2ıx3x1)(x1 −ıx2) = (2ıλpx3 −2ıx2 3)(1 + λp −x3) + (−2ıλpx1 −2x3x2 + 2λpx2 + 2ıx3x1)(x1 −ıx2) = 2ı(x3 −λp)(x3(−1 −λp + x3) + (x1 + ıx2)(x1 −ıx2)) = 2ı(1 −λp)(x3 −λp)(1 + λp −x3) using the commutation relations and the quadratic relation for the fuzzy sphere. By comparison, the coeﬃcient of s1 ∧s2 on the right hand side of the stated result using the commutation relations and the quadratic relation for the fuzzy sphere. By comparison, the coeﬃcient of s1 ∧s2 on the right hand side of the stated result is (1−λp) 4ı ((x3 −λp)(1 + λp −x3) (x3 −λp)(x1 + ıx2) (x3 + λp)(x1 −ıx2) (x3 + λp)(1 + λp + x3)) EVELYN LIRA-TORRES AND SHAHN MAJID 14 which thus veriﬁes the top left corner entry. Similarly for the other matrix entries. 5. Fuzzy monpole One then repeats for the coeﬃcient of s2 ∧s3 and of s3 ∧s1. Note that the factori- sation into matrices in the stated answer is not unique as P has a kernel; we have chosen a factorisation which is much simpler than taking the matrix coeﬃcients of dP ∧dP as the factor in front of P. □ □ Note that the right hand side in the proposition reduces as λp →0 to FP, where F = 1 2ı(x1s2 ∧s3 + x2s3 ∧s1 + x3s1 ∧s2) = 1 4ıϵijkxisj ∧sk = 1 4ıϵijkxidxj ∧dxk if we use the classical limit of the sl with θ ﬁxed as discussed after Lemma 2.1. This agrees with a classical monopole where the curvature is an imaginary multiple of the volume form. The last expression is indeed a multiple of the classical volume form by rotational invariance. 6. Concluding remarks In both cases, the other divergence is controlled by a cut-oﬀL, and in both cases the expectation value of an n-th power of the ﬁeld diverges as Ln with the result that ratios of expectation values can still be deﬁned as L →∞. In both cases, we found in this way a uniform relative uncertainly in the quantisation of the metric components (in our case, we quanitised the metric eigenvalues but one can also think of this as quantising diagonal metrics). There are many interesting directions that one could further explore. On the quantum gravity front, one could introduce matter and see how some kind of Ein- stein equation emerges out of quantum gravity with matter. A ﬁrst step here would QUANTUM GRAVITY AND RIEMANNIAN GEOMETRY ON THE FUZZY SPHERE 15 be to better understand the geometric approach to the stress-energy tensor. One can consider quantum matter ﬁelds on curved FLRW cosmologies R×S2 where the S2 is now fuzzy. Although the ﬁeld theory stress tensor is not clear, the analysis for a perfect ﬂuid is possible and will be done elsewhere in the spirit of [2], where R×Zn was done using quantum geometry on the polygon Zn (this work also solved Euclidean quantum gravity on Zn). Finally, one could look at quantum geodesics on the fuzzy sphere using the recent formalism in [8]. be to better understand the geometric approach to the stress-energy tensor. One can consider quantum matter ﬁelds on curved FLRW cosmologies R×S2 where the S2 is now fuzzy. Although the ﬁeld theory stress tensor is not clear, the analysis for a perfect ﬂuid is possible and will be done elsewhere in the spirit of [2], where R×Zn was done using quantum geometry on the polygon Zn (this work also solved Euclidean quantum gravity on Zn). Finally, one could look at quantum geodesics on the fuzzy sphere using the recent formalism in [8]. We have also constructed a natural fuzzy monopole and there are potentially many applications that could be related to that. In physical terms, this could be relevant to any quantum system where classically one has angular momentum at play, for example the eﬀective geometry around a quantised hydrogen atom. 6. Concluding remarks In mathematical terms, an important application would be towards the programme of ‘geometric realisation’ - constructing examples of Connes’ notion of spectral triple[10] or ‘axiomatic Dirac operator’ but in a geometric manner starting with the quantum diﬀerential structure, a spinor bundle with connection, a quantum metric and a Cliﬀord structure. This was done for the q-sphere in [7] and one might try to follow the same steps. Thus, the spinor bundle on the sphere should be of the form S = S+⊕S−where S+ is the charge 1 monopole line bundle as found in Section 5 and S−is its dual. The Cliﬀord structure is a ‘Cliﬀord action’ map Ω1 ⊗A S →S obeying certain axioms [7, 9] of compatibility with the ∗-structure, with the connection on S and with the quantum Levi-Civita connection. On the other hand, the Cliﬀord structure for the q-sphere case in [7] was found from the holomorphic structure of its 2D calculus, which does not apply here. This nevertheless merits further study and will be attempted elsewhere. If a geometrically-realised spectral triple can be constructed on the fuzzy sphere, it may (or may not) descend when λp = 1/n to the quotient cλ[S2] isomorphic to Mn, i.e. to the reduced matrix fuzzy spheres. This may then complement (or perhaps relate to) the ﬁnite fuzzy Dirac operators constructed in [3] using Connes formalism and starting from the reduced noncommutative torus. Conﬂict of interest. On behalf of all authors, the corresponding author states that there is no conﬂict of interest. 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https://openalex.org/W4296742933	https://vbn.aau.dk/ws/files/487328741/sustainability_14_11773.pdf	Latin	null	Day-Ahead Scheduling of Electric Vehicles and Electrical Storage Systems in Smart Homes Using a Novel Decision Vector and AHP Method	Sustainability	2,022	cc-by	12,848	Day-Ahead Scheduling of Electric Vehicles and Electrical Storage Systems in Smart Homes Using a Novel Decision Vector and AHP Method Published in: Sustainability DOI (link to publication from Publisher): 10.3390/su141811773 DOI (link to publication from Publisher): 10.3390/su141811773 Citation for published version (APA): Alilou , M., Gharehpetian, G. B., Ahmadiahangar, R., Rosin, A., & Anvari-Moghaddam, A. (2022). Day-Ahead Scheduling of Electric Vehicles and Electrical Storage Systems in Smart Homes Using a Novel Decision Vector and AHP Method. Sustainability, 14(18), 1-20. Article 11773. https://doi.org/10.3390/su141811773 General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. Citation for published version (APA): Alilou , M., Gharehpetian, G. B., Ahmadiahangar, R., Rosin, A., & Anvari-Moghaddam, A. (2022). Day-Ahead Scheduling of Electric Vehicles and Electrical Storage Systems in Smart Homes Using a Novel Decision Vector and AHP Method. Sustainability, 14(18), 1-20. Article 11773. https://doi.org/10.3390/su141811773 Aalborg Universitet Day-Ahead Scheduling of Electric Vehicles and Electrical Storage Systems in Smart Homes Using a Novel Decision Vector and AHP Method Alilou , Masoud ; Gharehpetian, Gevork B.; Ahmadiahangar, Roya; Rosin, Argo; Anvari- Moghaddam, Amjad Published in: Aalborg Universitet Day-Ahead Scheduling of Electric Vehicles and Electrical Storage Systems in Smart Homes Using a Novel Decision Vector and AHP Method Masoud Alilou 1, Gevork B. Gharehpetian 2, Roya Ahmadiahangar 3,4, Argo Rosin 3,4 and Amjad Anvari‐Moghaddam 5,* 1 Faculty of Electrical and Computer Engineering, University of Tabriz, Tabriz 5166616471, Iran 1 Faculty of Electrical and Computer Engineering, University of Tabriz, Tabriz 5166616471, Iran 2 Electrical Engineering Department, Amirkabir University of Technology, Tehran 1591634311, Iran 1 Faculty of Electrical and Computer Engineering, University of Tabriz, Tabriz 5166616471, Iran 2 Electrical Engineering Department, Amirkabir University of Technology, Tehran 1591634311, Iran 3 Department of Electrical Power Engineering & Mechatronics, Tallinn University of Technology, 19086 Tallinn, Estonia Electrical Engineering Department, Amirkabir University of Technology, Tehran 1591634311, Iran 3 Department of Electrical Power Engineering & Mechatronics, Tallinn University of Technology, 19086 Tallinn, Estonia 4 Smart City Center of Excellence (Finest Twins), Tallinn University of Technology, 19086 Tallinn, Estonia 5 Department of Energy (AAU Energy), Aalborg University, 9220 Aalborg, Denmark * Correspondence: aam@energy aau dk 4 Smart City Center of Excellence (Finest Twins), Tallinn University of Technology, 19086 Tallinn, Estonia 5 Department of Energy (AAU Energy), Aalborg University, 9220 Aalborg, Denmark * Correspondence: aam@energy.aau.dk Abstract: The two‐way communication of electricity and information in smart homes facilitates the optimal management of devices with the ability to charge and discharge, such as electric vehicles and electrical storage systems. These devices can be scheduled considering domestic renewable energy units, the energy consumption of householders, the electricity tariff of the grid, and other predetermined parameters in order to improve their efficiency and also the technical and economic indices of the smart home. In this paper, a novel framework based on decision vectors and the an‐ alytical hierarchy process method is investigated to find the optimal operation schedule of these devices for the day‐ahead performance of smart homes. The initial data of the electric vehicle and the electrical storage system are modeled stochastically. The aim of this work is to minimize the electricity cost and the peak demand of the smart home by optimal operation of the electric vehicle and the electrical storage system. Firstly, the different decision vectors for charging and discharg‐ ing these devices are introduced based on the market price, the produce power of the domestic photovoltaic panel, and the electricity demand of the smart home. Secondly, the analytical hierar‐ chy process method is utilized to implement the various priorities of decision criteria and calculate the ultimate decision vectors. Day‐Ahead Scheduling of Electric Vehicles and Electrical Storage Systems in Smart Homes Using a Novel Decision Vector and AHP Method Masoud Alilou 1, Gevork B. Gharehpetian 2, Roya Ahmadiahangar 3,4, Argo Rosin 3,4 and Amjad Anvari‐Moghaddam 5,* General rights C i h d - Users may download and print one copy of any publication from the public portal for the purpose of private study or research. - You may not further distribute the material or use it for any profit-making activity or commercial gain - Users may download and print one copy of any publication from the public portal for the purpose of private study or research. - You may not further distribute the material or use it for any profit-making activity or commercial gain - Users may download and print one copy of any publication from the public portal for the purpose of private study or re You may not further distribute the material or use it for any profit making activity or commercial gain You may not further distribute the material or use it for any profit making activity o - You may freely distribute the URL identifying the publication in the public portal - y y p g y - You may freely distribute the URL identifying the publication in the public portal - Take down policy If you believe that this document breaches copyright please contact us at vbn@aub.aau.dk providing details, and w the work immediately and investigate your claim. y this document breaches copyright please contact us at vbn@aub.aau.dk providing details, and we will remove access to ely and investigate your claim. Masoud Alilou 1, Gevork B. Gharehpetian 2, Roya Ahmadiahangar 3,4, Argo Rosin 3,4 and Amjad Anvari‐Moghaddam 5,* Finally, the operation schedule of the electric vehicle and the elec‐ trical storage system is selected based on the ultimate decision vectors considering the operational constraints of these devices and the constraints of charging and discharging priorities. The pro‐ posed method is applied to a sample smart home considering different priorities of decision crite‐ ria. Numerical results present that although the combination of decision criteria with a high rank of electricity demand has the highest improvement of technical and economic indices of the smart home by about 12 and 26%, the proposed method has appropriate performance in all scenarios for selecting the optimal operation schedule of the electric vehicles and the electrical storage system. Citation: Alilou, M.; Gharehpetian, G.B.; Ahmadiahangar, R.; Rosin, A.; Anvari‐Moghaddam, A. Day‐Ahead Scheduling of Electric Vehicles and Electrical Storage Systems in Smart Homes Using a Novel Decision Vector and AHP Method. Sustainability 2022, 14, 11773. https://doi.org/10.3390/su141811773 Academic Editor: Fausto Cavallaro Received: 22 August 2022 Accepted: 16 September 2022 Published: 19 September 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Keywords: analytical hierarchy process; decision vector; electric vehicles; electrical storage system; probabilistic model; renewable energy 1. Introduction Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/license s/by/4.0/). Developing new technologies, increasing environmental pollution, and the ten‐ dency of utilizing green energies cause growth in the penetration rate of electric vehicles (EVs) in the transportation network [1]. The concept of the smart home (SH) with two‐way electricity communication and a smart controllable system facilitates the usage of EVs with vehicle‐to‐home technology. In a SH, the operation schedule of an EV can be optimized properly for improving its performance [2]. In addition to EVs, the electrical www.mdpi.com/journal/sustainability Sustainability 2022, 14, 11773. https://doi.org/10.3390/su141811773 Sustainability 2022, 14, 11773 2 of 20 2 of 20 storage system (ESS) is another useful device that can be used to better match the gener‐ ation and consumption patterns of SHs [3]. Of course, finding the appropriate charging and discharging times of an EV and ESS based on the availability of the EV, the stored energy of the ESS, the electricity situation of the SH, the market price, and other prefer‐ ences are very important for improving the efficiency of EVs, ESSs, and SHs [4]. Photo‐ voltaic panels (PVs), which can be placed on a SH’s roof or in the unused parts of the yard, act as a domestic renewable energy source affecting the energy situation of the SH and the charging/discharging time of the EV and ESS [5]. The usage of a central man‐ agement system in a SH for considering various criteria such as the availability of PV power, market price, and electricity demand of the SH is very useful for selecting the proper operation schedule of the EV and ESS [6]. The management of EVs and ESSs has been studied in SHs in the last years. A liter‐ ature review based on the technologies and economic models of EVs and ESSs has been presented in ref. [7]. In this study, the authors reviewed the relevant literature on busi‐ ness models of EVs and also mentioned key points about each business model. Ref. [8] is another review paper about EVs. The main purpose of this study is the energy storage parts of EVs. In other words, the storage technologies of EVs were studied within the view of eco‐friendly and cost‐effective purposes. 1. Introduction Yang and Zhang presented a review of various types of EVs and the combination of them and different distributed energy sources in ref. [9]. Moreover, different scheduling methods of virtual power plants in the presence of EVs were compared with each other in this study. In Ref. [10], the EV routing problem has been comprehensively reviewed. The definition of the routing problem and the modeling of EVs for solving this problem have been presented in this study, and the developed solution approaches are also pondered. In ref. [11], Kajanova and Bracinik presented a method for controlling the charge time of EVs in a microgrid‐connected grid with renewable energy units. The generated power of local renewable energy units is used to charge EVs and also maximize social welfare. The proposed problem was solved using an optimal control method with the model predictive approach. The potential en‐ vironmental effect of using EV batteries as storage on the energy system as it moves to‐ wards the goal of net‐zero emissions in 2050 has been evaluated in ref. [12]. This trans‐ portation method is a significant step towards a more sustainable energy system to meet climate change mitigation. In ref. [13], a charging and navigation strategy for EVs has been investigated considering the difference in user time utility. The purpose of the proposed method is to find the fastest way for charging EVs. Moreover, it is considered that each vehicle driver can select different navigation routes according to his/her pref‐ erence. The authors of ref. [14] have presented a component sizing of a series hybrid EVs. The models of the combustion engine, the high voltage battery, and the electric motor have been investigated using experimental information. These experimental data are utilized to produce a robust artificial neural network model for EVs. In ref. [15], a method based on a genetic algorithm has been proposed to find the best location for finding EV charging stations. In the proposed method, the open data sources are analyzed during the optimization. Moreover, an agent‐based simulation framework is used around a fleet simulator. The study of energy systems, which is the combination of power generation systems and energy storage systems, has been investigated in ref. [16]. The obtained en‐ ergy from the power generation system is stored by the ESS to reduce the effect of peak loads on the distribution system. 1. Introduction The method for using an ESS has been proposed in ref. [17] in order to mitigate the intermittency of renewable energy units. In this study, the capacity of renewable distributed generation units is selected considering the minimiza‐ tion of the load of the network, while the size and the operational schedule of the ESS is optimized by meta‐heuristic algorithm and mixed‐integer linear programming. Mehr‐ jerdi and Rakhshani presented a new model of energy storage systems in buildings in ref. [18]. The operational schedule of the ESS is optimized in this study in order to reduce the daily energy cost of the building that is connected to the electrical grid. The mixed‐integer binary linear programming method is utilized to solve the proposed en‐ Sustainability 2022, 14, 11773 3 of 20 3 of 20 ergy management program. The utilization of ESS with optimal schedule besides PVs has been investigated in ref. [19] in order to improve the technical and economic objective functions. Different types of batteries have been studied and evaluated based on tech‐ no‐economic indices. The authors of ref. [20] proposed a frequency‐based energy man‐ agement method for optimizing the operational schedule of distributed generation units and ESS in a residential microgrid. The reduction in the total energy consumption of the microgrid and the control of the frequency and the peal load of the microgrid are the main purposes of this study that are improved considerably after applying the proposed method. In ref. [21], a review of smart home energy management systems has been pre‐ sented. The effect of demand response programs on home energy units has been evalu‐ ated in this study. A comprehensive scheduling for the day‐ahead operation of EVs has been proposed in order to maximize the cost‐benefit objective function in ref. [22]. In an‐ other study, the management of a residential system in the presence of PVs and ESS has been investigated [23]. In ref. [24], the authors proposed an energy management method for optimal operation of EVs in an SH. The reduction in the effect of the stochastic be‐ havior of renewable energy units on SHs’ energy is the main goal of this study. An op‐ timal home energy management method has been proposed in ref. [25] considering home energy sources, ESSs, and EVs. The minimization of the electricity cost and the peak‐to‐average rate is the purposes of the optimization. 1. Introduction In another study, the home en‐ ergy management system has been investigated in the presence of PVs, ESSs, and EVs in order to minimize the electricity cost, customer dissatisfaction, and the peak‐to‐average rate [26]. The authors of ref. [27] have proposed a home energy management system based on intelligent algorithms in order to optimize the techno‐economic objective func‐ tions. The optimal energy management of a residential microgrid has been investigated in ref. [28] in order to increase the efficiency of EVs. The energy management of EVs and ESSs in addition to renewable energy units in the residential microgrids has been studied in refs. [29] and [30]. In these papers, the authors proposed optimal operational programs in order to increase the performance of the microgrid. In ref. [31], the energy manage‐ ment of multi‐microgrids in the presence of SHs has been investigated. The management of EVs and ESSs in the energy hub platform has been studied in refs. [32–34]. The per‐ formance of these devices in the presence of demand response programs and local re‐ newable resources has been improved in these papers. In the mentioned papers, the operational schedule of EVs and ESSs has been achieved separately or simultaneously. In the common scheduling of an EV and an ESS, they are modeled mathematically, and then their charging and discharging times are op‐ timized in order to minimize or maximize technical and economic goals. In this study, we propose a novel approach based on innovative decision vectors and the AHP method to simultaneously optimize the operational schedule of an EV and ESS. In the proposed approach, their charging and discharging times are selected optimally considering the situation of the SH, domestic energy unit, and the economic parameters which are mod‐ eled in the new decision vectors. Indeed, in the proposed approach, their operational schedule is selected considering the predetermined vectors without the direct minimiza‐ tion or maximization of economic and technical objective functions. Although the tech‐ no‐economic indices are only considered for evaluating the approach, their value is re‐ duced after applying the proposed energy management strategy. Moreover, the opera‐ tional schedule of EVs and ESSs can be achieved using the proposed approach faster than the common methods. So, the current paper presents a novel framework for scheduling the charge and discharge times of EVs and the ESS in an SH. 1. Introduction The main highlights of this manuscript are as follows:  A new approach for the energy management of an EV and an ESS is proposed by considering the PVs, the variable load of the SH, and the electricity tariff.  A new approach for the energy management of an EV and an ESS is proposed by considering the PVs, the variable load of the SH, and the electricity tariff.  A novel decision vector is defined for the charging and discharging of EVs and ESSs based on the market price, the produced power of the PVs, and energy consumption of the householder. Sustainability 2022, 14, 11773 4 of 20 4 of 20  The analytical hierarchy process (AHP) method is utilized to implement various priorities of decision criteria and to calculate the ultimate decision vectors.  The analytical hierarchy process (AHP) method is utilized to implement various priorities of decision criteria and to calculate the ultimate decision vectors.  The optimal operation schedule of an EV and ESS is selected based on the ultimate decision vectors considering the operational constraints of these devices and the constraints of charging and discharging priorities.  The optimal operation schedule of an EV and ESS is selected based on the ultimate decision vectors considering the operational constraints of these devices and the constraints of charging and discharging priorities.  The main aim of the management problem is to minimize the electricity cost and peak demand of SHs during the day.  The main aim of the management problem is to minimize the electricity cost and peak demand of SHs during the day. So, in this paper, the AHP method is utilized to combine the decision vectors of charg‐ ing and discharging of EVs and ESSs, which are calculated based on the values of market price, the power of the PVs, and the demand of the SH at different times, to select the optimal operation schedule of the EV and ESS. It is worth mentioning that the electricity demand of the SH is variable, and the home buys its required energy from the distribution system with a variable market price. The technical and economic indices are considered for evaluating dif‐ ferent priorities and evaluating the performance of the proposed method in selecting the best charging and discharging time for private vehicles and batteries. 2. Proposed Management Method In this section, firstly, the energy structure of an SH, which consists of the load, PVs, EV, and ESS, is explained. Then, the proposed method for optimally scheduling an EV and ESS is introduced. 2.1. Energy Structure of the SH 𝑝ሺௌைாሻൌ𝑓்ீሺ𝑋; 𝜇ௌைா; 𝜎ௌைா ଶ ; 𝑥௠௜௡ ௌைா; 𝑥௠௔௫ ௌைாሻ (2) 𝑝ሺௌைாሻൌ𝑓்ீሺ𝑋; 𝜇ௌைா; 𝜎ௌைா ଶ ; 𝑥௠௜௡ ௌைா; 𝑥௠௔௫ ௌைாሻ (2) 𝑝ሺ௫ሻൌ𝑓்ீሺ𝑋; 𝜇௑; 𝜎௑ ଶ; 𝑥௠௜௡ ௑ ; 𝑥௠௔௫ ௑ ሻ (3) (3) According to these equations, the probability of each parameter (𝑥) is calculated using its average (𝜇௑), variance (𝜎௑ ଶ), minimum (𝑥௠௜௡ ௑ ), and maximum (𝑥௠௔௫ ௑ ). The com‐ bination of the Latin hypercube sampling algorithm and the K‐means method is utilized to calculate the stochastic amount of initial SOE of the ESS, the initial SOC, departure time, and arrival time of the EV. 2.1. Energy Structure of the SH The energy of the considered SH can be divided into five parts, including the elec‐ tricity demand of home appliances, the energy of the PVs, the energy of the EV, the en‐ ergy of the ESS, and the energy of the distribution system. The overall energy structure of the SH is shown in Figure 1. The total load of the SH is considered in the proposed method. The load is variable during the day. If the power of the PVs is unavailable, the SH buys all required energy from the distribution system with a variable market price. On the other hand, when solar irradiance is available and the PVs generate electricity, the SH buys the extra demand from the distribution system or sells the extra produced power to the distribution system. The produced power of the PVs at each hour (𝑃௉௏) can be calculated by Equation (1) [4]. Figure 1. The overall energy structure of the SH formatting 𝑃௉௏ൌ𝜂௉௏ൈ𝐴௉௏ൈ𝑟௦௜ (1) Figure 1. The overall energy structure of the SH formatting (1) 𝑃௉௏ൌ𝜂௉௏ൈ𝐴௉௏ൈ𝑟௦௜ 𝑃௉௏ൌ𝜂௉௏ൈ𝐴௉௏ൈ𝑟௦௜ Sustainability 2022, 14, 11773 5 of 20 5 of 20 where 𝜂௉௏ and 𝐴௉௏ show the efficiency and area of the PVs. 𝑟௦௜ is the value of solar ir‐ radiance. Moreover, it is considered that the PVs is operated with a battery in order to increase the stability and inject the predicted power at each hour. Another energy part of the SH is related to the hourly power of the EV and ESS. Although the details of the optimal operation schedule of them are explained completely in the next sections, the initial data of the EV and ESS and the method of their calculation are presented here. The initial state of energy (SOE) of the ESS, the initial state of charge (SOC), the departure time of the EV from the parking, and the arrival time to the SH are the parameters of the EV that are stochastic and variable daily. For this reason, the sto‐ chastic model of these parameters is defined using the truncated Gaussian function. Equation (2) is the probability distribution function of the SOE of the ESS, and Equation (3) shows the probability distribution function of the stochastic parameters of the EV [35]. 2.2. Method of Scheduling In the proposed method, the optimal operation schedule of the EV and ESS is cal‐ culated using the decision vector of charging and discharging based on various criteria, including the market price, the produced power of the PVs, and the electricity demand of the SH. The AHP method is used to implement the priority of criteria in selecting the best operation schedule of the EV and ESS. Their constraints are also considered during the management in order to notice the operation conditions of them. Finally, the technical and economical indices are utilized to evaluate the performance of the proposed method and also the different priorities of criteria in the AHP method. The flowchart of the pro‐ posed management method of the EV and ESS is demonstrated in Figure 2. The method of calculating the decision vectors of the charging and discharging of the EV and ESS based on the various criteria is explained in Section 3. Section 4 is related to the way of implementation of the AHP method. The constraints of the management problem are formulated in Section 5, and the evaluation indices are explained in Section 6. Sustainability 2022, 14, 11773 6 of 20 Figure 2. The flowchart of the proposed management method of the EV and ESS. To input daily market price To calculate the decision vectors based on the market price To define the various priorities of criteria (MP, PV, and Demand) To implement the AHP method based on various priorities for calculating the ultimate decision vectors To calculate stochastic parameters of EV and ESS and To schedule the operational plan of the EV and ESS based on the decision vectors of the AHP method in various priorities To reschedule the operational plan of EV and ESS considering the constraints (Constraints of EV, constraints of ESS, and constraints of charging and discharging priorities) To select the optimal operational schedule of the EV and ESS and also the best priority of the criteria To calculate the evaluation indices in various priorities To calculate daily produced power of the PV To calculate the decision vectors based on the photovoltaic panel To input daily demand of the SH To calculate the decision vectors based on the demand Figure 2. The flowchart of the proposed management method of the EV and ESS. 3.1. Based on the Market Price In the last years, the variable market price is implemented in the distribution sys‐ tems in order to encourage end‐users to reduce their consumption during peak hours. Based on the market price, it is proper that the EV or ESS charges when the market price is low. On the other hand, the hours with high market prices are the appropriate times for discharging. Therefore, the desirability of charging has an inverse relation with the market price. In other words, the desirability of charging is increased when the market price decreases. On the other hand, the desirability of discharging, which has a direct relation with the market price, is increased when the market price increases. Mathematically, the desirability of charging (𝑐௜_ெ௉) and discharging (𝑑௜_ெ௉) based on the market price is presented in Eqns. (6) and (7). 𝑐௜_ெ௉ൌ1 െ𝑀𝑃௜,௉௨ (6) 𝑑௜_ெ௉ൌ𝑀𝑃௜,௣௨ (7) 𝑐௜_ெ௉ൌ1 െ𝑀𝑃௜,௉௨ (6) 𝑑௜_ெ௉ൌ𝑀𝑃௜,௣௨ (7) 𝑀𝑃௜௉௨ൌ 𝑀𝑃௜ (8) 𝑐௜_ெ௉ൌ1 െ𝑀𝑃௜,௉௨ (6) (6) ௜_ெ௉ ௜,௉௨ ( ) 𝑑௜_ெ௉ൌ𝑀𝑃௜,௣௨ (7) 𝑑௜_ெ௉ൌ𝑀𝑃௜,௣௨ (7) (7) where 𝑀𝑃௜,௉௨ൌ 𝑀𝑃௜ 𝑀𝑃௠௔௫ (8) (8) Here, 𝑀𝑃௜ is the market price at hour i and 𝑀𝑃௠௔௫ shows the daily maximum market price. In Figure 3, the hourly desirability of charging and discharging based on the sample market price is demonstrated. 0 0.2 0.4 0.6 0.8 1 1.2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Market price ($) Desirability of charging Desirability of discharging 0 0.2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Figure 3. The hourly desirability of charging and discharging of the EV and ESS based on the sample market price. 3. Decision Vectors In the proposed method, the decision vectors are defined for charging and dis‐ charging of the EV and ESS based on the market price, the produced power of the PVs, and the load of the SH. The daily decision vectors of the charging and discharging of the EV and ESS based on various parameters are defined as follows: 𝐷𝑉஼ൌሾ𝑐ଵ, 𝑐ଶ, … , 𝑐௜, … 𝑐்ିଵ, 𝑐்ሿ (4) 𝐷𝑉஽ൌሾ𝑑ଵ, 𝑑ଶ, … , 𝑑௜, … 𝑑்ିଵ, 𝑑்ሿ (5) (4) (5) In these equations, 𝐷𝑉஼ and 𝐷𝑉஽ are the decision vectors of charging and dis‐ charging, respectively. 𝑐௜ shows the desirability rate of charging at hour i, which is a number between 0 and 1. On the other hand, 𝑑௜, which has a number between 0 and 1, is the desirability rate of discharging at hour i. The method of calculating these decision vectors at each hour based on the different parameters is presented in the following. Sustainability 2022, 14, 11773 7 of 20 7 of 20 3.2. Based on the Power of the PVs Domestic PVs are one of the eco‐friendly methods for decreasing the dependence of the SH on the energy of the distribution system. Based on the produced power of the PVs, it is reasonable that the EV or ESS charges when the produced power of the PVs is high. On the other hand, the hours that the power of the PVs is low or its power is unavailable are the appropriate times for discharging. pp p g g Thus, the desirability of charging has a direct relationship with the produced power of the PVs. In other words, the desirability of charging is increased when the power of the PVs increases. On the other hand, the desirability of discharging, which has an inverse relationship with the power of the PVs, is increased when the market price declines. Mathematically, the desirability of charging (𝑐௜_௉௏) and discharging (𝑑௜_௉௏) based on the produced power of the PVs is presented in Equations (9) and (10). Sustainability 2022, 14, 11773 8 of 20 𝑐௜_௉௏ൌ𝑃_𝑃𝑉௜,௉௨ (9) 𝑑௜_௉௏ൌ1 െ𝑃_𝑃𝑉௜,௣௨ (10) 𝑐௜_௉௏ൌ𝑃_𝑃𝑉௜,௉௨ (9) 𝑑௜_௉௏ൌ1 െ𝑃_𝑃𝑉௜,௣௨ (10) 𝑃𝑃𝑉 𝑃_𝑃𝑉௜ (11) (9) ௜_௉௏ ௜,௉௨ ( ) 𝑑௜_௉௏ൌ1 െ𝑃_𝑃𝑉௜,௣௨ (10) (10) where where 𝑃_𝑃𝑉௜,௣௨ൌ 𝑃_𝑃𝑉௜ 𝑃_𝑃𝑉௠௔௫ (11) (11) Here, 𝑃_𝑃𝑉௜ is the produced power of the PVs at hour i and 𝑃_𝑃𝑉௠௔௫ shows the daily maximum power of the PVs. Here, 𝑃_𝑃𝑉௜ is the produced power of the PVs at hour i and 𝑃_𝑃𝑉௠௔௫ shows the daily maximum power of the PVs. The hourly desirability of charging and discharging based on the sample produced power of the PVs is shown in Figure 4. Figure 4. The hourly desirability of charging and discharging of the EV and ESS based on the sample power of the PVs. 0 0.5 1 1.5 2 2.5 3 3.5 4 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Power of PV (kW) Desirability of charging Desirability of discharging Figure 4. The hourly desirability of charging and discharging of the EV and ESS based on the sample power of the PVs. 3.3. Based on the Load of the Home Another important subject in the scheduling of the EV is the load of the home. Ac‐ cording to the hourly variation of the demand of the SH and considering the hours with low, average, and peak demand, it is reasonable that the EV or ESS are charged during the off‐peak times. So, based on the home’s electricity demand, it is proper that they charge when the demand is low. On the other hand, the hours with high electricity de‐ mand are the appropriate times for discharging. Therefore, the desirability of charging has an inverse relation with the load of the home. In other words, the desirability of charging is increased when the demand de‐ creases. On the other hand, the desirability of discharging, which has a direct relationship with the load of the home, is raised when the value of the demand increases. Mathematically, the desirability of charging (𝑐௜_஽௘௠௔௡ௗ) and discharging (𝑑௜_஽௘௠௔௡ௗ) based on the demand of the SH is presented in Eqns. (12) and (13). 𝑐௜_஽௘௠௔௡ௗൌ1 െ𝑃_𝐷𝑒𝑚𝑎𝑛𝑑௜,௉௨ (12) 𝑑௜_஽௘௠௔௡ௗൌ𝑃_𝐷𝑒𝑚𝑎𝑛𝑑௜,௣௨ (13) 𝑐௜_஽௘௠௔௡ௗൌ1 െ𝑃_𝐷𝑒𝑚𝑎𝑛𝑑௜,௉௨ (12) (12) (13) where 𝑃_𝐷𝑒𝑚𝑎𝑛𝑑௜,௣௨ൌ 𝑃_𝐷𝑒𝑚𝑎𝑛𝑑௜ 𝑃_𝐷𝑒𝑚𝑎𝑛𝑑௠௔௫ (14) (14) Here, 𝑃_𝐷𝑒𝑚𝑎𝑛𝑑௜ is the electricity demand of the SH at hour i, and 𝑃_𝐷𝑒𝑚𝑎𝑛𝑑௠௔௫ shows the daily maximum demand. In Figure 5, the hourly desirability of charging and discharging based on the sample demand of the SH is demonstrated. 9 of 20 Sustainability 2022, 14, 11773 Figure 5. The hourly desirability of charging and discharging of the EV and ESS based on the sample electricity demand. 0 1 2 3 4 5 6 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Demand (kW) Desirability of charging Desirability of discharging Demand (kW) Desirability of charging Desirability of discharging Figure 5. The hourly desirability of charging and discharging of the EV and ESS based on the sample electricity demand. EVs have some constraints that should be considered during the selection of their operating schedule. The constraints are presented in the following. 4. Analytical Hierarchy Process Method The AHP method is the practical method for selecting the best compromise solution when there are some decision criteria [36]. Although in the proposed management method the best charge and discharge schedule of the EV and ESS can be selected inde‐ pendently based on the mentioned criteria, including the market price, the produced power of the PVs, and the demand of the SH, the AHP method is utilized to select their best operation schedule considering all decision criteria. The ultimate decision vectors of charging and discharging are calculated using the AHP method after defining initial vectors based on the market price, the produced power of the PVs, and the demand of the SH. In the AHP method, firstly, the judgment matrix is created based on the priority of decision criteria. Then, the arithmetic mean method is used to calculate the final weight of each index ( 𝑤௜). Finally, the desirability of charging (𝑐௜_஺ு௉) and discharging (𝑑௜_஺ு௉) at hour i is calculated by Eqns. (15) and (16) [36]. 𝑐௜ಲಹುൌ൫ 𝑤௜ಾುൈ𝑐௜ಾು൯൅൫ 𝑤௜ುೇൈ𝑐௜ುೇ൯൅൫ 𝑤௜ವ೐೘ೌ೙೏ൈ𝑐௜ವ೐೘ೌ೙೏൯ (15) 𝑑௜_஺ு௉ൌ൫ 𝑤௜ಾುൈ𝑑௜ಾು൯൅൫ 𝑤௜ುೇൈ𝑑௜ುೇ൯൅൫ 𝑤௜ವ೐೘ೌ೙೏ൈ𝑑௜ವ೐೘ೌ೙೏൯ (16) (15) (16) For selecting the best combination or priorities of the mentioned criteria in the AHP method, 13 cases are considered. The priority of the criteria in these cases is presented in Table 1. These cases, which are all possible priorities, are defined in order to select the best case based on the numerical results. In the first three cases, only one decision index is considered; zero (0) means that the criterion is not considered. In other cases, 1 and 3 have the highest and lowest priorities, respectively. The judgment matrix is defined based on the priorities, and then the final weights are calculated using the arithmetic mean method. In Table 2, the judgment matrix and final weight of criteria in various cases are presented. Table 1. The priority of the criteria in various cases. Cases Priority of Each Criterion Market Price Power of PVs Demand of Home 1 1 0 0 2 0 1 0 3 0 0 1 4 1 1 1 5 1 2 2 6 1 2 3 7 1 3 2 Table 1. The priority of the criteria in various cases. Table 1. The priority of the criteria in various cases. 5.1. Constraints of EVs 5. Constraints of Management 5.1. Constraints of EVs 4. Analytical Hierarchy Process Method Sustainability 2022, 14, 11773 10 of 20 8 2 1 2 9 2 1 3 10 3 1 2 11 2 2 1 12 2 3 1 13 3 2 1 Table 2. The judgment matrix and final weight of criteria in different cases. Table 2. The judgment matrix and final weight of criteria in different cases. Table 2. The judgment matrix and final weight of criteria in different cases. Cases Judgment Matrix Final Weight MP PVs Demand 1 MP 1.00 0.00 0.00 1.000 2 PVs 0.00 1.00 0.00 1.000 3 Demand 0.00 0.00 1.00 1.000 4 MP 1.00 1.00 1.00 0.333 PVs 1.00 1.00 1.00 0.333 Demand 1.00 1.00 1.00 0.333 5 MP 1.00 2.00 2.00 0.500 PVs 0.50 1.00 1.00 0.250 Demand 0.50 1.00 1.00 0.250 6 MP 1.00 2.00 3.00 0.545 PVs 0.50 1.00 1.50 0.272 Demand 0.33 0.66 1.00 0.181 7 MP 1.00 3.00 2.00 0.545 PVs 0.33 1.00 0.66 0.181 Demand 0.50 1.50 1.00 0.272 8 MP 1.00 0.50 1.00 0.250 PVs 2.00 1.00 2.00 0.500 Demand 1.00 0.50 1.00 0.250 9 MP 1.00 0.50 1.50 0.272 PVs 2.00 1.00 3.00 0.545 Demand 0.66 0.33 1.00 0.181 10 MP 1.00 0.33 0.66 0.181 PVs 3.00 1.00 2.00 0.545 Demand 1.50 0.50 1.00 0.272 11 MP 1.00 1.00 0.50 0.250 PVs 1.00 1.00 0.50 0.250 Demand 2.00 2.00 1.00 0.50 12 MP 1.00 1.50 0.50 0.272 PVs 0.66 1.00 0.33 0.181 Demand 2.00 3.00 1.00 0.545 13 MP 1.00 0.66 0.33 0.181 PVs 1.50 1.00 0.50 0.272 Demand 3.00 2.00 1.00 0.545 5. Constraints of Management 5.2.1. The Limit of Hourly Charging and Discharging 5.2.1. The Limit of Hourly Charging and Discharging 5.2.1. The Limit of Hourly Charging and Discharging The value of hourly charging and discharging of the ESS should be in a range be‐ tween its minimum and maximum rates. The ranges of hourly charging and discharging of the ESS are presented in Eqns. (21) and (22). In these equations, 𝐸𝑆𝑆௖_௜ and 𝐸𝑆𝑆ௗ_௜ are the charged and discharged energy of the ESS at hour i. The parameters 𝐸𝑆𝑆஼_௠௔௫ and 𝐸𝑆𝑆ௗ_௠௔௫ show the maximum rate of charge and discharge of the ESS at each hour [16]. 0 ൑𝐸𝑆𝑆௖_௜൑𝐸𝑆𝑆஼_௠௔௫ (21) 0 ൑𝐸𝑆𝑆௖_௜൑𝐸𝑆𝑆஼_௠௔௫ (21) 0 ൑𝐸𝑆𝑆ௗ_௜൑𝐸𝑆𝑆ௗ_௠௔௫ (22) 0 ൑𝐸𝑆𝑆௖_௜൑𝐸𝑆𝑆஼_௠௔௫ (21) (21) (22) 5.1.3. The Limit of SOC It should be considered that the SOC of the EV at hour i (𝑆𝑂𝐶௜) does not violate its range between the minimum SOC (𝑆𝑂𝐶௠௜௡) and the maximum SOC (𝑆𝑂𝐶௠௔௫) of the EV according to Equation (20) [36]. 𝑆𝑂𝐶௠௜௡൑𝑆𝑂𝐶௜൑𝑆𝑂𝐶௠௔௫ (20) (20) 5.1.1. The SOC of the EV at Departure Time It is considered that the EV should have been fully charged at the departure time. Equation (17) shows this constraint [36]. 𝑆𝑂𝐶஽்ൌ𝑆𝑂𝐶௠௔௫ (17) (17) 𝑆𝑂𝐶஽்ൌ𝑆𝑂𝐶௠௔௫ Here, 𝑆𝑂𝐶஽் is the SOC of the EV at the departure time and 𝑆𝑂𝐶௠௔௫ is the maxi‐ mum SOC. 5.1.2. The Limit of Hourly Charging and Discharging The value of hourly charging and discharging of the EV should be in a range be‐ tween their minimum and maximum rate. The ranges of hourly charging and discharg‐ ing are presented in Eqns. (18) and (19) [36]. 0 ൑𝐸𝑉௖_௜൑𝐸𝑉஼_௠௔௫ (18) 0 ൑𝐸𝑉௖_௜൑𝐸𝑉஼_௠௔௫ (18) 0 ൑𝐸𝑉ௗ_௜൑𝐸𝑉ௗ_௠௔௫ (19) 0 ൑𝐸𝑉௖_௜൑𝐸𝑉஼_௠௔௫ (18) (18) 0 ൑𝐸𝑉ௗ_௜൑𝐸𝑉ௗ_௠௔௫ (19) (19) These equations present that the value of charge (𝐸𝑉௖_௜)/discharge (𝐸𝑉ௗ_௜) of the EV should be in the range between zero and the maximum rate of charge (𝐸𝑉஼_௠௔௫)/discharge (𝐸𝑉ௗ_௠௔௫). 5.2. Constraints of ESS The considered constraints of the ESS during the proposed energy management method are explained in the following. 5.1. Constraints of EVs EVs have some constraints that should be considered during the selection of their operating schedule. The constraints are presented in the following. Sustainability 2022, 14, 11773 11 of 20 11 of 20 5.1.1. The SOC of the EV at Departure Time 6. Evaluation Indices The technical and economic indices are considered for evaluating the performance of the proposed management method in finding the best operation schedule of the EV and ESS. The electricity cost of the SH is the economic index, and the peak demand is the considered technical index. 5.3.2. Priority of EV in Charging It is considered that the EV has the priority of charging when the calculated decision vector suggests charge mode for the EV and ESS. Equation (25) shows this constraint. 𝐼ாௌௌൌ0 𝑖𝑓 𝐼ா௏ൌ1 (25) (25) 5.3.3. Priority of ESS in Discharging It is considered that the ESS has the priority of discharging when the calculated de‐ cision vector suggests discharge mode for the EV and ESS. This constraint is considered by Equation (26) 𝐼ா௏ൌ0 𝑖𝑓 𝐼ாௌௌൌെ1 (26) (26) 5.3. Constraints of Charging and Discharging Priorities Three following constraints are also considered in order to find the optimum opera‐ tional schedule of the EV and ESS. 5.3.1. Inactivity of EV and ESS at the Same Time 5.3.1. Inactivity of EV and ESS at the Same Time It is considered that the performance of the EV and ESS (their charging and dis‐ charging actions) should not be at the same time in order to achieve more linear electric‐ ity variations of the SH. Mathematically, this constraint can be evaluated by Equation (24). (24) \|𝐼ா௏\| ൅\|𝐼ாௌௌ\| ൑1 (24) \|𝐼ா௏\| ൅\|𝐼ாௌௌ\| ൑1 Here, 𝐼ா௏ and 𝐼ாௌௌ show the operation mode of EV and ESS. Their value is −1, 0, and 1 when the EV or ESS is in discharge, idle, and charge mode. 5.2.2. The Limit of SOE It should be considered that the SOE of the ESS at hour i (𝑆𝑂𝐸௜) does not violate its range between the minimum SOE (𝑆𝑂𝐸௠௜௡) and the maximum SOE (𝑆𝑂𝐸௠௔௫) of the ESS according to Equation (23) [16]. 𝑆𝑂𝐸௠௜௡൑𝑆𝑂𝐸௜൑𝑆𝑂𝐸௠௔௫ (23) (23) 𝑆𝑂𝐸௠௜௡൑𝑆𝑂𝐸௜൑𝑆𝑂𝐸௠௔௫ 𝑆𝑂𝐸௠௜௡൑𝑆𝑂𝐸௜൑𝑆𝑂𝐸௠௔௫ (23) Sustainability 2022, 14, 11773 12 of 20 12 of 20 5.3. Constraints of Charging and Discharging Priorities 6.2. Peak Demand 6.2. Peak Demand Equation (32) is utilized for calculating the peak demand of the SH or the maximum dependence of the SH on the energy of the grid during a day. 𝑃𝐷ௌுൌ𝑚𝑎𝑥௜ୀଵ ଶସ ሼ𝑆𝐻௉೔ሽ (32) (32) Here, 𝑃𝐷ௌு is the daily peak demand, and 𝑆𝐻௉೔ is the demand of the SH at hour i. Here, 𝑃𝐷ௌு is the daily peak demand, and 𝑆𝐻௉೔ is the demand of the SH at hour i. 6.1. Electricity Cost For calculating this index, firstly, the hourly power situation of the SH should be calculated by Equation (27). 𝑆𝐻௉೔ൌ𝑆𝐻஽௘௠௔௡ௗ೔൅𝑆𝐻ா௏೔𝑚ா௏೔൅𝑆𝐻ாௌௌ೔𝑚ாௌௌ೔െ𝑆𝐻௉௏೔ (27) (27) In this equation, 𝑆𝐻஽௘௠௔௡ௗ೔, 𝑆𝐻ா௏೔, 𝑆𝐻ாௌௌ೔, and 𝑆𝐻௉௏೔ are the demand of the SH, the power of the EV, the power of the ESS, and the produced power of the PVs at hour i, re‐ spectively. 𝑚ா௏೔ and 𝑚ாௌௌ೔, which show the modes of the EV and ESS, are equal to +1 and −1 when the EV and ESS are in charge and discharge modes, respectively. 𝑆𝐻௉೔ shows the power situation of the SH at hour i. If the value of the power situation is higher than zero, the SH has to buy electricity from the distribution system. The cost of the bought energy at hour i (𝐶𝑜௜) is calculated by Equation (28). Here, 𝑀𝑃௜ is the market price at hour i. 𝐶𝑜௜ൌ𝑆𝐻௉೔ൈ𝑀𝑃௜ if 𝑆𝐻௉೔൐0 (28) (28) On the other hand, if the value of the power situation is lower than zero, the SH can sell the extra energy to the distribution system. Equation (29) presents the profit of the Sustainability 2022, 14, 11773 13 of 20 13 of 20 sold energy at hour i (𝑃𝑟௜). Here, 𝑡𝑟௜ is the tariff of the electricity that the distribution company buys from the end‐user at hour i. sold energy at hour i (𝑃𝑟௜). Here, 𝑡𝑟௜ is the tariff of the electricity that the distribution company buys from the end‐user at hour i. 𝑃𝑟௜ൌ𝑆𝐻௉೔ൈ𝑡𝑟௜ if 𝑆𝐻௉೔൏0 (29) (29) Thus, the electricity cost of the SH (𝐸𝐶ௌு) can be calculated by Equation (30). Thus, the electricity cost of the SH (𝐸𝐶ௌு) can be calculated by Equation (30). 𝐸𝐶ௌுൌ෍𝐶𝑜௜൅𝑃𝑟௜ ଶସ ௜ୀଵ (30) (30) where where ቊ 𝐶𝑜௜ൌ0 𝑖𝑓 𝑆𝐻௉೔൏0 𝑃𝑟௜ൌ0 𝑖𝑓 𝑆𝐻௉೔൐0 (31) (31) 7. Numerical Analysis 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Market price ($/kWh) Hour Figure 8. The variation of the solar irradiance during the day. The total capacity of the ESS is 10 kWh. The charging and discharging efficiency of the ESS is 0.95%. The mean value of the initial SOE of the ESS is 50 percent of the total SOE, while the lower limit of the SOE of the ESS is 20 percent of the total SOE to avoid deep discharging. The hourly charging and discharging limit of the battery of the ESS is 2.5 kW [40]. As mentioned above, firstly, the stochastic parameters of the EV and ESS are calcu‐ lated by the combination of the Latin hypercube sampling algorithm and the K‐means method. According to the results of the probabilistic program, the initial SOE of the ESS is 52.33%, the initial SOC of the EV is 44.73%, and the hours of departure and arrival of the EV are 7 and 19, respectively. p y In this step, the decision vectors are calculated using the AHP method considering various preferences of different criteria. As mentioned above (Table 2), 13 cases are con‐ sidered for evaluating the priorities and selecting the best operation schedule of the EV and ESS. Moreover, the results of the proposed method are also compared with the mentioned method in ref. [41]. The operation schedules of the EV and ESS in different cases are presented in Tables 3 and 4, respectively. In these tables, the positive and negative values show the charging and discharging modes, respectively, while the idle mode is represented by zero. As shown in these tables, the operation times are different based on preferences of market price, availability of PVs, and demand. In cases that the market price has the highest priority, the EV and ESS tend to charge when the market price is low and to discharge when the market price is high. In cases that the demand has the highest priority, they tend to charge when the electricity load of the SH is low and to discharge when the load is high. 7. Numerical Analysis In this section, the proposed management method of the EV and ESS is evaluated in a sample SH. It is worth mentioning that the time horizon of the analysis is the day‐ahead. The electricity demand of the SH is shown in Figure 6 [37]. The owner of the SH buys its required electricity from the distribution system with a variable market price that is demonstrated in Figure 7. On the other hand, the tariff of the bought energy from the SH by the distribution company is the average of the daily market price [38]. y p y g y p It is considered that the SH has the capacity for allocating 10 PVs with the technol‐ ogy of 335W SolarPower X21. Figure 8 shows the variation of the solar irradiance during the day [39]. The type of EV is a CHEVROLET VOLT. The capacity of its battery is 16 kWh. The maximum charge and discharge rate is 3.3 kWh with an efficiency of 95%. It is considered that the EV should be fully charged at the departure time. Moreover, it should not be discharged lower than 30% of its capacity because of the health and longevity of the bat‐ tery and the welfare of the consumer. The mean values of the initial SOC, departure time, and arrival time are 50%, 8 o’clock, and 19 o’clock, respectively [39]. Figure 6. The variation of the electricity demand of the SH. 0 1 2 3 4 5 6 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Demand (kW) Hour Figure 6. The variation of the electricity demand of the SH. 14 of 20 14 of 20 Sustainability 2022, 14, 11773 Figure 7. The market price of the distribution system. Figure 8. The variation of the solar irradiance during the day. 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Market price ($/kWh) Hour 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Solar irradiance (kW/m2) Hour Figure 7. The market price of the distribution system. 7. Numerical Analysis In cases that the availability of the PVs has the highest preference, the EV and ESS tend to charge when the rate of the produced power of the PVs is high. Of course, be‐ cause of the EV being out of the home in the middle time of the day, the EV tends to charge at hours that the PVs are available or close to the available times of the PVs. To‐ Sustainability 2022, 14, 11773 15 of 20 15 of 20 tally, the charging time of the EV mostly happens during the night, when the market price and demand are low, while the charging time of the ESS mostly happens during midday, when the power of the PVs is available. On the other hand, the ESS can be dis‐ charged at the end of the day to reduce the dependence of the SH on the energy of the distribution system. In the following, the performance of different cases, and also the proposed method, are pondered using the results of the evaluation indices. Table 3. The operation schedule of the EV. Hour Power of the EV at Each Hour in Various Cases Method of Ref. 7. Numerical Analysis [41] 1 2 3 4 5 6 7 8 9 10 11 12 13 1 0.00 0.00 3.30 0.00 0.00 0.00 0.00 0.00 0.00 0.00 3.30 3.30 3.30 2.71 2 0.00 0.00 3.30 2.71 0.00 0.00 0.00 2.71 0.00 3.30 3.30 3.30 3.30 0.00 3 2.71 0.00 2.71 3.30 3.30 3.30 3.30 3.30 2.71 3.30 2.71 2.71 2.71 3.30 4 3.30 0.00 0.00 3.30 3.30 3.30 3.30 3.30 3.30 2.71 0.00 0.00 0.00 3.30 5 3.30 2.71 0.00 0.00 2.71 2.71 2.71 0.00 0.00 0.00 0.00 0.00 0.00 0.00 6 0.00 3.30 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 7 0.00 3.30 0.00 0.00 0.00 0.00 0.00 0.00 3.30 0.00 0.00 0.00 0.00 0.00 8 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 9 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 10 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 11 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 12 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 13 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 14 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 15 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 16 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 17 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 18 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 19 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 20 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 21 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 22 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 23 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 24 3.30 0.00 3.30 0.00 0.00 0.00 3.30 0.00 0.00 0.00 0.00 3.30 0.00 3.30 Table 3. The operation schedule of the EV. Table 3. The operation schedule of the EV. Table 3. The operation schedule of the EV. 7. Numerical Analysis Hour Power of the EV at Each Hour in Various Cases Method of Ref. [41] 1 2 3 4 5 6 7 8 9 10 11 12 13 1 0.00 0.00 3.30 0.00 0.00 0.00 0.00 0.00 0.00 0.00 3.30 3.30 3.30 2.71 2 0.00 0.00 3.30 2.71 0.00 0.00 0.00 2.71 0.00 3.30 3.30 3.30 3.30 0.00 3 2.71 0.00 2.71 3.30 3.30 3.30 3.30 3.30 2.71 3.30 2.71 2.71 2.71 3.30 4 3.30 0.00 0.00 3.30 3.30 3.30 3.30 3.30 3.30 2.71 0.00 0.00 0.00 3.30 5 3.30 2.71 0.00 0.00 2.71 2.71 2.71 0.00 0.00 0.00 0.00 0.00 0.00 0.00 6 0.00 3.30 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 7 0.00 3.30 0.00 0.00 0.00 0.00 0.00 0.00 3.30 0.00 0.00 0.00 0.00 0.00 8 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 9 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 10 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 11 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 12 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 13 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 14 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 15 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 16 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 17 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 18 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 19 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 20 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 21 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 22 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 23 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 24 3.30 0.00 3.30 0.00 0.00 0.00 3.30 0.00 0.00 0.00 0.00 3.30 0.00 3.30 Table 4. The operation schedule of the ESS. 7. Numerical Analysis Hour Power of the ESS at Each Hour in Various Cases Method of Ref. [41] 1 2 3 4 5 6 7 8 9 10 11 12 13 1 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 2.375 2 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 2.375 3 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 4 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 5 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 −2.375 6 2.375 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 7 2.375 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 2.375 8 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 9 0.000 0.000 2.375 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 2.375 10 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 2.375 11 0.000 0.000 2.375 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 Table 4. The operation schedule of the ESS. 7. Numerical Analysis Sustainability 2022, 14, 11773 16 of 20 16 of 20 12 0.000 0.000 2.375 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 2.375 0.000 0.000 13 0.000 2.375 0.000 2.375 2.375 2.375 2.375 2.375 2.375 2.375 2.375 2.375 2.375 0.000 14 0.000 2.375 0.000 2.375 2.375 2.375 2.375 2.375 2.375 2.375 2.375 2.375 2.375 0.000 15 0.000 2.375 0.000 2.375 2.375 2.375 2.375 2.375 2.375 2.375 2.375 0.000 2.375 0.000 16 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 17 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 18 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 19 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 −2.375 20 0.000 0.000 −2.37 5 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 −2.375 21 −2.37 5 0.000 −2.37 5 −2.37 5 −2.37 5 −2.37 5 −2.37 5 0.000 0.000 0.000 −2.37 5 −2.37 5 −2.37 5 0.000 22 −2.37 5 −2.37 5 −2.37 5 −2.37 5 −2.37 5 −2.37 5 −2.37 5 −2.37 5 −2.37 5 −2.37 5 −2.37 5 −2.37 5 −2.37 5 0.000 23 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 2.375 24 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 2.375 23 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 2.375 24 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 2.375 The hourly power situation of the SH in different cases and based on the proposed method of ref. [41] is demonstrated in Figure 9. In this figure, the SH has to buy electricity from the distribution system with the market price when the value of the power is higher than zero. On the other hand, the owner of the SH sells energy to the distribution system with the predetermined tariff. Thus, the daily electricity cost is calculated using the hourly costs or profits. In Table 5, the amounts of the evaluation indices are presented. 7. Numerical Analysis -2 0 2 4 6 1 3 5 7 9 11 13 15 17 19 21 23 Power (kW) Hour Case 1 -2 0 2 4 6 8 1 3 5 7 9 11 13 15 17 19 21 23 Power (kW) Hour Case 2 -2 0 2 4 6 1 3 5 7 9 11 13 15 17 19 21 23 Power (kW) Hour Case 3 -2 0 2 4 6 1 3 5 7 9 11 13 15 17 19 21 23 Power (kW) Hour Case 4 -2 0 2 4 6 1 3 5 7 9 11 13 15 17 19 21 23 Power (kW) Hour Case 5 -2 0 2 4 6 1 3 5 7 9 11 13 15 17 19 21 23 Power (kW) Hour Case 6 -2 0 2 4 6 1 3 5 7 9 11 13 15 17 19 21 23 Power (kW) Hour Case 7 -2 0 2 4 6 1 3 5 7 9 11 13 15 17 19 21 23 Power (kW) Hour Case 8 -2 0 2 4 6 1 3 5 7 9 11 13 15 17 19 21 23 Power (kW) Hour Case 1 -2 0 2 4 6 1 3 5 7 9 11 13 15 17 19 21 23 Power (kW) Hour Case 3 -2 0 2 4 6 8 1 3 5 7 9 11 13 15 17 19 21 23 Power (kW) Hour Case 2 -2 0 2 4 6 1 3 5 7 9 11 13 15 17 19 21 23 Power (kW) Hour Case 4 -2 0 2 4 6 1 3 5 7 9 11 13 15 17 19 21 23 Power (kW) Hour Case 6 -2 0 2 4 6 1 3 5 7 9 11 13 15 17 19 21 23 Power (kW) Hour Case 8 -2 0 2 4 6 1 3 5 7 9 11 13 15 17 19 21 23 Power (kW) Hour Case 3 -2 0 2 4 6 1 3 5 7 9 11 13 15 17 19 21 23 Power (kW) Hour Case 5 -2 0 2 4 6 1 3 5 7 9 11 13 15 17 19 21 23 Power (kW) Hour Case 8 Case 8 17 of 20 Sustainability 2022, 14, 11773 Figure 9. Hourly power situation of the SH. 7. Numerical Analysis -2 0 2 4 6 8 1 3 5 7 9 11 13 15 17 19 21 23 Power (kW) Hour Case 9 -2 0 2 4 6 1 3 5 7 9 11 13 15 17 19 21 23 Power (kW) Hour Case 11 -2 0 2 4 6 1 3 5 7 9 11 13 15 17 19 21 23 Power (kW) Hour Case 13 -2 0 2 4 6 1 3 5 7 9 11 13 15 17 19 21 23 Power (kW) Hour Case 10 -2 0 2 4 6 1 3 5 7 9 11 13 15 17 19 21 23 Power (kW) Hour Case 12 -2 0 2 4 6 8 1 3 5 7 9 11 13 15 17 19 21 23 Power (kW) Hour Method of Ref. [41] Case 10 Figure 9. Hourly power situation of the SH. In cases where only one decision criterion is considered (Cases 1–3), case 1 has the lowest electricity cost, while case 3 has the lowest peak demand. This difference is be‐ cause of the different decision vectors. Cases 5 and 6 have the lowest electricity cost, with USD 5.549, and cases 11 and 13 have the lowest peak demand, with 4.83 kW. For selecting the best case, the AHP method is utilized again. In this step, the weight of each evaluat‐ ing index (electricity cost and peak demand) is considered equal to 0.5. Therefore, cases 11 and 13 are selected as the best combination state of the decision criteria. In this case, the peak demand of the SH is 4.835 kW, while the daily electricity cost is USD 5.723. It is worth mentioning that, in these cases, the demand has the highest priority. In cases where only one decision criterion is considered (Cases 1–3), case 1 has the lowest electricity cost, while case 3 has the lowest peak demand. This difference is be‐ cause of the different decision vectors. Cases 5 and 6 have the lowest electricity cost, with USD 5.549, and cases 11 and 13 have the lowest peak demand, with 4.83 kW. For selecting the best case, the AHP method is utilized again. In this step, the weight of each evaluat‐ ing index (electricity cost and peak demand) is considered equal to 0.5. Therefore, cases 11 and 13 are selected as the best combination state of the decision criteria. 7. Numerical Analysis In cases where only one decision criterion is considered (Cases 1–3), case 1 has the lowest electricity cost, while case 3 has the lowest peak demand. This difference is be‐ cause of the different decision vectors. Cases 5 and 6 have the lowest electricity cost, with USD 5.549, and cases 11 and 13 have the lowest peak demand, with 4.83 kW. For selecting the best case, the AHP method is utilized again. In this step, the weight of each evaluat‐ ing index (electricity cost and peak demand) is considered equal to 0.5. Therefore, cases 11 and 13 are selected as the best combination state of the decision criteria. In this case, the peak demand of the SH is 4.835 kW, while the daily electricity cost is USD 5.723. It is worth mentioning that, in these cases, the demand has the highest priority. -2 0 2 4 6 8 1 3 5 7 9 11 13 15 17 19 21 23 Power (kW) Hour Case 9 -2 0 2 4 6 1 3 5 7 9 11 13 15 17 19 21 23 Power (kW) Hour Case 10 -2 0 2 4 6 1 3 5 7 9 11 13 15 17 19 21 23 Power (kW) Hour Case 11 -2 0 2 4 6 1 3 5 7 9 11 13 15 17 19 21 23 Power (kW) Hour Case 12 -2 0 2 4 6 1 3 5 7 9 11 13 15 17 19 21 23 Power (kW) Hour Case 13 -2 0 2 4 6 8 1 3 5 7 9 11 13 15 17 19 21 23 Power (kW) Hour Method of Ref. [41] Figure 9. Hourly power situation of the SH. -2 0 2 4 6 8 1 3 5 7 9 11 13 15 17 19 21 23 Power (kW) Hour Case 9 -2 0 2 4 6 1 3 5 7 9 11 13 15 17 19 21 23 Power (kW) Hour Case 11 -2 0 2 4 6 1 3 5 7 9 11 13 15 17 19 21 23 Power (kW) Hour Case 13 Figure 9. Hourly power situation of the SH. 7. Numerical Analysis In this case, the peak demand of the SH is 4.835 kW, while the daily electricity cost is USD 5.723. It is worth mentioning that, in these cases, the demand has the highest priority. Table 5. The value of evaluation indices of the SH. Cases Evaluation Index Electricity Cost (USD) Peak Demand (kW) 1 5.654 5.288 2 6.048 6.048 3 5.960 5.035 4 5.597 5.135 5 5.549 5.135 6 5.549 5.135 7 5.831 5.135 8 5.978 5.135 9 5.998 6.048 10 5.991 5.035 11 5.723 4.835 12 6.022 5.035 13 5.723 4.835 The method of Ref. [41] 6.489 6.548 Table 5. The value of evaluation indices of the SH. The considered cases are also pondered based on different points of view. The owner of the SH has a different view than the owner of the distribution system about the Sustainability 2022, 14, 11773 18 of 20 18 of 20 operational schedule of the EV and ESS. The lowest electricity cost is the most important subject for the owner of the SH. So, based on the consumer’s point of view, the opera‐ tional schedules of the EVs and ESSs in cases 5 and 6 with the lowest electricity cost are the best results. On the other hand, cases 11 and 13, with the lowest peak demand, are the proper results based on the producer’s point of view. operational schedule of the EV and ESS. The lowest electricity cost is the most important subject for the owner of the SH. So, based on the consumer’s point of view, the opera‐ tional schedules of the EVs and ESSs in cases 5 and 6 with the lowest electricity cost are the best results. On the other hand, cases 11 and 13, with the lowest peak demand, are the proper results based on the producer’s point of view. The electricity cost and peak demand of the SH after utilizing the method of ref. [41] are USD 6.489 and 6.548 kW, respectively. So, according to the results of the case 13 (also case 11) as the best result, the proposed management method reduces the electricity cost by about 11.79% and improves the peak demand by about 26.16% more than ref. [41]. Although the proposed method is utilized for day‐ahead management, and the running time of the program is not so important, the proposed method is much faster than the other method. 8. Conclusions In this paper, a novel method was investigated to properly schedule EVs and ESSs in SHs. The combination of decision vectors and the AHP method was utilized to select the best operation schedule based on the market price, the power of the PVs, and demand. The performance of the proposed method and different priorities of decision criteria were evaluated using economic and technical indices. Numerical results, obtained from the implementation of the proposed method on a sample SH, show that the management method can properly choose the operation schedule of the EV and ESS in the SH. When the decision criteria, including market price, PVs, and demand, are considered separately (without the AHP method), the electricity cost of the SH is the lowest when the operation schedule is selected using the decision vectors of the market price, while the peak demand is the lowest when the demand of the SH is only considered. The SH has the best performance when the operational schedules of the EV and ESS are selected using the priorities of demand, PVs, and market prices equal to one, two, and three. According to the results, the charging time of the EV mostly happens during the night, when the market price and demand are low, while the charg‐ ing time of the ESS mostly happens during midday, when the power of the PVs is avail‐ able. On the other hand, the ESS can be discharged at the end of the day to reduce the dependence of the SH on the energy of the distribution system. The proposed method has a better performance than another method of energy management, so that the elec‐ tricity cost and peak demand of the SH are reduced by about 12 and 26% using the pro‐ posed method. Moreover, it finds better and more correct results about 99% sooner than the other method. Hence, it can be said that the efficiency of the SH is improved signifi‐ cantly by implementing the proposed energy management in order to select the opera‐ tion schedule of the EV and ESS in the SH. Author Contributions: Conceptualization, M.A.; Formal analysis, G.B.G., R.A., A.R. and A.A.‐M.; Funding acquisition, A.R. and A.A.‐M.; Methodology, M.A.; Resources, R.A., A.R. and A.A.‐M.; Software, M.A.; Supervision, G.B.G., R.A. and A.R.; Writing—original draft, M.A.; Writ‐ ing—review & editing, G.B.G., R.A., A.R. and A.A.‐M. 7. Numerical Analysis The running time of the proposed method is 0.07802 s, while the running time of the method in ref. [27] is 29.686587 s. Thus, the proposed method finds the best operation schedule of the EV and ESS about 99.74% sooner than ref. [41]. Therefore, the proposed method has proper performance in selecting the optimal operation schedule of EVs and ESSs in SHs for improving the technical and economic indices of SHs. References 1. Teng, F.; Ding, Z.; Hu, Z.; Sarikprueck, P. Technical review on advanced approaches for electric vehicle charging demand management, part i: Applications in electric power market and renewable energy integration. IEEE Trans. Ind. Appl. 2020, 56, 5684–5694. 2. Zolfaghari, M.; Gharehpetian, G.B.; Shafiekhah, M.; Catalão, J. Comprehensive review on the strategies for controlling the interconnection of AC and DC microgrids. Int. J. Electr. Power Energy Syst. 2022, 136, 107742. 3. Arani, A.; Karami, H.; Gharehpetian, G.B.; Hejazi, M. Review of Flywheel Energy Storage Systems structures and applications in power systems and microgrids. Renew. Sustain. 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Conclusions All authors have read and agreed to the published version of the manuscript. Author Contributions: Conceptualization, M.A.; Formal analysis, G.B.G., R.A., A.R. and A.A.‐M.; Funding acquisition, A.R. and A.A.‐M.; Methodology, M.A.; Resources, R.A., A.R. and A.A.‐M.; Software, M.A.; Supervision, G.B.G., R.A. and A.R.; Writing—original draft, M.A.; Writ‐ ing—review & editing, G.B.G., R.A., A.R. and A.A.‐M. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported in part by the Estonian Research Council under Grant PSG739, by the European Commission through the H2020 project Finest Twins (grant No. 856602) and by the Estonian Ministry of Education and Research and European Regional Fund (grant 2014‐2020.4.01.20‐0289). 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https://openalex.org/W4295533097	http://ejournal.kopertais4.or.id/tapalkuda/index.php/qodiri/article/download/4827/3388	Indonesian	null	Penggunaan Media WhatshApp pada Pembelajaran Maharah Kalam Bahasa Arab pada Mahasiswa PBA IAI Al-Qodiri Jember	Al Qodiri : Jurnal Pendidikan, Sosial dan Keagamaan	2,022	cc-by	4,020	1 Ela Isnani Munawwaroh, Problematika Pembelajaran Maharah Qiraah Daring: Studi Kasus Terhadap Mahasiswa PBA IAIN Syaikh Abdurrahman Siddik Bangka Belitung, Jurnal Alibbaa’: Jurnal Pendidikan Bahasa Arab Vol. 2. No. 2 Juli 2021 doi: https://doi.org/10.19105/alb.v2i2.4755 hlm. 93. Abstract Online Arabic classes, including maharah kalam, are available. This circumstance prevents pupils from meeting offline and makes it tough for them to hold an Arabic conversation. One of these is to use the simple Whatsapp messaging service, which everyone has on their phones. This is a qualitative descriptive research. The goal of this study is to define WhatsApp as a learning media maharah kalam. Interviews, observation, and documentation were used to gather information. The findings of this study show that learning mahrah kalam via WhatsApp during this online period might improve communication between instructors and students, as well as amongst students who are conversing in Arabic. Furthermore, various tools present in the WhatsApp medium, particularly the voice note and video call functions, assist in learning Maharah Kalam. Penggunaan Media WhatshApp pada Pembelajaran Maharah Kalam Bahasa Arab pada Mahasiswa PBA IAI Al-Qodiri Jember Oleh: Muhammad ‘Ainul Yaqin Institut Agama Islam Al Qodiri Jember ayaqin28@gmail.com Oleh: Muhammad ‘Ainul Yaqin Institut Agama Islam Al Qodiri Jember ayaqin28@gmail.com Volume 20 Nomor 2 Agustus 2022: DOI: https://doi.org/10.53515/qodiri.2022.20.2.318-32 Submission: 23-07-2022 Revised: 27-07-2022 Accepted: 19-08-2022 Published: 29-08-2022 Volume 20 Nomor 2 Agustus 2022: DOI: https://doi.org/10.53515/qodiri.2022.20.2.318-326 Article History Submission: 23-07-2022 Revised: 27-07-2022 Accepted: 19-08-2022 Published: 29-08-2022 Al Qodiri: Jurnal Pendidikan, Sosial dan Keagamaan is licensed under a Creative Commons Attribution-Share Alike 4.0 International License. Copyright @ 2020 IAI Al Qodiri Jember. All Rights Reserved p-ISSN 2252- 4371\| e-ISSN 2598-8735 2 Linur, R., & Mubarak, M. R. (2020). Facebook Sebagai Alternatif Media Pengembangan Mahara Kitabah. Jurnal Naskhi: Jurnal Kajian Pendidikan Dan Bahasa Arab, 2(1), 8–18. https://doi.org/10.47435/naskhi.v2i1.154 3 Ahmadi, A., & Ilmiani, A. M. (2020). The Use of Teaching Media in Arabic Language Teaching During Covid-19 Pandemic. Dinamika Ilmu: Jurnal Pendidikan, 20 (2), 307–322. https://doi.org/10.21093/di.v20i2.2515 4 Audina, N. A., & Muassomah, M. (2020). Instagram: Alternatif Media dalam Pengembangan Maharah Al-Kitabah. Al-Ta’rib : Jurnal Ilmiah Program Studi Pendidikan Bahasa Arab IAIN Palangka Raya, 8(1), 77– 90. https://doi.org/10.23971/altarib.v8i1.1986 q Penggunaan Media WhatshApp pada Pembelajaran Maharah Kalam Bahasa 319 Muhammad ‘Ainul Yaqin 9 Muhammad Ainul Yaqin Penggunaan Media WhatshApp pada Pembelajaran Maharah Kalam Bahasa Arab pada Mahasiswa PBA IAI Al-Qodiri Jember Al Qodiri: Jurnal Pendidikan, Sosial dan Keagamaan is licensed under a Creative Commons Attribution-Share Alike 4.0 International License. Copyright @ 2020 IAI Al Qodiri Jember. All Rights Reserved p-ISSN 2252- 4371\| e-ISSN 2598-8735 5 Utomo, Supri Wahyudi dan Ubaidillah, Moh. 2018. “Pemanfaatan Aplikasi Whatsapp Pada Pembelajaran Berbasis Masalah Untuk Mata Kuliah Akuntasi Internasional Di Universitas PGRI Madiun”, K- JTP (Kwangsan-Jurnal Teknologi Pendidikan), Vol. 06, No. 02, hh. 203-204. http://dx.doi.org/10.31800/jtp.kw.v6n2.p199--211. PENDAHULUAN Aplikasi ini mempunyai banyak fitur obrolan seperti obrolan groub atau group chat dengan batas pengguna maksimal 20 Muhammad ‘Ainul Yaqin Penggunaan Media WhatshApp pada Pembelajaran Maharah Kalam Bahasa Arab pada Mahasiswa PBA IAI Al-Qodiri Jember 20 Muhammad ‘Ainul Yaqin Penggunaan Media WhatshApp pada Pembelajaran Maharah Kalam Bahasa Arab pada Mahasiswa PBA IAI Al-Qodiri Jember 320 Penggunaan Media WhatshApp pada Pembelajaran Maharah Kalam Bahasa Arab pada Mahasiswa PBA IAI Al-Qodiri Jember 250 anggota, yang biasa dipergunakan untuk mengobrol via online tanpa harus bertemu secara langsung dan tidak terdapat Batasan waktu.5 Hemat peneliti dan selaku pengguna media Whatsapp untuk kebutuhan dalam proses kegiatan belajara mengajar, bahwa di media ini ada beberapa fitur dan fungsi seperti photo sending, video sending, text voice chat, document sending dan location sending. Dari peneliti rasakan dalam penggunannya media ini dapat mengkontruksi budaya pada generasi melek teknologi serta lebih tepatnya digunakan untuk media komunikasi yang bersifat individu ataupun group. Media ini juga menjadi sumber informasi dan sarana untuk mempererat hubungan kepada sesama dan dapat dikatakan bahwa media ini juga bersifat kekinian dan dapat menyesuaikan kebutuhan masyarakat. 250 anggota, yang biasa dipergunakan untuk mengobrol via online tanpa harus bertemu secara langsung dan tidak terdapat Batasan waktu.5 Hemat peneliti dan selaku pengguna media Whatsapp untuk kebutuhan dalam proses kegiatan belajara mengajar, bahwa di media ini ada beberapa fitur dan fungsi seperti photo sending, video sending, text voice chat, document sending dan location sending. Dari peneliti rasakan dalam penggunannya media ini dapat mengkontruksi budaya pada generasi melek teknologi serta lebih tepatnya digunakan untuk media komunikasi yang bersifat individu ataupun group. Media ini juga menjadi sumber informasi dan sarana untuk mempererat hubungan kepada sesama dan dapat dikatakan bahwa media ini juga bersifat kekinian dan dapat menyesuaikan kebutuhan masyarakat. Selain itu, media whatsapp dapat membantu interaksi antara pengajar dan peserta didik, termasuk di masa pandemi atau daring seperti sekarang. Sehingga keberadaan whatshapp dimanfaatkan sebagai media pembelajaran yang dapat langsung berinteraksi kepada peserta didik. WhatsApp menjadi media sosial dan media digital yang membantu terjadinya tanya jawab, dialog, dan diskusi antar anggota grub. Sehingga setiap anggota grup dapat mengenal dan bisa mendapatkan solusi dari bahan pembicaraan yang diperbincangkan. Berdasarkan latar belakang yang dijelaskan di atas, maka penelitian ini membatasi masalah pada: Pertama, bagaimana tahapan yang dilakukan agar Whatsapp dapat digunakan sebagai media pembelajaran maharah kalam? Kedua, apakah dampak dari penggunaan Whatsapp terhadap pembelajaran maharah kalam? 0 u a ad u aq Penggunaan Media WhatshApp pada Pembelajaran Maharah Kalam Bahasa 320 Muhammad ‘Ainul Yaqin PENDAHULUAN Pembelajaran di masa pandemi ini menuntun seluruh lapisan civitas akademika pada suatu kebiasaan baru, yaitu pembelajaran daring (dalam jaringan), atau yang dikenal dengan pembelajaran online. Namun demikian, pembelajaran daring juga menyebabkan munculnya permasalahan baru, karena tidak semua pembelajaran bisa dilaksanakan secara daring.1 Dalam bahasa Arab, ada beberapa keterampilan berbahasa yang menjadi target bagi siapa saja yang akan mempelajarinnya. Keterampilan tersebut terbagi menjadi empat yang tersusun secara sistematis dan tidak seharusnya diacak dalam mempelajarinnya. Ada 4 (empat) keterampilan berbahasa yang dimaksud, diantarannya: maharah istma’, (keterampilan 9 Muhammad ‘Ainul Yaqin Penggunaan Media WhatshApp pada Pembelajaran Maharah Kalam Bahasa Arab pada Mahasiswa PBA IAI Al-Qodiri Jember 9 Muhammad ‘Ainul Yaqin 319 mendengar), maharah kalam (keterampilan berbicara), maharah qiroah (keterampilan membaca), dan maharah kitabah (keterampilan menulis). mendengar), maharah kalam (keterampilan berbicara), maharah qiroah (keterampilan membaca), dan maharah kitabah (keterampilan menulis). Pada aspek kegiatan belajar mengajar bahasa Arab terdapat banyak unsur yang perlu diperhatikan, diantarannya kurikulum, metode, pengajar, evaluasi dan termasuk penggunaan media pembelajarannya. Para pengajar dibidang bahasa Arab banyak berinovasi untuk dapat menghadirkan media pembelajaran yang efektif, serta membisakan. Dalam masa wabah covid-19 proses pembelajaran dilakukan secara jaringan atau yang kita kenal dengan singkatan daring, ia termasuk pembelajaran yang menggunakan jaringan internet dan beberapa media teknologi yang dapat diakses sewaktu-waktu dimanapun dan kapanpun tempatnya, dengan catatan ada koneksi internet yang dapat dikembangkan secara produktif dan bermutu. Ada beberapa problematika dalam kegiatan belajar mengajar bahasa Arab diantarannya perbedaan karakteristik bahasa Arab dengan bahasa Indonesia, kurangnya minat serta stimulus bersemangat pada diri peserta didik dalam mendalami bahasa Arab. Ada beberapa peneliti juga menyampaikan hasil risetnya bahwa penggunaan media pembelajaran Bahasa Arab memiliki pengaruh besar serta memberikan keefektifan dan keefesienan dalam ranah Pendidikan. Ada beberapa hasil penelitian Mubarak & Linur, Ahmadi & Ilhami, Audina & Muassomah bahwa media pembelajaran yang dapat digunakan untuk kegiatan belajar mengajar bahasa Arab secara online diantarannya Google Meet, Google Classroom, Facebook, Instagram, Youtube. Dari beberapa penelitian tersebut bahwa media pembelajaran dalam proses pembelajaran bahasa Arab daring adalah media komunikasi Whatsapp.234 Beberapa hasil penelitian seperti Supri Wahyu Utomo dan Moh Ubaidillah bahwa media Whatsapp adalah sebuah aplikasi yang memiliki fitur yang cepat, lengkap, praktis dan mudah dioperasikan hanya dengan menggunakan Smartphone. Al Qodiri: Jurnal Pendidikan, Sosial dan Keagamaan is licensed under a Creative Commons Attribution-Share Alike 4.0 International License. Copyright @ 2020 IAI Al Qodiri Jember. All Rights Reserved p-ISSN 2252- 4371\| e-ISSN 2598-8735 6 Jumiatmoko, Whatsapp Messenger Dalam Tinjauan Manfaat dan Adab, (Wahana Akademika, 2016), hlm. 12. Metode Penelitian Penelitian ini termasuk dalam field research atau penelitian lapangan. Untuk mendapatkan data, peneliti berada di lokasi penelitian, yaitu di Program Studi Pendidikan Bahasa Arab Fakultas Tarbiyah Institut Agama Islam Al Qodiri Jember. Penelitian ini bersifat deskriptif dengan mengungkap fakta-fakta yang ada di lapangan yang bertujuan untuk memberikan deskripsi dari Whatsapp sebagai media pembelajaran maharah kalam. Sumber data dari penelitian ini terdiri dari dua sumber, yaitu data primer dalam penelitian ini adalah 20 mahasiswa Pendidikan bahasa Arab semester V Institut Agama Islam Al Qodiri Jember. Sedangkan data sekunder diperoleh melalui berbagai literature, seperti buku-buku dan jurnal- jurnal ilmiah yang terkait dengan tema penelitian. Data bersumber dari Observasi, dokumentasi serta wawancara secara mendalam. Pengumpulan data didapatkan melalui observasi pada kegiatan pembelajaran maharah kalam di Program Studi Pendidikan Bahasa Arab. Data juga didapatkan melalui wawancara kepada 1 Muhammad ‘Ainul Yaqin Penggunaan Media WhatshApp pada Pembelajaran Maharah Kalam Bahasa Arab pada Mahasiswa PBA IAI Al-Qodiri Jember 1 Muhammad ‘Ainul Yaqin 321 mahasiswa PS 5 yang belajar maharah kalam serta data yang berupa dokumentasi diperoleh dari kegiatan pembelajaran maharah kalam dengan menggunakan Whatsapp. Data didapatkan dinalisis melalui reduks data, penyajian data dan verifikasi. mahasiswa PS 5 yang belajar maharah kalam serta data yang berupa dokumentasi diperoleh dari kegiatan pembelajaran maharah kalam dengan menggunakan Whatsapp. Data didapatkan dinalisis melalui reduks data, penyajian data dan verifikasi. a. Pembahasan Media whatsapp menjadi bagian teknologi yang banyak digunakan oleh banyak orang, termasuk di kalangan pelajar dan mahasiswa. Whatsapp memiliki fitur-fitur yang menarik bagi para penggunannya. Media ini digunakan sebagai alat komunikasi berbasis teknologi pengiriman pesan berbantu data pada internet.6 Dalam kehidupan sehari-hari Whatsapp dapat membantu interaksi antar individu dengan tanpa bertemu langsung. Media Whatsapp dapat membantu interaksi antara pengajar dan peserta didik, termasuk di masa pandemi atau daring seperti sekarang ini. Sehingga, keberadaan whatsapp dimanfaatkan sebagai media pembelajaran. Whatsapp tidak hanya dapat mengirim atau menerima pesan melalui chatting atau tertulis, tetapi juga dapat berupa foto, audio dan video. Selain dapat melakukan interaksi secara inividu, aplikasi ini juga dapat digunakan dalam sebuah grup yang dapat menampung 250 orang. Whatsapp sebagai bagian dari fitur yang ada pada aplikasi Whatsapp yang digunakan untuk optimalisasi terjalinnya interaksi dan komunikasi yang lebih efektif. Dalam Whatsapp ini hanya terdiri dari 8 (delapan) orang saja. Dengan jumlah anggota grup yang terbatas, peserta dalam grup tidak hanya dapat melakukan komunikasi secara tertulis melalui chatting, tetapi juga dapat berkirim pesan melalui suara atau voice note, saling telepon, bahkan juga bertemu secara virtual melalui fitur video call. Whatsapp menjadi media sosial dan media digital yang membantu tejadinya tanya jawab, dialog dan diskusi antar anggota grup. Sehingga, setiap anggota grup dapat mengenal dan bisa mendapatkan solusi dari bahan pembicaraan yang diperbincangkan. b. Maharah Kalam Maharah kalam dalam bahasa Arab merupakan unsur penting dalam proses pengembangan keterampilan berbicara bahasa Arab untun penutur Non Arab. Dengan keterampilan ini, orang dapat berbicara dengan pikiran, perasaan yang akan diungkapkan bahkan ide-ide produktif dapat diungkapkan. Tujuan dalam keterampilan ini, untuk komunikasi yang baik dengan menggunakan bahasa yang baik pula. Al Qodiri: Jurnal Pendidikan, Sosial dan Keagamaan is licensed under a Creative Commons Attribution-Share Alike 4.0 International License. Copyright @ 2020 IAI Al Qodiri Jember. All Rights Reserved p-ISSN 2252- 4371\| e-ISSN 2598-8735 Ada beberapa jenis maharah kalam diantarannya: Ada beberapa jenis maharah kalam diantarannya: 1. Dialog (hiwar) 2. Bercerita (qishosh) 22 Muhammad ‘Ainul Yaqin Penggunaan Media WhatshApp pada Pembelajaran Maharah Kalam Bahasa Arab pada Mahasiswa PBA IAI Al-Qodiri Jember 3. Diskusi (al Munaqashah) 4. Debat (al-Mujadalah) 5. Berbicara sendiri (takallum an Nafs) Dari beberapa jenis tersebut dalam pengembangannya untuk berbicara bahasa Arab, harus ada kebiasaan rutin untuk dapat melatih keterampilan ini dengan menyesuaikan karakter serta level peserta didik/mahasiswa dalam berbicara berbahasa Arab. Begitu juga pengajar juga dapat mengembangkan media-media pembelajaran agar nuansa pembelajaran menjadi baik dan membisakan untuk berbicara bahasa Arab. Pemilihan media Whatshapp sebagai Media Pembalajaran Maharah Kalam Maharah kalam bagi mahasiswa di Program Studi Pendidikan Bahasa Arab semester lima bukan hal yang baru, dikarenakan mahasiswa yang ada di program studi ini berlatar belakang alumni pesantren di Kabupaten Jember. Hasil wawancara dengan para mahasiswa yang saat ini di semester lima bahwa maharah kalam dengan menggunakan media whatsapp menjadikan mereka berani untuk menyampaikan tanya jawab dan tidak canggung atau malu jika harus berpraktik berbicara berbahasa Arab, baik secara berpasangan dengan teman mereka. Selain itu, mahasiswa terbantu dengan adannya Whatsapp group dikarenakan mahasiswa dapat melakukan tatap muka secara langsung (meskipun dalam jaringan) dengan teman-temannya dan mereka praktik berbicara, baik dialog maupun monolog dapat diberikan komentar atau feedback secara langsung oleh dosen atau mahasiswa lainnya. Mahasiswa juga merasa lebih nyaman dan efektif, dikarenakan pembelajaran dilakukan dengan jumlah peserta yang minim dan terbatas. 2. Diskusi materi dan persiapan Pembelajaran al-kalam dilakukan secara daring, berbeda dengan luring. Ketika luring, mahasiswa dapat langsung membagi materi dialog berbahasa Arab dengan temannya secara langsung. Sedangkan dengan daring ini, mahasiswa dapat memanfaatkan whatsapp sebagai sarana untuk melakukan persiapan dan mendiskusikan materi yang akan dibicarakan. Seperti pada materi mendeskipsikan gambar yang harus diceritakan oleh semua anggota grup secara bergantian, maka tahap persiapan kalam didiskusikan dan dipersiapkan dengan menggunakan whatshapp agar Ketika penyampaian praktik kalam kepada dosen dapat maksimal. 3. Kerjasama Pada aspek ini, anggota diajak untuk menjawab pertanyaan yang ada pada materi atau penjelasan dosen. Sebagaimana contoh, dosen memberikan enam pertanyaan tentang الجامعة yang disampaikan seperti cerita sederhana, maka ketua dan para anggota whatsapp bekerjasama dengan baik agar keenam pertanyaan tersebut dapat diubah menjadi sebuah cerita sederhana. 4. Memahami matari pembelajaran Maharah al-Kalam 4. Memahami matari pembelajaran Maharah al-Kalam Dalam urgensi serta tujuan media itu sendiri adalah untuk dimanfaatkan agar nuansa pembelajaran lebih mudah dan membisakan. Media whatshapp dapat mendukung para mahasiswa untuk memahami materi maharah al-kalam yang dipelajari. Bahkan, dengan media ini, mahaiswa langsung dapat berkomunikasi dan berinteraksi dengan teman- temannya secara langsung untuk berbicara berbahasa Arab. Dalam urgensi serta tujuan media itu sendiri adalah untuk dimanfaatkan agar nuansa pembelajaran lebih mudah dan membisakan. Media whatshapp dapat mendukung para mahasiswa untuk memahami materi maharah al-kalam yang dipelajari. Bahkan, dengan media ini, mahaiswa langsung dapat berkomunikasi dan berinteraksi dengan teman- temannya secara langsung untuk berbicara berbahasa Arab. 5. Berani untuk berbicara bahasa Arab Dengan media ini, mahasiswa sudah berani untuk menyampaikan kalam mereka dihadapan teman dan dosennya. Hal ini disebabkan adannya keterbatasan jumlah orang yang mendengarkan kalam yang disampaikan serta mahasiswa merasa kenal dengan para anggota whatsapp. Sehingga dipertemuan berikutnya mahasiswa semakin antusias untuk mengikuti pembelajaran maharah kalam serta mempraktekkannya di depan teman-teman dan dosen. 5. Berani untuk berbicara bahasa Arab Dengan media ini, mahasiswa sudah berani untuk menyampaikan kalam mereka dihadapan teman dan dosennya. Hal ini disebabkan adannya keterbatasan jumlah orang yang mendengarkan kalam yang disampaikan serta mahasiswa merasa kenal dengan para anggota whatsapp. Sehingga dipertemuan berikutnya mahasiswa semakin antusias untuk mengikuti pembelajaran maharah kalam serta mempraktekkannya di depan teman-teman dan dosen. 5. Berani untuk berbicara bahasa Arab Dengan media ini, mahasiswa sudah berani untuk menyampaikan kalam mereka dihadapan teman dan dosennya. Hal ini disebabkan adannya keterbatasan jumlah orang yang mendengarkan kalam yang disampaikan serta mahasiswa merasa kenal dengan para anggota whatsapp. Sehingga dipertemuan berikutnya mahasiswa semakin antusias untuk mengikuti pembelajaran maharah kalam serta mempraktekkannya di depan teman-teman dan dosen. Terbentuk Komunikasi yang Efektif antar Mahasiswa Ada hasil observasi dan wawancara bahwa ada beberapa stimulus-respon antara mahasiswa dan dosen dalam menggunakan media whatsapp ini, diantarannya adalah: 1. Setiap mahasiswa menyimpan nomor whatshapp dosen dan teman-teman 1. Setiap mahasiswa menyimpan nomor whatshapp dosen dan teman teman 2. Mahasiswa dalam satu kelas dibagi menjadi beberapa kelompok 2. Mahasiswa dalam satu kelas dibagi menjadi beberapa kelompo 3. Dosen dan para anggota whatsapp memilih satu orang sebagai ketua atau koordinatir kelompok. 3. Dosen dan para anggota whatsapp memilih satu orang sebagai ketua atau koordinatir kelompok. Ada beberapa manfaat dalam penggunaan media whatsapp untuk pengembangan maharah kalam, diantanrannya: 1. Adannya jalinan interaksi antar mahasiswa Maharah al-Kalam bahasa Arab keterampilan ini membutuhkan interaksi yang bai kantar sesame mahasiswa. Berdasarkan hasil wawancara, mahasiswa mengatakan bahwa penggunaan media whatsapp ini dapat mempererat hubungan para mahasiswa sehingga antara mahasiswa yang sudah mengenal bahasa Arab dapat dengan mudah untuk mengembankan lagi keterampilan berbicara ini dengan membiasakan diri bercakap-cakap Al Qodiri: Jurnal Pendidikan, Sosial dan Keagamaan is licensed under a Creative Commons Attribution-Share Alike 4.0 International License. Copyright @ 2020 IAI Al Qodiri Jember. All Rights Reserved p-ISSN 2252- 4371\| e-ISSN 2598-8735 323 Muhammad ‘Ainul Yaqin Penggunaan Media WhatshApp pada Pembelajaran Maharah Kalam Bahasa Arab pada Mahasiswa PBA IAI Al-Qodiri Jember berhasa Arab dengan dosen dan teman-teman lainnya. Dan juga membantu ke teman lainnya yang gaya berbicara bahasa Arabnya masih kategori rendah atau dibawah minimal. berhasa Arab dengan dosen dan teman-teman lainnya. Dan juga membantu ke teman lainnya yang gaya berbicara bahasa Arabnya masih kategori rendah atau dibawah minimal. manfaatkan VC (vidio call) dan VN (Voice Note) Pada Proses Berbicara Bahasa Ara Kita ketahui bersama bahwa media whatsapp dilengkapi beberapa fitur seperti VC dan VN. Kedua fasilitas tersebut semua pengguna media ini memilikinnya. Dengan fasiltas Al Qodiri: Jurnal Pendidikan, Sosial dan Keagamaan is licensed under a Creative Commons Attribution-Share Alike 4.0 International License. Copyright @ 2020 IAI Al Qodiri Jember. All Rights Reserved p-ISSN 2252- 4371\| e-ISSN 2598-8735 24 Muhammad ‘Ainul Yaqin Penggunaan Media WhatshApp pada Pembelajaran Maharah Kalam Bahasa Arab pada Mahasiswa PBA IAI Al-Qodiri Jember 324 tersebut tenaga pendidikan dan mahasiswa dapat memanfaatkannya dengan baik serta dapat membiasakan berbicara berbahasa Arab. Penggunaan VC dapat mengungkapkan sebuah ungkapan pendek, pembetulan kalimat yang telah diungkapkan atau kalimat bertanya. Ungkapan tersebut seperti kalimat هل تذهب اىل اجلامعة atau kalimat اجلامعة يف نفس املعهد القادري وفيه ا مطعم ومكان التعليم املناسب من فصلك اىل جامعتنا sebagai kalimat pembetulan yang sebelumnya tanpa ada kata من فضلك atau kalimat tanya. Seperti: ماذا حتب من اجلامعة القادري kalimat tersebut disampaikan melalui VN dan sebaliknya anggota grup tersebut diberikan kesempatan untuk membalasnya secara lisan melalui VN juga. Adapun fitur VC digunakan untuk melakukan interaksi yang lebih lama dari pada VN. Hal ini disebabkan adannya tatap muka secara virtual antara anggota grup kecil di kelompok tersebut dan dengan atau tanpa dosen. Penggunaan VC memacu mereka untuk saling bertanya dan menjawab atau berdialog, bahkan bercerita secara online dengan menggunakan bahasa Arab. Apabila VC juga didampingi oleh dosen, maka mahasiswa semakin bersemangat karena mereka mendapatkan feedback, tanggapan dan koreksi secara langsung dari kalam yang telah diungkapkan. Sehingga pembelajaran mahrah kalam dapat mudah dipraktekkan dan dibiasakan di rumahnya masing-masing. q Penggunaan Media WhatshApp pada Pembelajaran Maharah Kalam Bahasa 324 Muhammad ‘Ainul Yaqin Dampak dari Penggunaan Whatsapp terhadap pembelajaran maharah kalam Dengan media ini mahasiswa lebih mudah dan focus dalam belajar maharah kalam karena di dalam grub tersebut memiliki anggota dan pembahasan yang terbatas. 6. Dengan media ini mahasiswa lebih mudah dan focus dalam belajar maharah kalam karena di dalam grub tersebut memiliki anggota dan pembahasan yang terbatas. Sedangkan dampak negative dari penggunaan whatsapp sebagai media pembelajaran maharah kalam di masa daring adalah terkait dengan kedala teknis, yaitu jaringan atau signal internet yang tekadang kurang stabil, sehingga berpengaruh pada durasi kelancaran penyampaian kalam bahasa Arab. Sedangkan dampak negative dari penggunaan whatsapp sebagai media pembelajaran maharah kalam di masa daring adalah terkait dengan kedala teknis, yaitu jaringan atau signal internet yang tekadang kurang stabil, sehingga berpengaruh pada durasi kelancaran penyampaian kalam bahasa Arab. Dampak dari Penggunaan Whatsapp terhadap pembelajaran maharah kalam Media ini digunakan dalam pembelajaran maharah kalam tentu mengandung dampak dalam proses pembelajaran bagi mahasiswa di Program Studi Pendidikan Bahasa Arab Fakultas Tarbiyah IAI Al-Qodiri Jember. Berdasarkan hasil wawancara kepada para mahasiswa terkait dengan pendapat mereka tentang penggunannya sebagai media pembelajaran maharah kalam di masa daring, maka terdapat dampak yang positif dan negative. Dampak positif yang disebutkan lebih variatif dari pada dampak negative. Adapun dampak positif dari penggunaan whatshapp sebagai media pembelajaran maharah al-kalam di masa daring diantarannya sebagaimana berikut; 1. Adanya jalinan interaksi dan kerjasama antar mahasiswa, sehingga antara mahasiswa yang sudah mengenal dan baik berbicara bahasa Arabnya. Dan juga mahasiswa yang berpotensi dalam kalam dapat membantu pada mahasiswa yang belum dapat melakukan kalam dengan baik. 2. Mahasiswa dapat memanfaatkan whatshapp sebagai sarana untuk melakukan persiapkan dan mendiskusikan materi yang akan dibicarakan. 3. Secara psikologis, mahasiswa tidak merasa malu, takut dan ragu untuk berbicara dengan bahasa Arab, bahkan sebaliknya merasa senang dan percaya diri. 4. Dengan whatshapp ini mahasiswa berani untuk menyampaikan kalam mereka dihadapan teman dan dosen pengampu. Hal ini disebabkan adannya keterbatasan jumlah orang yang mendengarkan kalam yang disampaikan serta mahasiswa merasa kenal dengan para anggota whatshapp baik melalui video call maupun voice note. Al Qodiri: Jurnal Pendidikan, Sosial dan Keagamaan is licensed under a Creative Commons Attribution-Share Alike 4.0 International License. Copyright @ 2020 IAI Al Qodiri Jember. All Rights Reserved p-ISSN 2252- 4371\| e-ISSN 2598-8735 5 Muhammad ‘Ainul Yaqin Penggunaan Media WhatshApp pada Pembelajaran Maharah Kalam Bahasa Arab pada Mahasiswa PBA IAI Al-Qodiri Jember 325 5. Whatshapp menjadi media untuk menerima penjelasan, masukan dan koreksi secara langsung, baik dari teman maupun dari dosen. 5. Whatshapp menjadi media untuk menerima penjelasan, masukan dan koreksi secara langsung, baik dari teman maupun dari dosen. 6. Dengan media ini mahasiswa lebih mudah dan focus dalam belajar maharah kalam karena di dalam grub tersebut memiliki anggota dan pembahasan yang terbatas. Sedangkan dampak negative dari penggunaan whatsapp sebagai media pembelajaran maharah kalam di masa daring adalah terkait dengan kedala teknis, yaitu 6. Dengan media ini mahasiswa lebih mudah dan focus dalam belajar maharah kalam karena di dalam grub tersebut memiliki anggota dan pembahasan yang terbatas. Sedangkan dampak negative dari penggunaan whatsapp sebagai media pembelajaran maharah kalam di masa daring adalah terkait dengan kedala teknis, yaitu jaringan atau signal internet yang tekadang kurang stabil, sehingga berpengaruh pada durasi kelancaran penyampaian kalam bahasa Arab. 6. Kesimpulan Berdasarkan analis terhadap data-data dan hasil, maka kesimpulan dari penelitian ini adalah terdapat beberapa tahapan yang harus dilakukan untuk menggunakan whatsapp sebagai media pada pembelajaran maharah kalam yaitu melalui tiga tahapan 1) Persiapan, 2) Pelaksanaan, 3) Tahap penutup. Pada tahap persiapan dilakukan saving nomer whatsapp kemudian membagi kelompok grub mahasiswa. Pemilihan koordinator mata kuliah, mengaktifkan whatshapp dan sosialisasi rencana pembelajaran semester (rps) serta pemberian materi dan tujuan pembelajaran. Diskusi rencana pembelajaran semester dan mahasiswa dapat mempertanyakan RPS tersebut. Persiapan untuk mempraktekkan maharah kalam dengan menggunakan fitur whatshapp voice note atau video call. Pada tahap oenutup diberikan feedback dan kesempatan untuk bertanya terkait materi yang telah dipelajari. Adapun dampak dalam penggunaan whatsapp sebagai media pada pembelajaran maharah kalam lebih cenderung pada aspek positif, yaitu: adanya interaksi yang aktif antara dosen dan mahasiswa, terjadinya diskusi antar mahasiswa, mahasiswa dapat berbicara percaya diri untuk berbicara bahasa Arab, sebagai media yang sesuai dengan kondisi mahasiswa pada program studi ini. Ada dampak negatif hanya kelemahan pada kekuatan sinyal internet. Al Qodiri: Jurnal Pendidikan, Sosial dan Keagamaan is licensed under a Creative Commons Attribution-Share Alike 4.0 International License. Copyright @ 2020 IAI Al Qodiri Jember. All Rights Reserved p-ISSN 2252- 4371\| e-ISSN 2598-8735 26 Muhammad ‘Ainul Yaqin Penggunaan Media WhatshApp pada Pembelajaran Maharah Kalam Bahasa Arab pada Mahasiswa PBA IAI Al-Qodiri Jember 326 6 Muhammad ‘Ainul Yaqin Penggunaan Media WhatshApp pada Pembelajaran Maharah Kalam Bahasa Arab pada Mahasiswa PBA IAI Al-Qodiri Jember Daftar Pustaka Ahmadi, A., & Ilmiani, A. M. (2020). The Use of Teaching Media in Arabic Language Teaching During Covid- 19 Pandemic. Dinamika Ilmu: Jurnal Pendidikan, 20 (2), 307–322. https://doi.org/10.21093/di.v20i2.2515 Audina, N. A., & Muassomah, M. (2020). Instagram: Alternatif Media dalam Pengembangan Maharah Al- Kitabah. Al-Ta’rib : Jurnal Ilmiah Program Studi Pendidikan Bahasa Arab IAIN Palangka Raya, 8(1), 77–90. https://doi.org/10.23971/altarib.v8i1.1986 Linur, R., & Mubarak, M. R. (2020). Facebook Sebagai Alternatif Media Pengembangan Mahara Kitabah. Jurnal Naskhi: Jurnal Kajian Pendidikan Dan Bahasa Arab, 2(1), 8–18. https://doi.org/10.47435/naskhi.v2i1.154 Munawwaroh, Ela Isnani, Problematika Pembelajaran Maharah Qiraah Daring: Studi Kasus Terhadap Mahasiswa PBA IAIN Syaikh Abdurrahman Siddik Bangka Belitung, Jurnal Alibbaa’: Jurnal Pendidikan Bahasa Arab Vol. 2. No. 2 Juli 2021 doi: https://doi.org/10.19105/alb.v2i2.4755 p g Jumiatmoko, Whatsapp Messenger Dalam Tinjauan Manfaat dan Adab, Wahana Akademika, 2016. Utomo, Supri Wahyudi dan Ubaidillah, Moh. 2018. “Pemanfaatan Aplikasi Whatsapp Pada Pembelajaran Berbasis Masalah Untuk Mata Kuliah Akuntasi Internasional Di Universitas PGRI Madiun”, K-JTP (Kwangsan-Jurnal Teknologi Pendidikan), Vol. 06, No. 02, hh. 203-204. http://dx.doi.org/10.31800/jtp.kw.v6n2. Al Qodiri: Jurnal Pendidikan, Sosial dan Keagamaan is licensed under a Creative Commons Attribution-Share Alike 4.0 International License. Copyright @ 2020 IAI Al Qodiri Jember. All Rights Reserved p-ISSN 2252- 4371\| e-ISSN 2598-8735 Al Qodiri: Jurnal Pendidikan, Sosial dan Keagamaan is licensed under a Creative Commons Attribution-Share Alike 4.0 International License. Copyright @ 2020 IAI Al Qodiri Jember. All Rights Reserved p-ISSN 2252- 4371\| e-ISSN 2598-8735 Al Qodiri: Jurnal Pendidikan, Sosial dan Keagamaan is licensed under a Creative Commons Attribution-Share Alike 4.0 International License. Copyright @ 2020 IAI Al Qodiri Jember. All Rights Reserved p-ISSN 2252- 4371\| e-ISSN 2598-8735 A
https://openalex.org/W3018575995	https://journal.transpl.ru/vtio/article/download/1158/920	Russian	null	Global organ shortage: an analysis of national self‑sufficiency strategies	Vestnik transplantologii i iskusstvennyh organov	2,020	cc-by	7,037	Глобальный дефицит донорских органов: анализ национальных стратегий самообеспечения О.Н. Резник1, 2, Д.В. Михель3 О.Н. Резник , Д.В. Михель 1 ГБУ «Санкт-Петербургский научно-исследовательский институт скорой помощи имени И.И. Джанелидзе», Санкт-Петербург, Российская Федерация 2 ФГБОУ ВО «Первый Санкт-Петербургский государственный медицинский университет имени И.П. Павлова», Санкт-Петербург, Российская Федерация 3 ФГБОУ ВО «Российская академия народного хозяйства и государственной службы при Президенте Российской Федерации», Москва, Российская Федерация С позиций социально-гуманитарного знания в статье анализируется проблема глобального дефицита донорских органов. Рассматриваются основные представления международного медицинского сообщес‑ тва о дефиците органов и основные предложения по его преодолению. Особое внимание уделяется трем наиболее показательным национальным стратегиям самообеспечения донорскими органами – американ‑ ской, испанской и иранской. Обсуждается вопрос о влиянии культурных различий и фактора социально- экономического неравенства на сложившиеся практики донорства органов на примере Мексики, Турции, Пакистана и Бангладеш. Ключевые слова: дефицит донорских органов, ВОЗ, национальные стратегии самообеспечения, США, Испания, Иран, Мексика, Турция, Пакистан, Бангладеш. Global organ shortage: an analysis of national self-sufficiency strategies O.N. Reznik1, 2, D.V. Mikhel3 1 I.I. Dzhanelidze St. Petersburg Research Institute of Emergency Care, St. Petersburg, Russian Federation 2 I.P. Pavlov First St. Petersburg State Medical University, St. Petersburg, Russian Federation 3 Russian Presidential Academy of National Economy and Public Administration, Moscow, Russian Federation From the standpoint of socio-humanitarian knowledge, the paper analyzes the problem of global organ shortage. The basic ideas of the international medical community about organ shortage and the main proposals for overco‑ ming it are considered. Special emphasis is placed on the three most revealing national self-sufficiency strategies adopted by donor agencies – American, Spanish and Iranian strategies. The issue of influence of cultural diffe‑ rences and socio-economic inequality on established organ donation practices is discussed using Mexico, Turkey, Pakistan and Bangladesh as examples. Keywords: organ shortage, WHO, national self-sufficiency strategies, USA, Spain, Iran, Mexico, Turkey, Pakistan, Bangladesh. For correspondence: Reznik Oleg Nikolaevich. Address: 3, Budapeshtskaya str., St. Petersburg, 192242, Russian Federation. E-mail: onreznik@gmail.com Для корреспонденции: Резник Олег Николаевич. Адрес: 192242, Санкт-Петербург. ул. Будапештская, д. 3. E-mail: onreznik@gmail.com E-mail: onreznik@gmail.com For correspondence: Reznik Oleg Nikolaevich. Address: 3, Budapeshtskaya str., St. Petersburg, 192242, Russian Federation E-mail: onreznik@gmail.com ВЕСТНИК ТРАНСПЛАНТОЛОГИИ И ИСКУССТВЕННЫХ ОРГАНОВ ВЕСТНИК ТРАНСПЛАНТОЛОГИИ И ИСКУССТВЕННЫХ ОРГАНОВ том XXII № 1–2020 Reznik Oleg Nikolaevich. Address: 3, Budapeshtskaya str., St. Petersburg, 192242, Russian Federation. mail.com ВОЗ О ДЕФИЦИТЕ ОРГАНОВ С момента, когда трансплантация органов ста‑ ла превращаться в успешное средство спасения че‑ ловеческих жизней, международное медицинское сообщество в лице ВОЗ начало проявлять самое пристальное внимание к различным вопросам, ка‑ сающимся организации практик трансплантаций и донорства. В 1987 г. 40-я Всемирная ассамблея здра‑ воохранения приняла решение разработать «Руково‑ дящие принципы для трансплантаций человеческих органов», в которых бы подчеркивались достигнутый в сфере трансплантаций прогресс и несовместимость торговли органами от живых доноров с базовыми гуманистическими ценностями, Всеобщей деклара‑ цией прав человека и духом Устава ВОЗ [1]. Многие из идей, озвученных во время Глобаль‑ ной консультации в 2007 г., были представлены в рамках Стамбульской декларации 2008 года, по священной вопросам предотвращения торговли ор‑ ганами и трансплантационного туризма. В тексте декларации было выражено несколько важных под‑ ходов к рассматриваемому вопросу. «Всем странам необходимы правовая и профессиональная базы для управления процессами донорства и трансплантации органов, также как и надзорная система, гарантиру‑ ющая безопасность донора и реципиента, соблюде‑ ние стандартов и запрещение деятельности, проти‑ воречащей этическим принципам». «Каждая страна должна гарантировать развитие и осуществление программ, предупреждающих дефицит донорских органов, и обеспечивать своим гражданам предо‑ ставление донорских органов для трансплантации в пределах своего населения или за счет региональной кооперации». Авторы декларации призывали всех участников международного общения расширить «терапевтический потенциал посмертного донорс‑ тва», «минимизировать использование живых доно‑ ров», устранять «недоверие, барьеры и неправильные представления, мешающие развитию эффективного посмертного донорства», улучшать инфраструк‑ туру здравоохранения. В рамках декларации было сформулировано шесть принципов, один из которых гласил: «Страны, регионы и сопредельные области должны пытаться достигнуть самообеспечения в ор‑ ганном донорстве, обеспечивая достаточное коли‑ чество органов для своих жителей, используя также региональную кооперацию» [6]. В 1991 г. ВОЗ одобрила «Руководящие принци‑ пы по трансплантации человеческих органов» [2], в которых отмечалось наличие дефицита донорских органов и утверждалось, что «спрос никогда не бу‑ дет удовлетворен предложением». Особое внимание обращалось на проблему торговли органами от не‑ родственных доноров и выражалась серьезная оза‑ боченность судьбами различных уязвимых групп, ко‑ торые становятся жертвами этой торговли. С целью пресечения этой торговли выдвигались следующие принципы: 1) органы следует получать прежде всего от умерших доноров; 2) живые доноры должны быть генетически связаны с реципиентами; 3) не должно быть купли или продажи органов [3]. В дальнейшем ВОЗ неоднократно возвращалась к вопросу о дефиците органов, неизменно связывая его с проблемами коммерческого трафика органов, который рассматривался как серьезное препятствие для нормального развития трансплантации во всем мире. Так, в 2004 г. ВВЕДЕНИЕ ских учреждениях по всему миру. Вследствие этого уже с 1990-х гг. главной проблемой, препятствующей эффективно предоставлять трансплантационную ме‑ дицинскую помощь нуждающимся в ней пациентам, стал дефицит донорских органов. Это явление носит не страновой, а глобальный характер. Феномен гло‑ бального дефицита органов требует специального Прогресс в области трансплантационной медици‑ ны, связанный с развитием методов хирургического вмешательства, послеоперационной реабилитации и использованием эффективных иммуносупрессивных препаратов, привел к тому, что пересадки органов стали рутинной медицинской практикой в клиниче 174 ОБЗОРЫ ЛИТЕРАТУРЫ изучения, как и дефицит органов в пределах конк‑ ретной национальной юрисдикции. изучения, как и дефицит органов в пределах конк‑ ретной национальной юрисдикции. что «трансплантация включает в себя не только ме‑ дицинские, но и юридические и этические аспекты и затрагивает экономические и психологические проблемы» [4]. что «трансплантация включает в себя не только ме‑ дицинские, но и юридические и этические аспекты и затрагивает экономические и психологические проблемы» [4]. В рамках предлагаемой статьи планируется обсу‑ дить следующие вопросы: В рамках предлагаемой статьи планируется обсу‑ дить следующие вопросы: р [ ] В марте 2007 г. в Женеве состоялась вторая Гло‑ бальная консультация по вопросам транспланта‑ ции, на которой ВОЗ представила план обновления «Руководящих принципов», а также было решено создать Глобальный форум по трансплантации под руководством ВОЗ для содействия и поддержки раз‑ вивающихся стран, приступающих к проведению программ по трансплантации, и работы по созданию единой координирующей системы для клеток, тканей и органов. Во время женевской консультации было отмечено, что в 2005 г. году в мире было пересажено 66 000 почек, но это удовлетворило лишь 10% всех потребностей. Кроме того, сообщалось, что посред ством трансплантационного туризма осуществлялось 10% глобальной практики в области трансплантации. Эксперты ВОЗ подчеркивали, что «решающее значе‑ ние для того, чтобы общество могло воспользоваться теми преимуществами, которые может предоставить трансплантация как терапия», имеют «качество, бе‑ зопасность, эффективность и прозрачность» [5]. – Что представляет собой дефицит органов с точки зрения международного медицинского сообщес‑ тва? – Какие стратегии преодоления дефицита органов предлагаются в настоящее время? – Как на практики самообеспечения органов влияют культурные различия и социально-экономическое неравенство? Американская стратегия Опираясь на принципы Стамбульской декларации и 63-й Всемирной ассамблеи, ведущие эксперты ВОЗ вскоре опубликовали специальный текст, призыва‑ ющий правительства всех стран добиваться более строгого контроля в достижении самообеспечения в сфере органного донорства и трансплантации. Авто‑ ры публикации специально подчеркивали «необходи‑ мость новой парадигмы национального самообеспе‑ чения» и вновь утверждали, что «каждая страна или регион должны стремиться к обеспечению доста‑ точного количества органов от своего собственного населения, руководствуясь этическими принципами ВОЗ». Опубликованный материал также содержал в себе разнообразную информацию о том, как обстоит дело с самообеспечением донорскими органами в различных странах мира [8]. Наряду с Советским Союзом США были одним из пионеров в области пересадки органов, и в этой стра‑ не создана одна из первых в мире успешных систем органного донорства, которая позволила осущест‑ влять заготовку органов в национальном масштабе и оперативно перераспределять их между медицин‑ скими учреждениями. В 1968 г. там появилась пер‑ вая организация, профессионально занимающаяся донорством органов – NEOB (New England Organ Bank), в штате которой состояли особые специалис‑ ты, занимающиеся выявлением доноров в находя‑ щихся в этом регионе больницах, их ведением после диагностики смерти мозга, получением согласия на изъятие органов у родственников донора и их пси‑ хологической поддержкой, получением и транспор‑ тировкой органов, контролем качества выполняемых работ, организацией информационных кампаний для общественности, контактами со СМИ и т. д. Вслед за NEOB появились еще 57 таких же организацией, за‑ нимающихся заготовкой органов (Organ Procurement Organizations) [9, 10]. В целом на протяжении конца 1980-х – начала 2010-х гг. ВОЗ постоянно придерживалась той линии поведения, которая была нацелена на решение про‑ блемы глобального дефицита органов. Постепенно пришло осознание того, что преодолению дефицита мешает много явлений – рассогласованность наци‑ онального законодательства о донорстве и транс‑ плантации, недобросовестность некоторых участ‑ ников мирового трансплантационного сообщества, торговля органами и трансплантационный туризм. Практически с самого начала также было осознано наличие серьезных различий между развитыми и раз‑ вивающимися странами в организации ими эффек‑ тивных и справедливых систем донорства, а также выявлены противоречия по вопросам приемлемости использования умерших или живых доноров и от‑ ношения к купле-продаже органов. Признавая эти различия и противоречия, ведущие эксперты ВОЗ, тем не менее, все более утверждаются во мнении, что Важный шаг был сделан в 1984 г., когда Конгресс принял Национальный закон о трансплантации орга‑ нов (National Organ Transplant Act), после чего Ми‑ нистерство здравоохранения и социального обслужи‑ вания поручило специально назначенной группе из 25 специалистов – Task Force on Organ Procurement and Transplantation – упорядочить работу всех ре‑ гиональных OPOs. ВОЗ О ДЕФИЦИТЕ ОРГАНОВ 57-я Всемирная ассамблея здра‑ воохранения, учитывая уже наработанный опыт и новые тенденции в практике трансплантации, реко‑ мендовала переработать «Руководящие принципы» 1991 года. Специальное внимание обращалось не только на торговлю органами, но и на такое явле‑ ние, как трансплантационный туризм. Говоря о «все большей нехватке имеющегося человеческого мате‑ риала для трансплантации», ВОЗ также признала, 175 ВЕСТНИК ТРАНСПЛАНТОЛОГИИ И ИСКУССТВЕННЫХ ОРГАНОВ том XXII № 1–2020 В 2010 г. 63-я Всемирная ассамблея здравоохра‑ нения одобрила новые «Руководящие принципы», в которых нашли воплощение идеи Стамбульской декларации. Особое внимание было вновь уделено проблемам трансплантационного туризма и торговли органами, которая ставилась в тесную связь с неза‑ конной торговлей людьми. В одиннадцати предло‑ женных мировому сообществу принципах подчерки‑ вались необходимость законного получения органов от доноров, четкое разделение ролей и обязанностей между лечащими врачами и трансплантологами, при‑ оритет посмертного донорства над прижизненным, а в рамках прижизненного – родственного над нерод ственным, запрещение получения донорских органов от несовершеннолетних, кроме тех случаев, что раз‑ решены законом, безвозмездный характер донорства, в равной мере одинаковость заботы о здоровье как реципиентов, так и доноров, прозрачность и подкон‑ трольность всех процедур, связанных с донорством и трансплантацией, обеспечение личной автономности и конфиденциальности доноров и реципиентов [7]. преодоление дефицита органов должно стать общей целью для всех участников международного обще‑ ния, а средством для этого должна быть деятельность по самообеспечению донорскими органами в масш‑ табах каждой страны или региона. НАЦИОНАЛЬНЫЕ СТРАТЕГИИ САМООБЕСПЕЧЕНИЯ Неустранимый дефицит органов не только оста‑ ется постоянной темой для осмысления со стороны международного медицинского сообщества, но и от‑ правной точкой для выработки различных стратегий по его преодолению. Предложенная ВОЗ концепция национального самообеспечения органами является рамочной, но у каждой страны или группы стран могут быть собственные подходы к ее практичес‑ кой реализации. Ниже будут рассмотрены некоторые наиболее известные стратегии самообеспечения до‑ норскими органами. Испанская стратегия С 1990 г. наиболее успешный опыт самообеспече‑ ния донорскими органами демонстрирует Испания, где под руководством нефролога Рафаэля Матесан‑ ца в 1989 г. была создана Национальная организа‑ ция по трансплантациям (Organización Nacional de Trasplantes), возглавившая всю работу по органному донорству на основе новой модели получения донор‑ ских органов – трансплантационной координации. Впервые в мире в Испании была налажена практика, когда выявление потенциальных доноров и их веде‑ ние было поручено особым специалистам – транс‑ плантационным координаторам, чья деятельность осуществлялась на строго подотчетной и оплачи‑ ваемой основе. Назначаемые из числа больничных врачей, прежде всего из числа врачей отделений ин‑ тенсивной терапии (реанимации), трансплантацион‑ ные координаторы смогли обеспечить эффективное взаимодействие между лечащими врачами и транс‑ плантационными бригадами, тогда как в прежние десятилетия это не удавалось. Была выстроена вся цепочка трансплантационной координации – от боль‑ ничного до регионального и национального уровня, а также продуманы все технические и логистические аспекты быстрого получения доступа к донорам и донорским органам. Признавая значимость прижиз‑ ненного донорства, испанские специалисты, тем не менее, сосредоточились на улучшении работы в сфе‑ ре организации посмертного донорства, и уже в те‑ чение нескольких лет страна вышла на первое место в мире по этим показателям. Вследствие этого ВОЗ признала опыт Р. Матесанца и его коллег образцовым и рекомендовала его остальным странам. Наряду с общенациональными организациями OPTN и UNOS в США в те же самые годы был начат процесс создания региональных объединений, кото‑ рые включали в себя трансплантационные центры, центры заготовки органов, представителей местного бизнеса и медицинской администрации штата и были ориентированы на то, чтобы вывести деятельность по получению и распределению органов на более высокий уровень, добиться материальной заинте‑ ресованности от вовлеченных в нее медицинских учреждений, а также оптимизировать отбор канди‑ датов на получение органов, в особенности таких дефицитных, как трансренальные органы. Наиболее известным из таких региональных объединений стал OSOTC (Ohio Solid Organ Transplantation Consortium), созданный в штате Огайо в 1984 г. [13]. В связи с ростом количества медицинских реше‑ ний о проведении лечения посредством трансплан‑ тации и увеличением спроса на донорские органы американская национальная система органного до‑ норства уже в начале 1990-х гг. начала работать с перегрузками, и это потребовало последующей ее адаптации к новым вызовам. Начав борьбу за уве‑ личение источников донорских органов, американ‑ ские специалисты наряду с донорами со смертью мозга стали более активно использовать доноров с расширенными критериями, маргинальных доноров, доноров с остановкой сердца, а также стали более широко привлекать различные категории прижизнен‑ ных доноров. В 2000-е гг. Американская стратегия Благодаря этому была создана единая национальная система трансплантации и донорства – OPTN (Organ Procurement and Transplan‑ tation Network), все звенья которой связаны в единую компьютеризированную сеть. Администрированием этой сети и всей OPTN с 1986 г. стала заниматься не‑ 176 ОБЗОРЫ ЛИТЕРАТУРЫ ской трансплантационной медицине: использовать максимум имеющихся возможностей и создавать новые возможности. ской трансплантационной медицине: использовать максимум имеющихся возможностей и создавать новые возможности. коммерческая научно-образовательная организация UNOS (United Network for Organ Sharing), надзор над которой взял на себя Департамент трансплантации Министерства здравоохранения США. Организаци‑ онно UNOS включает в себя все трансплантационные центры и OPOs, управляемые через Совет директо‑ ров и специальные комитеты [11, 12]. Испанская стратегия в профессиональный язык американских врачей-трансплантологов вошли такие понятия, как «прижизненные неродственные доно‑ ры», «прижизненные доноры, состоящие в правовой и эмоциональной связи с реципиентами», «целенап‑ равленные и нецеленаправленные прижизненные доноры», «доноры – добрые самаритяне» [14, 15]. Кроме работы по улучшению ситуации с донор ством органов в своей стране испанские специалисты также активно включились в сферу международной экспертно-аналитической и пропагандистской де‑ ятельности по вопросам органного донорства. При участии испанской Национальной организации стали публиковаться различные общеевропейские и меж‑ дународные документы по донорству органов – ру‑ ководящие принципы, статистическая информация, декларации, директивы. Благодаря настойчивым усилиям Р. Матесанца впервые в мире в испанское уголовное законодательство была введена статья, за‑ прещающая торговлю органами и строго наказываю‑ щая лиц, покупающих органы за границей. Называя трансплантационный туризм преступной и амораль‑ ной деятельностью, Р. Матесанц при этом указал на то, что почву для его существования создают сами врачи, прежде всего в развитых странах – США, Япо‑ Стремление использовать весь спектр возможнос‑ тей для получения новых органов позволяет считать американскую стратегию самообеспечения одной из самых агрессивных в мире. Примечательно, что термин «агрессивная заготовка органов», который начал продвигаться в научной литературе в середине 2000-х гг., предназначается для обозначения такти‑ ки «агрессивного манипулирования (management) телом донора с целью получения максимального ко‑ личества органов для трансплантации» [16]. Однако представляется, что это понятие верно не только для характеристики конкретных медицинских ситуаций, но и для общей тенденции, сложившейся в американ‑ 177 ВЕСТНИК ТРАНСПЛАНТОЛОГИИ И ИСКУССТВЕННЫХ ОРГАНОВ том XXII № 1–2020 вомерную и справедливую [20, 21], хотя ни ВОЗ, ни большинство западных специалистов не считают ее таковой. нии, Израиле и странах Европы, поддерживающие своих пациентов, которые отправляются на поиски органов [17]. Принятие платной модели приобретения донор‑ ских органов (почек) позволило Ирану всего через десять лет избавиться от длинных очередей в боль‑ ничных листах ожидания, а также покончить с неле‑ гальной торговлей органами. С введением платной системы доноры почек стали получать за проданный орган по 3500 долларов (в конце 1990-х гг.), хотя позднее стоимость органа стала сокращаться – 1265 в 2002 г. и 900 в 2011 г. Кроме того, правительство пре‑ доставило донорам бесплатную медицинскую стра‑ ховку. Поддержав куплю-продажу органов, иранское государство добилось существенного сокращения средств на высокотехнологичную медицинскую по‑ мощь: сократились расходы на содержание пациен‑ тов в центрах диализа. Как западные, так и иранские специалисты уделяют внимание вопросу о мотивах, которыми руководствуются иранские граждане, про‑ дающие свои органы. Считается, что эти мотивы сме‑ шанные – как финансовые, так и альтруистические. Испанская стратегия При этом продажа органов не является делом только бедных людей. По разным оценкам, доля совершен‑ но неграмотных людей, продающих свои органы, варьируется от 2,7 до 29%, тогда как количество лю‑ дей со школьным образованием (сроком 6–12 лет) колеблется от 71 до 90,8%. В 2000 г. в Иране был принят закон, позволяющий использовать органы от пациентов с зарегистрированной смертью мозга, и к началу 2010-х гг. число полученных почек от та‑ ких доноров составило 12%. Тем не менее основным источником донорских органов в начале 2010-х гг. в Иране продолжали оставаться неродственные живые доноры [22–25]. Испанская национальная стратегия самообеспе‑ чения, предполагающая активную пропаганду идеи посмертного донорства среди населения с привле‑ чением СМИ, церкви и учреждений образования, тем не менее, уступает американской в степени аг‑ рессивности. Так, испанские врачи-трансплантоло‑ ги почти вдвое реже, чем их американские коллеги, используют потенциал прижизненного донорства и почти не используют возможности прижизненного неродственного донорства (в 2017 г. было проведено всего 14 пересадок почки от неродственных доноров, что составило 0,3 случая на каждый миллион насе‑ ления, тогда как в США было выполнено 1124 такие операции, или 3,5 случая на миллион) [18]. Тем не менее среди испанских специалистов идея «агрессив‑ ной заготовки органов» также встречает поддержку. Примером этого служит предложение Диего Гра‑ ции – наиболее авторитетного испанского медицин‑ ского философа и биоэтика – перейти от концепции добровольного альтруистического пожертвования к концепции обязательного гражданского долга пере‑ давать свои органы после смерти. По его мнению, «органы умерших людей являются общественным благом», и поэтому распоряжаться ими должен не автономный индивид, а так называемый «супер-поль‑ зователь» (supererogatory) – все общество в целом или государство. Д. Грация называет такой подход «радикальным решением» проблемы дефицита ор‑ ганов, но подчеркивает, что правовые основания для этого пока отсутствуют [19]. Иранская стратегия При сравнении иранской стратегии самообеспече‑ ния с американской и испанской нетрудно увидеть, что она явным образом сфокусирована на исполь‑ зовании потенциала прижизненных неродственных доноров. Однако у нас нет оснований говорить о за‑ ложенной в ней идеологии «агрессивной заготовки органов». В отличие от своих американских и ис‑ панских коллег иранские врачи-трансплантологи, очевидно, не используют весь спектр существующих возможностей в сфере донорства. Представляется, что их выбор предопределен господствующими куль‑ турными нормами исламского общества, где значи‑ тельная часть населения настороженно относится к практике посмертного изъятия органов. Еще одна стратегия самообеспечения связана с легализацией платных форм получения донорских органов. Официально она осуществляется лишь в одной стране – Иране, где она была введена в 1988 г. сразу после окончания ирано-иракской войны и в условиях международной политической изоляции. Не вполне ясен вопрос о роли авторитарного теокра‑ тического режима в принятии этой модели донорской системы в этом исламском государстве, но вполне возможно, что выбор в пользу легализации купли- продажи органов был своего рода реакцией на от‑ вергнутые Ираном ценности западной либеральной демократии и всего, что с этим ассоциируется. Как бы то ни было, легализация купли-продажи орга‑ нов сыграла важную роль в развитии национальной трансплантационной программы. Покупателем всех донорских органов в Иране стало государство, что сняло целый ряд вопросов морально-этического и правового порядка. Большинство иранских специ‑ алистов оценивает эту модель как абсолютно пра‑ Три приведенных выше примера не исчерпывают всего разнообразия существующих сегодня наци‑ ональных стратегий самообеспечения донорскими органами, но при этом их можно считать весьма пока‑ зательными. Американская стратегия «агрессивной заготовки органов» в силу той роли, которую играет в мире американская трансплантационная медицина, 178 ОБЗОРЫ ЛИТЕРАТУРЫ во многом является образцом для других стран, хотя и не признана в качестве таковой ВОЗ. Стремление использовать весь спектр существующих возмож‑ ностей в привлечении донорских ресурсов является нормой, которой руководствуется медицинское со‑ общество большинства развитых стран. Испанская стратегия самообеспечения, признанная ВОЗ в ка‑ честве образцовой модели, тоже имеет много при‑ верженцев, в том числе, по-видимому, и потому, что ей свойственна меньшая агрессивность. Иранская же стратегия официально не имеет аналогов за пре‑ делами этой страны, но устойчивый интерес к ней и обилие публикаций о роли иранского опыта, похоже, уготовили и этой стратегии шанс на сохранение в будущем. умерших доноров вдвое с лишним меньше: 7,2 [18]. В стране не существует запрета на использование доноров со смертью мозга, однако низкий уровень развития посмертного донорства обусловлен прежде всего культурными особенностями местной жизни. Медицинский антрополог М. Кроули-Матока предложила одно из самых интересных объяснений мексиканского феномена. Иранская стратегия В своем многолетнем ис‑ следовании она оттолкнулась от одного случая, ко‑ торый можно считать парадигмальным. Родители од‑ ного юноши, которому требовалась пересадка почки, отправились в Германию, на родину его матери. Там и врачи, и родня по материнской линии стали настаи‑ вать на том, чтобы провести пересадку от умершего донора. Однако семья приняла другое решение и вер‑ нулась в Мексику, где немецкая мать пожертвовала сыну собственную почку. Этот поступок не только получил одобрение со стороны мексиканской родни и врачей, но и был воспринят всеми как совершенно оправданный и закономерный: женщина-мать, родив‑ шая ребенка, в случае необходимости всегда должна пожертвовать ему часть своего тела. КУЛЬТУРНЫЕ РАЗЛИЧИЯ И СОЦИАЛЬНО‑ЭКОНОМИЧЕСКОЕ НЕРАВЕНСТВО Международное медицинское сообщество в лице ВОЗ связывает возможности развития трансплан‑ таций и сокращение дефицита донорских органов прежде всего с увеличением масштабов посмертно‑ го донорства. При этом эксперты ВОЗ справедливо указывают на то, что в некоторых частях мира сама идея посмертного донорства вызывает «культур‑ ное сопротивление». Речь идет в первую очередь о странах Азии, Латинской Америки и Африки, т. е. в основном о развивающихся странах. Признавая это, ВОЗ настаивает на важности «качественных иссле‑ дований для понимания «немедицинских» причин этого сопротивления» [26, 27]. р у Согласно выводам исследовательницы, данная логика тесно связана с представлениями о сексуаль‑ ности. Если зачатие ребенка всегда является след ствием половой связи, и следовательно, следствием моральной нечистоты женщины-матери, то дето‑ рождение предстает как моральное искупление. То же касается и прижизненного донорства почки: оно выступает материнским искуплением за плотский грех. Размышляя над этой логикой, исследователь‑ ница обратилась также к особенностям мексиканской религиозности, которая, в сущности, сложилась еще в XVI в., когда древняя Мексика была завоевана ев‑ ропейцами. Именно в этот период, после морального унижения, которому подверглись коренные мекси‑ канцы, они впервые приняли религию своих завоева‑ телей – католицизм. Примечательно, что источником религиозного чувства коренных жителей страны стал образ Девы Марии Гваделупской, которая с тех пор является наиболее почитаемым христианским сим‑ волом в Новом Свете. В образе мексиканской Мадон‑ ны соединились черты материнской жертвенности и культурно-религиозный выбор самих мексиканцев, которые с середины XVI в. превратились в единую нацию коренных жителей и их завоевателей. Кроме того, по мысли Кроули-Матока, в общественном со‑ знании мексиканцев глубоко укоренился и еще один женский образ, Малинче – индианки знатного рода, которая была в качестве рабыни отдана правителю ацтеков, а затем подарена им предводителю конкис‑ тадоров Эрнану Кортесу. Став наложницей Кортеса, она также стала и лучшей его шпионкой, которая помогла испанцам завоевать Мексику. Если довести до логического завершения обе эти значимые для современных мексиканцев истории, то становится Мексика Среди развивающихся стран Латинской Америки случай наиболее явного влияния культуры на донор ские практики демонстрирует Мексика. Страна с пре‑ обладанием католического населения также управля‑ ется правительством, которое на протяжении многих десятилетий пытается проводить в жизнь политику модернизации, но при этом находится в положении безнадежно отстающего от своего северного соседа, США. В стране достаточно высоко развита транс‑ плантационная медицина, и по общему количеству пересадок почки Мексика в 2017 г. занимала чет‑ вертое место среди всех стран Латинской Америки: 24,5 трансплантации на миллион населения. Между тем наиболее часто осуществляются пересадки с ис‑ пользованием живых доноров, тогда как посмертное донорство развито очень слабо. По уровню исполь‑ зования посмертных донорских органов Мексика за‑ нимает одно из последних мест в регионе, зато по ко‑ личеству пересадок почек с использованием живых доноров она уверенно занимает первое место: 17,3. При этом общее количество трансплантаций почек от 179 ВЕСТНИК ТРАНСПЛАНТОЛОГИИ И ИСКУССТВЕННЫХ ОРГАНОВ том XXII № 1–2020 дивида, сколько семьи. Все органы так или иначе перераспределяются в рамках семейной структуры, и действует моральная экономия семейного выжи‑ вания. более понятным и тезис о естественности материн‑ ской жертвы. Всякая женщина-мать в Мексике – это сначала Малинче, предательница своего народа, со‑ вершающая плотский грех с врагом, а затем – Дева Мария Гваделупская, мать Божества, приносящая искупительную жертву [28, 29]. В целом можно считать, что большинство об‑ ществ с традиционными взглядами, или развиваю‑ щихся обществ, с большей готовностью признает прижизненное донорство, чем посмертное. В отли‑ чие от европейцев и североамериканцев, которые придерживаются рационалистических представ‑ лений о человеке, подавляющее большинство на‑ селения развивающихся стран отвергает материа‑ листические воззрения на тело и душу. На Западе человек мыслится как разумное существо, а органом разума считается мозг. Если мозг умирает, то уми‑ рает и сам человек. Не то же самое в традиционных обществах, где смерть мозга вовсе не есть смерть человека. В пространстве живого религиозного со‑ знания смерть есть всегда нечто большее, переход в новое состояние, как и сам человек, чье тело даже в его посмертном состоянии мыслится как святыня и собственность Высшего Существа. Для большинства мексиканцев неприемлемой яв‑ ляется идея посмертного донорства. Кроули-Матока связывает это с верой мексиканцев в посмертное вос‑ кресение и их представлениями о том, что тело после смерти должно оставаться целым, без повреждений. Но наряду с аргументами, относящимися к сфере духовной культуры, по мысли исследовательницы, важную роль также играют и факторы материаль‑ но-культурного порядка. Мексика В частности, она отмечает неразвитость материальной инфраструктуры мекси‑ канских больниц, отсутствие в большинстве из них палат реанимации и очевидную нехватку аппаратов искусственной вентиляции легких. Кроме того, в стране не хватает специалистов, способных поста‑ вить достоверный диагноз «смерть мозга». В работе Кроули-Матока показано также, что естественность материнского донорства для мекси‑ канцев хорошо согласуется с имеющимися фактами, хотя в отдельных случаях может показаться, что это не так. В частности, собранная исследовательницей статистика показывает, что значительно чаще, чем матери, донорами друг для друга становятся братья и сестры. За двенадцать лет наблюдений было вы‑ явлено 168 случаев жертвования органов сестрами и 160 случаев – братьями (всего 328 случаев). В свою очередь, дети жертвовали органы для родителей 63 раза (31 раз – дочери и 32 – сыновья), а родители для детей 76 раз (46 – матери и 30 – отцы). Супруги жертвовали органы друг для друга 42 раза, причем 35 раз – жены для мужей и только 7 – мужья для жен. Такое же неравномерное распределение донорских органов между полами, как между супругами, на‑ блюдается и между братьями и сестрами. Сестры для братьев выступали донорами 93 раза, а братья для сестер вдвое меньше, 46 раз. Более охотно сиблинги жертвовали своими органами для родственников того же пола: братья для братьев – 115 раз, сестры для сестер – 46 раз. Несмотря на то что все эти факты, приводимые Кроули-Матока, говорят о несколько большей жертвенности мексиканских женщин, чем мексиканских мужчин [28], мы склонны акцентиро‑ вать внимание на главном выводе всей ее работы: в Мексике донорство органов по целому ряду причин является делом семейным. Именно семья, поскольку она является опорой для всех своих членов, является главным потребителем донорских органов. Мекси‑ канцам не свойствен крайний индивидуализм. Хотя одни члены семьи с готовностью жертвуют свои ор‑ ганы для других, но, в сущности, дело всегда идет о том, чтобы обеспечить не столько выживание ин‑ Турция Это неминуемо привело к эксцессам в практике приобретения донорских органов. Как по‑ казано в исследовании А. Санала, на рубеже 1990-х и 2000‑х гг. в частных турецких клиниках распростра‑ нилась подпольная торговля органами, в которую оказались вовлечены некоторые местные доктора. Источниками органов становились трупы бедняков, умерших в психиатрических лечебницах, лица, по‑ кончившие самоубийством, и жертвы крупных зем‑ летрясений. Нередко донорские органы приобрета‑ лись у бедняков, которых специально привозили в турецкие клиники из Индии, Ирака и других мест. Оценивая масштабы скандала в турецкой трансплан‑ тационной медицине этого времени, Санал называ‑ ет его значительным, а предприимчивых турецких докторов, вроде печально известного «доктора S.», называет «техно-Робин Гудами», которые участвуют перераспределении дефицитных богатств (органов) от бедных к богатым [30, 31]. Турции – по крайней мере, до 2010-х гг. – не удалось создать прозрачную систему органного донорства, а все существующие практики донорства население воспринимало скеп‑ тически. почек только от родственных доноров, и активно поощряется мысль, что донорство есть моральная обязанность каждого человека перед членом своей семьи. При этом явно выраженный дефицит донор‑ ских органов толкает многих пакистанцев к тому, чтобы искать возможность получить транспланта‑ ционную помощь за границей [32–34]. Медицинское сообщество в лице ВОЗ и СМИ уже четверть века настойчиво говорит о глобальном дефиците органов, и этот дефицит действительно существует там, где осуществляются транспланта‑ ции. Однако парадоксальным образом этот дефи‑ цит может принимать разные проявления. В странах Южной Азии, прежде всего в Индии, а также Шри Ланка и Бангладеш, на протяжении длительного вре‑ мени наблюдается дефицит иного рода – «не дефицит органов, а дефицит пациентов» [35]. Целый ряд ис‑ следований последних лет, которые были проведены медицинскими антропологами, указывает на то, что на практики донорства может накладывать большой отпечаток не только культурная среда, но и социаль‑ но-экономическая ситуация. Бангладеш Бангладеш – одна из самых бедных стран в мире. Из 150 миллионов ее населения 78% живут менее чем на два доллара США в день. В начале 2000-х гг. она присвоила себе печальную славу еще одного «миро‑ вого базара органов». Ее столица, Дакка – это место, где число желающих продать свои органы (почку, роговицу глаза, часть печени) неизмеримо превыша‑ ет число тех, кто готов их купить. Именно поэтому стоимость продаваемых органов имеет тенденцию снижаться, а циничные и предприимчивые брокеры и покупатели органов бесстыдно обманывают бед‑ няков, желающих их продать. Турция Турция – крупная страна, где на протяжении поч‑ ти векового периода у власти находится светское пра‑ вительство, а большинство населения исповедует ислам, причем половина в весьма умеренной форме. Географическое положение делает ее наполовину европейской страной, и это примечательным обра‑ зом связано с той политикой модернизации, которую власти осуществляют в самых разных аспектах хо‑ зяйственной и культурной жизни. Опыт Турции сви‑ детельствует о том, что большинство населения не‑ охотно соглашается с возможностью использования органов человека после его смерти. Данные за 2017 г. свидетельствуют о крайне низком уровне донорской активности для случаев пересадки сердца, легких и поджелудочной железы, т. е. органов, которые можно изъять только у мертвого донора. Напротив, количес‑ тво трансплантаций с использованием доли печени от живого донора достаточно высоко – 13,5 человека на миллион населения, а количество изъятий почки от живого донора – 32,8 человека на миллион населе‑ ния; это один из самых высоких показателей в мире (в Европе такие же показатели только в Нидерлан‑ дах). При этом уровень активности для посмертного донорства почки в Турции весьма невысокий – 8,6 (в Европе ниже только в православных Греции, Бол‑ гарии, Сербии и Молдавии, а также в России) [18]. Специальные исследования показывают, что преобладание традиционных взглядов в турецком обществе сказалось на неудачах национальной трансплантационной программы в 1990-е гг. По‑ ощряя различные формы модернизации, турецкое 180 ОБЗОРЫ ЛИТЕРАТУРЫ правительство тогда же инициировало создание сети частных клиник для представителей среднего класса и богатых иностранцев, в которых начали пересажи‑ вать органы. Это неминуемо привело к эксцессам в практике приобретения донорских органов. Как по‑ казано в исследовании А. Санала, на рубеже 1990-х и 2000‑х гг. в частных турецких клиниках распростра‑ нилась подпольная торговля органами, в которую оказались вовлечены некоторые местные доктора. Источниками органов становились трупы бедняков, умерших в психиатрических лечебницах, лица, по‑ кончившие самоубийством, и жертвы крупных зем‑ летрясений. Нередко донорские органы приобрета‑ лись у бедняков, которых специально привозили в турецкие клиники из Индии, Ирака и других мест. Оценивая масштабы скандала в турецкой трансплан‑ тационной медицине этого времени, Санал называ‑ ет его значительным, а предприимчивых турецких докторов, вроде печально известного «доктора S.», называет «техно-Робин Гудами», которые участвуют перераспределении дефицитных богатств (органов) от бедных к богатым [30, 31]. Турции – по крайней мере, до 2010-х гг. – не удалось создать прозрачную систему органного донорства, а все существующие практики донорства население воспринимало скеп‑ тически. правительство тогда же инициировало создание сети частных клиник для представителей среднего класса и богатых иностранцев, в которых начали пересажи‑ вать органы. Список литературы / References 1. World Health Assembly Resolution 40.13. Develop‑ ment of guiding principles for human organ transplants, 4–15 May 1987. [Online]. Available: http://apps.who. int/medicinedocs/documents/s15559e/s15559e.pdf (Ac‑ cessed: 14.08.2019). 2. World Health Assembly Resolution 44.25. Human organ transplantation, 6–16 May 1991. [Online]. Available: http://apps.who.int/medicinedocs/documents/s15559e/ s15559e.pdf (Accessed: 14.08.2019). 3. Capron A. 1991 Guiding principles: roots and impli‑ cations. Ethics. Access and safety in tissue and organ transplantation: issues of global concern. Madrid, Spain, 6–9 October 2003. Report. Geneva: WHO, 2004: 9. [On‑ line]. Available: http://www.who.int/transplantation/en/ Madrid_Report.pdf?ua=1 (Accessed: 14.08.2019). 4. World Health Assembly Resolution 57.18. Human or‑ gan and tissue transplantation, 22 May 2004. [Online]. Available: http://www.who.int/gb/ebwha/pdf_files/ WHA57/A57_R18-en.pdf. (Accessed: 1.08.2019). 5. 5. WHO proposes global agenda on transplantation. WHO. Media centre. 30 March 2007. [Online]. Available: ht‑ tps://www.who.int/mediacentre/news/releases/2007/ pr12/en/ (Accessed: 1.08.2019).i Пакистан Со‑ вершенно никто не смог воспользоваться деньгами, чтобы хоть как-то улучшить свою жизнь, а многим их не хватило даже на покрытие всех долгов. Доноры, продавшие свои почки, неизменно вспоминали о дне своей операции как о самом черном дне жизни. Один из тех, с кем беседовал ученый, сказал, что чувствует себя после операции «человеком лишь наполовину» [36, 37]. также серьезные психологические проблемы. У не‑ которых в силу всего этого вскоре распался брак. Со‑ вершенно никто не смог воспользоваться деньгами, чтобы хоть как-то улучшить свою жизнь, а многим их не хватило даже на покрытие всех долгов. Доноры, продавшие свои почки, неизменно вспоминали о дне своей операции как о самом черном дне жизни. Один из тех, с кем беседовал ученый, сказал, что чувствует себя после операции «человеком лишь наполовину» [36, 37]. Национальный закон о трансплантации органов в Бангладеш был принят в 1999 г., и согласно этому закону, в стране официально запрещена торговля ор‑ ганами. За нарушение закона преступникам грозит крупный штраф и тюремный срок. Однако в реаль‑ ности за это никого не преследуют, а объявления о готовности купить тот или иной донорский орган регулярно встречаются в пяти крупнейших газетах страны. По мнению Монируззамана, применительно к Бангладеш даже сам термин «донорство» выгля‑ дит неуместно. Органы здесь не дарят, а продают в открытую. Все это является не только следствием нищеты огромных масс населения, но и того, что антрополог называет «бионасилием». Наиболее бо‑ гатые покупатели органов предпочитают вывозить своих «доноров» за границу, обычно в Индию или Сингапур, и уже там делать операцию. Те, кто не так богат, пользуются услугами местных больниц, таких как больница медицинского университета Шейха Муджиба в Дакке. Самые же богатые летят самолетом в Америку, везя с собой купленный орган. Горячо одобряемая мировым медицинским сообщес‑ твом альтруистическая философия, согласно которой жизнь одних людей может быть спасена за счет жиз‑ ни других, здесь выглядит совершенно иначе: жизнь богатых продлевается за счет жизни бедных. По заме‑ чанию Н. Шейпер-Хьюз, в современном глобальном мире вслед за капиталами донорские органы также перемещаются из бедных стран в богатые, с Юга на Север, из третьего мира в первый мир; имеет место «медицинский апартеид» [38, 39]. Публикация подготовлена в рамках поддержанно- го РНФ научного проекта № 17-18-01444, 2019 год. Авторы заявляют об отсутствии конфликта интересов.l ф р The authors declare no conflict of interest. Пакистан Пакистан – одна из наиболее быстро развиваю‑ щихся стран в Азии. Здесь также у власти находится светское правительство, поддерживаемое военными, а многомиллионное население исповедует ислам. Трансплантационная программа, осуществляемая в стране с 1985 г., связана исключительно с прижизнен‑ ным донорством. В начале 2000-х гг. доля пациентов с тяжелыми формами заболеваний почек достигала 100 человек на каждый миллион населения, при этом в стране ежегодно проводилось порядка 600–700 опе‑ раций. Посмертное донорство отсутствует, поскольку законодательно запрещены трансплантации, связан‑ ные с использованием доноров со смертью мозга. При этом, хотя мусульманское духовенство и ученые из мусульманских академических центров признают, что посмертное донорство не противоречит исламу, в массовом религиозном сознании оно отвергается и воспринимается как надругательство над мертвым телом. Не создает оснований для развития посмерт‑ ного донорства и господство в сознании населения семейно-ориентированного коллективизма, который подавляет всякую автономию личности и ее права. В Пакистане успешно работает крупный транс‑ плантационный центр – Sindh Institute of Urology and Transplantation, организаторам которого удалось адаптировать к своим интересам культурные ценнос‑ ти пакистанцев: в нем осуществляются пересадки Медицинский антрополог М. Монируззаман в середине 2000-х гг. предпринял длительное иссле‑ дование в Дакке, в ходе которого ему удалось соб‑ рать интервью с тридцатью местными мужчинами и тремя женщинам, продавшими свой орган, а также пообщаться с врачами-урологами и нефрологами, участвующими в трансплантациях. Полученные сведения нельзя назвать иначе как удручающими. Почти все люди, продавшие свои органы, остались разочарованными. В большинстве случаев покупа‑ тели органов даже не заплатили им всю обещанную сумму – около 1400 долларов. У всех после операции на теле остался огромный шрам, которого могло бы не быть, если бы операция делалась лапароскопи‑ ческим методом. Почти никто не получил должной медицинской помощи после изъятия органа и после очень быстрой выписки из больницы был вынужден вернуться в совершенно антисанитарные условия. У большинства начались проблемы со здоровьем, а 181 ВЕСТНИК ТРАНСПЛАНТОЛОГИИ И ИСКУССТВЕННЫХ ОРГАНОВ том XXII № 1–2020 то, что в этом вопросе между профессионалами и обществом консенсус не всегда достигается. Обще‑ ство не обязательно принимает философию (этику) альтруистического донорства, во всяком случае в полном объеме. Данное обстоятельство делает не‑ обходимым более обстоятельное изучение того, как общество относится к донорству органов. В этой связи глобальная проблема дефицита донорских ор‑ ганов не может рассматриваться только как проблема профессионального медицинского сообщества. Она требует междисциплинарного изучения, совместной работы врачей и ученых-гуманитариев. также серьезные психологические проблемы. У не‑ которых в силу всего этого вскоре распался брак. ЗАКЛЮЧЕНИЕ 6. The declaration of Istanbul on organ trafficking and transplant tourism (2008 edition). [Online]. Available: https://www.declarationofistanbul.org/images/Policy_ Documents/2008_Edition_of_the_Declaration_of_Is‑ tanbul_Final.pdf (Accessed: 1.08.2019). Концепт «дефицит донорских органов» прочно вошел в профессиональный дискурс транспланта‑ ционной медицины и стал своеобразным отраже‑ нием текущего кризиса той философии, что лежит в основе современной трансплантационной прак‑ тики. Это философия альтруизма, которая впервые была разработана еще в XIX в. О. Контом. Руко‑ водствуясь философией альтруистического донор ства, представители трансплантационной медицины предлагают и остальным членам общества разделить идеалы этой философии – добровольность, безвоз‑ мездность, солидарность и т. д. Однако ситуация с глобальным дефицитом органов явно указывает на 7. World Health Assembly Resolution 63.22, WHO guiding principles on human cell, tissue and organ transplantati‑ on, 21 May 2010. 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https://openalex.org/W2030558355	https://bmcstructbiol.biomedcentral.com/counter/pdf/10.1186/1472-6807-7-29	English	null	X-ray sequence and crystal structure of luffaculin 1, a novel type 1 ribosome-inactivating protein	BMC structural biology	2,007	cc-by	9,407	BioMed Central BioMed Central Open Acc Research article X-ray sequence and crystal structure of luffaculin 1, a novel type 1 ribosome-inactivating protein Xiaomin Hou1,2, Minghuang Chen1,2, Liqing Chen3, Edward J Meehan3, Jieming Xie4 and Mingdong Huang1,2 Address: 1State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, The Chinese Academy of Sciences, 155 Yang Qiao Xi Lu, Fuzhou, Fujian, 350002, China, 2Graduate School of Chinese Academy of Sciences, The Chinese Academy of Sciences, Beijing 10039, China, 3Laboratory for Structural Biology, Department of Chemistry, Graduate Programs of Biotechnology, Chemistry and Materials Science, University of Alabama in Huntsville, Huntsville, AL 35899, USA and 4Fujian Medical University, Fuzhou 350004, China Email: Xiaomin Hou - houxiaomin@fjirsm.ac.cn; Minghuang Chen* - cmh@fjirsm.ac.cn; Liqing Chen - chenlq@uah.edu; Edward J Meehan - meehane@uah.edu; Jieming Xie - xiejm1@sina.com; Mingdong Huang* - mhuang@fjirsm.ac.cn * Corresponding authors Received: 26 October 2006 Accepted: 30 April 2007 Received: 26 October 2006 Accepted: 30 April 2007 Received: 26 October 2006 Accepted: 30 April 2007 Published: 30 April 2007 BMC Structural Biology 2007, 7:29 doi:10.1186/1472-6807-7-29 BMC Structural Biology 2007, 7:29 doi:10.1186/1472-6807-7-29 This article is available from: http://www.biomedcentral.com/1472-6807/7/29 © 2007 Hou et al; licensee BioMed Central Ltd. © 2007 Hou 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. BMC Structural Biology Open Access Background Here, we demonstrate that the evaluation of the chemical environments of these pairs can help to break down such ambiguity and 86% (see Table 2) of the amino acids were assigned with confidence on the basis of the electron den- sity and the chemical environment evaluation of the resi- dues. RIPs have received wide attentions due to their potential therapeutic applications in medicine and transgenic rea- gents in agriculture. In medicine, they have been found to possess various pharmacological activities including abor- tifacient [17], antifungal [18], anti-tumor [19,20], antivi- rus and HIV-1 integrase inhibitory activity [21,22]. Plants transfected with RIP genes exhibit broad-spectrum resist- ance to viral and fungal infection [23,24] in the plant defense system. We identified a new type 1 RIP, luffaculin 1 [25]. It is a basic protein with a pI of 8.86 by IEF analysis and has a molecular mass of about 28 kDa based on the mobility on SDS-PAGE. Luffaculin 1 not only possesses rRNA N-gly- cosidase activity as expected [26], but also inhibits prolif- eration of tumor cells, induces apoptosis [27] and differentiation on tumor cells [28]. The ribosome-inactivating proteins (RIPs) are RNA N-gly- cosidases [5,6] that inactivate ribosome by cleaving a sin- gle N-C glycosidic bond between adenine and ribose at A4324 in the 28S eukaryotic mammalian rRNA or at A2660 in the 23S Escherichia coli rRNA. The cleaved N-C glycosidic bond is located in a loop containing a GAGA sequence and highly conserved in rRNAs from bacteria, plants and animals. The removal of one adenine from rRNA by RIPs prevents the binding of elongation factor II (EF-2) to the 60S subunit, resulting in the termination of protein translation. In E. coli, the cleavage by RIP affects the combination of EF-Tu and EF-G. Both EF-G and EF-Tu protect bases in the universally conserved loop around position 2660 of 23S rRNA. This loop is also the site of action of cytotoxins that alter the structure of a region of rRNA that interacts with EF-Tu and EF-G and thus abolish protein synthesis. RIPs from plants can be classified into three types based on the structure of the genes and mature proteins [7]. Type 1 RIPs, such as trichosanthin [8], bryo- din [9], α, β-momorcharin [10,11], luffin a and b [12] and cucurmosin [13], have alkaline isoelectric points and molecular weights ranging from 26 to 31 kDa. Background potent ability to inhibit protein synthesis in the cell free system but are relatively non-toxic to the intact cells. Type 2 RIPs, such as ricin [14] and abrin [15], consist of two chains, chain A and chain B, linked by disulfide bridges. The A chain is homologous to type 1 RIPs and possesses the ribosome-inactivating activity; the B chain, containing a lectin domain, binds to galactosyl-terminated receptors on the target cell surface, facilitating the entry of the A chain into the cytoplasm of the cell. Thus, some, but not all, type 2 RIPs are more potent toxin than type 1 RIPs because type 1 RIPs have difficulty in entering into cells. Type 3 RIP includes JIP60 [16] (jasmonate-induced pro- tein) from maize, which consists of an N-terminal domain similar to type 1 RIPs and an unrelated C-termi- nal domain of unknown function. Most RIPs are glyco- proteins, with varying amount and type of sugars. g The amino-acid sequence is the most basic but critical information for proteins. The primary structure of the pro- tein can be obtained through Edman degradation, mass spectrometry or cDNA method. These methods, especially cDNA method, have been applied widely, but they still have their own limitations [1]. Edman degradation is expensive and cannot deal with cases where the N-termi- nal amino-acid of protein is blocked. cDNA sequencing is the most popular sequencing method nowadays, but it cannot identify the amino-acid post translational modifi- cations. Mass spectrometry has made dramatic advance in the last decade in its technology [2], but still has difficul- ties to give the full-length sequence and to distinguish the residue pair Ile/Leu. X-ray sequencing method, based on electron density, is another method to determine the pro- tein sequence. Although this method has limited usage, it is a useful addition to the sequencing methods in some cases, e.g., where cDNA is not readily available. This method has been used to determine the sequence of PAP- Saci [3] and trichomaglin [4]. A major problem of this method is the difficulty to distinguish residue pairs Asp/ Asn, Glu/Gln, and Val/Thr. Moreover, weak electron den- sity of some residues located at the molecular surface also gives rise to uncertainty for the X-ray sequence analysis. Background They typi- cally contain a single polypeptide chain and have the Here, we report the high resolution (1.4 Å) crystal struc- ture of luffaculin 1 and the protein sequence derived from this crystal structure. The structural comparison with other RIPs provides a structural basis to understand their possible biological activity. The amino-acid sequence of luffaculin 1 has not been determined by the traditional cDNA method. We demonstrated that the primary struc- ture of luffaculin 1 can be derived with a high degree of confidence from the high-resolution electron density. The existence of two independent luffaculin 1 molecules in the asymmetric unit allows the cross-validation of this X- ray sequence, further increasing the reliability of the sequence assignment. Abstract Background: Protein sequence can be obtained through Edman degradation, mass spectrometry, or cDNA sequencing. High resolution X-ray crystallography can also be used to derive protein sequence information, but faces the difficulty in distinguishing the Asp/Asn, Glu/Gln, and Val/Thr pairs. Luffaculin 1 is a new type 1 ribosome-inactivating protein (RIP) isolated from the seeds of Luffa acutangula. Besides rRNA N-glycosidase activity, luffaculin 1 also demonstrates activities including inhibiting tumor cells' proliferation and inducing tumor cells' differentiation. Results: The crystal structure of luffaculin 1 was determined at 1.4 Å resolution. Its amino-acid sequence was derived from this high resolution structure using the following criteria: 1) high resolution electron density; 2) comparison of electron density between two molecules that exist in the same crystal; 3) evaluation of the chemical environment of residues to break down the sequence assignment ambiguity in residue pairs Glu/Gln, Asp/Asn, and Val/Thr; 4) comparison with sequences of the homologous proteins. Using the criteria 1 and 2, 66% of the residues can be assigned. By incorporating with criterion 3, 86% of the residues were assigned, suggesting the effectiveness of chemical environment evaluation in breaking down residue ambiguity. In total, 94% of the luffaculin 1 sequence was assigned with high confidence using this improved X-ray sequencing strategy. Two N-acetylglucosamine moieties, linked respectively to the residues Asn77 and Asn84, can be identified in the structure. Residues Tyr70, Tyr110, Glu159 and Arg162 define the active site of luffaculin 1 as an RNA N-glycosidase. Conclusion: X-ray sequencing method can be effective to derive sequence information of proteins. The evaluation of the chemical environment of residues is a useful method to break down the assignment ambiguity in Glu/Gln, Asp/Asn, and Val/Thr pairs. The sequence and the crystal structure confirm that luffaculin 1 is a new type 1 RIP. Page 1 of 10 (page number not for citation purposes) Page 1 of 10 (page number not for citation purposes) BMC Structural Biology 2007, 7:29 http://www.biomedcentral.com/1472-6807/7/29 http://www.biomedcentral.com/1472-6807/7/29 Residue Tyr 141 of the molecule B, located in a turn connecting α6 helix and α7 helix, and residue Asn 235 of the molecule A, located in a turn connecting α9 helix and α10 helix, lie in the generously allowed region. Data collection and refinement statistics are summarized in Table 1. active site cleft. Helices α1 and α3 are part of the crossover connections between the parallel strands of the β-sheet. Helices α7 and α8 are contiguous in sequence and a single residue (Phe 163) assumes a non-helical dihedral confor- mation, introducing a bend between the two helices. Two β-strands of C-terminal domain (β9 and β10) are con- nected by a loop whose length varied among different RIPs. The crystals of luffaculin 1 contain two enzyme molecules (A and B) in the asymmetric unit. A comparison of mole- cules A and B shows that the overall structures of these two molecules are almost identical (Fig. 2) with rmsd of 0.181 Å for 221 Cα atoms. Some deviation between two molecules occurs at the terminal of the α9-helix that is involved in the crystal packing. The electron density of luffaculin 1 clearly indicated the existence of two well-defined N-acetylglucosamines (NAGs), each covalently linked to an Asn residue at posi- tions 77 and 84, respectively. Both N-acetylglucomamines protrude from the molecular surface and do not have extensive interaction with the protein. These saccharide moieties of luffaculin 1 are also distant from the active site (Fig. 1), suggesting that these saccharide moieties may not involve in the enzymatic activity. The glycosylation in pro- tein has been recognized to play important roles in vari- ous functions, including protein folding in the endoplasmic reticulum, transport and secretion, anchor- ing of proteins to target sites, protection from protease, and increased protein conformational stability [31,32]. Values in parentheses refer to the highest resolution shell (1.45-1.40) Å. a Rmerge = Σ\|Ii-<I>\|/ΣIi, where Ii is the intensity of the ith observation and <I> is the mean intensity of the reflections. http://www.biomedcentral.com/1472-6807/7/29 http://www.biomedcentral.com/1472-6807/7/29 of 0.03 (Table 1). The current model of luffaculin 1, con- taining two molecules in the asymmetric unit, was refined to an R factor of 0.213 and Rfree of 0.232. Most residues in the model fit the electron density quite well, except for some residues in loop regions that do not have good qual- ity of electron density. Residues 28, 206, 215–220 of mol- ecule B were omitted from the final model due to lack of the electron density. The quality of the stereochemistry of the final protein structure was analyzed by PROCHECK package [29]. The root mean square deviations (rmsd) of bond length and bond angles are 0.007 Å and 1.153°, respectively. The Ramachandran plot shows 91.3% of the residues in the most favored region and 7.6% in the addi- tional allowed region. Residues Asn 77 of molecules A and B, which are linked to an N-acetylglucosamine, respectively, lie just outside the generously allowed region. Residue Tyr 141 of the molecule B, located in a turn connecting α6 helix and α7 helix, and residue Asn 235 of the molecule A, located in a turn connecting α9 helix and α10 helix, lie in the generously allowed region. Data collection and refinement statistics are summarized in Table 1. of 0.03 (Table 1). The current model of luffaculin 1, con- taining two molecules in the asymmetric unit, was refined to an R factor of 0.213 and Rfree of 0.232. Most residues in the model fit the electron density quite well, except for some residues in loop regions that do not have good qual- ity of electron density. Residues 28, 206, 215–220 of mol- ecule B were omitted from the final model due to lack of the electron density. The quality of the stereochemistry of the final protein structure was analyzed by PROCHECK package [29]. The root mean square deviations (rmsd) of bond length and bond angles are 0.007 Å and 1.153°, respectively. The Ramachandran plot shows 91.3% of the residues in the most favored region and 7.6% in the addi- tional allowed region. Residues Asn 77 of molecules A and B, which are linked to an N-acetylglucosamine, respectively, lie just outside the generously allowed region. Results and discussion Quality of the model The crystals of luffaculin 1 belong to space group P1 and diffract to 1.4 Å resolution with synchrotron X-ray radia- tion. A high quality data set was collected to a complete- ness of 86.7% and redundancy of 1.9, yielding an Rmerge Page 2 of 10 (page number not for citation purposes) Page 2 of 10 (page number not for citation purposes) BMC Structural Biology 2007, 7:29 Structure description 227 (exclude 28, 29, 97, 109, 111, 190, 216, 217, 218, 220, 225, 228, 223, 237) Neither molecule A nor B has clear density map, but the sequence is highly conserved among RIPs 4 (A&B109, A&B179, A&B111, A&B223) Neither molecule A nor B has clear density map, and the sequence is not conserved among RIPs 6 (A&B29, A&B97, A&B217, A&B218, A&B225, A&B228) These residues belong to residue pairs (Asn/Asp, Gln/Glu, Val/Thr) and cannot be distinguished by X-ray crystallography. *These two residues are conserved, but the electron density of both molecules A and B isn't clear, so we cannot classify these two residues to Table 2: Assignment of the sequence of luffaculin 1 by X-ray sequencing method (total number of residues: 241) Sequence assignment method Number of identified residues 1 Both molecule A and B have clear electron density and can be assigned unambiguously 151 160 (9 additional residues were assigned: A123, A185, A206, A215, A219, A221, A222, A234 and B209) 2 Using method 1 plus electron density of the second molecule for those residues where the first molecule has weak electron density, 66% of residues (160) can be deduced 3 Using methods 1 and 2 plus chemical environment evaluation without using sequence comparison, 86% of residues (208) can be deduced. 208 (exclude 28, 29, 33, 48, 56, 93, 97, 109, 111, 122, 132, 142, 156, 157, 169, 177,179, 190, 197, 202, 204, 205, 213, 216, 217, 218, 220, 223, 225, 226, 228, 237, 241) 4 Using method 3 plus the sequence comparison, 94% of residues (227) can be deduced. 2 2 227 (exclude 28, 29, 97, 109, 111, 190, 216, 217, 218, 220, 225, 228, 223, 237) 227 (exclude 28, 29, 97, 109, 111, 190, 216, 217, 218, 220, 225, 228, 223, 237) Neither molecule A nor B has clear density map, but the sequence is highly conserved among RIPs 4 (A&B109, A&B179, A&B111, A&B223) 4 (A&B109, A&B179, A&B111, A&B223) Neither molecule A nor B has clear density map, and the sequence is not conserved among RIPs 6 (A&B29, A&B97, A&B217, A&B218, A&B225, A&B228) These residues belong to residue pairs (Asn/Asp, Gln/Glu, Val/Thr) and cannot be distinguished by X-ray crystallography. *These two residues are conserved, but the electron density of both molecules A and B isn't clear, so we cannot classify these two residues to G 4 Structure description Fig. 1 shows a ribbon representation of luffaculin 1. The structure of luffaculin 1 contains two domains: a large N- terminal domain composed of eight α-helices and eight β- strands, and a smaller C-terminal domain consisting of two α-helices (α9 and α10) and two β-strands (β9 and β10). The secondary structure of luffaculin 1 is typical of type 1 RIPs: six β-strands of N-terminal domain (β1, β4, β5, β6, β7 and β8) form a mixed β-sheet. Eight helices of N-terminal have canonical geometry [30] and enclose the Table 1: Data collection and model refinement statistics for luffaculin 1 Space group P1 Cell parameters a = 39.135 Å, b = 46.813 Å, c = 83.571 Å, α = 89.068°, β = 80.009°, γ = 72.143° Resolutions (Å) 1.4 Completeness (%) 86.7 (62.0) Redundancy 1.9 (1.7) Rmerge (%)a 0.03 (0.115) Unique reflections 94795 I/σ(I) 21.8 (4.6) Resolution range 50.00-1.40 (1.45-1.40) Rwork 0.213 (0.250) Rfree 0.232 (0.275) No. of water molecules 492 No. of carbohydrates 4 per asymmetric unit No. of polyethylene glycols 3 R.m.s.deviations from ideal geometry Bond lengths (Å) 0.007 Bond angles (°) 1.153 Values in parentheses refer to the highest resolution shell (1 45-1 40) Å Table 1: Data collection and model refinement statistics for luffaculin 1 Page 3 of 10 (page number not for citation purposes) Page 3 of 10 (page number not for citation purposes) http://www.biomedcentral.com/1472-6807/7/29 BMC Structural Biology 2007, 7:29 Table 2: Assignment of the sequence of luffaculin 1 by X-ray sequencing method (total number of residues: 241) Sequence assignment method Number of identified residues 1 Both molecule A and B have clear electron density and can be assigned unambiguously 151 2 Using method 1 plus electron density of the second molecule for those residues where the first molecule has weak electron density, 66% of residues (160) can be deduced 160 (9 additional residues were assigned: A123, A185, A206, A215, A219, A221, A222, A234 and B209) 3 Using methods 1 and 2 plus chemical environment evaluation without using sequence comparison, 86% of residues (208) can be deduced. 208 (exclude 28, 29, 33, 48, 56, 93, 97, 109, 111, 122, 132, 142, 156, 157, 169, 177,179, 190, 197, 202, 204, 205, 213* 216, 217, 218, 220, 223, 225, 226, 228, 237, 241) 4 Using method 3 plus the sequence comparison, 94% of residues (227) can be deduced. X-ray sequence of luffaculin 1 luffin a belong to the same genus (Luffa) but different spe- cies in the Cucurbitaceae family, thus they are expected to share high sequence homology. For structural determina- tion of luffaculin 1, we used luffin a to build a homology model for molecular replacement method. Although the sequence of luffaculin 1 is not yet known, the high resolu- tion of electron density, combined with the known sequence of homolog luffin a, has allowed us to under- take an 'X-ray sequencing' method with a high degree of confidence. For this purpose, we used annealed compos- ite and σ-weighted 2Fo-Fc omit maps [33] throughout this work to reduce model bias. The electron density allowed us to identify differences between luffaculin 1 and its molecular replacement model, luffin a. For exam- ple, residues 3 and 64 were reported as Arg and Val in luf- fin a, whereas in luffaculin 1 these residues were recognized unambiguously as Ser and Ile, respectively (Fig. 3a). The sequence of luffaculin 1 was unknown. Only the first five residues in N-terminal region were determined to be DVSFS by N-terminal sequencing. Both luffaculin 1 and Overall structure of luffaculin 1 in stereo representation Figure 1 Overall structure of luffaculin 1 in stereo representa- tion. The two domains at the N- and C-terminal were colored in cyan and violet, respectively. Stick representations are residues Tyr70, Tyr110, Glu159 and Arg162 in the active site, and the two N-acetylglucosamines that are each cova- lently linked to Asn 77 and 84, respectively. All figures except for Fig. 4 were prepared using Pymol [46]. Fig. 4 shows the final X-ray sequence of luffaculin 1, its alignment with other RIPs, the correlation coefficient (real space fit [34]) between the electron density and the assigned sequence, and the evaluation of chemical envi- ronments (hydrophobic interactions, hydrogen bonds and salt bridges) of the residues that cannot be distin- guished by electron density (i.e. Asp/Asn, Glu/Gln, and Val/Thr pairs). The reliability of this X-ray sequence assignment is summarized in Table 2. Fig. 4 shows the final X-ray sequence of luffaculin 1, its alignment with other RIPs, the correlation coefficient (real space fit [34]) between the electron density and the assigned sequence, and the evaluation of chemical envi- ronments (hydrophobic interactions, hydrogen bonds and salt bridges) of the residues that cannot be distin- guished by electron density (i.e. Asp/Asn, Glu/Gln, and Val/Thr pairs). 1 Both molecule A and B have clear electron density and can be assigned unambiguously 151 http://www.biomedcentral.com/1472-6807/7/29 Residues not present in the molecule B are outlined in the boxes. trated in Fig. 3b for residues 46, 94 and 129. The two pro- tein molecules (A and B) in the crystal should have the identical amino-acid sequence. This redundancy provides an additional level of validation on the electron-density- based sequence assignment (Fig. 3a and 3b). In the case that electron density is disordered or weak in one mole- cule, the electron density of the other molecule allows the identification of the sequence (9 residues are in such case, see Table 2 and Fig. 3c). Fig. 3c shows an example where residue 185 has weak electron density in molecule B but can be clearly identified as Ile in molecule A. 66% of resi- dues (160 out of a total of 241 residues, see Table 2) of luf- faculin 1 can be identified with confidence purely based on the high resolution electron density. Similar to our results, a previous study [4] suggested that 60% of resi- dues can be identified reliably based on electron density. Four residues (109, 111, 179 and 223) have weak electron density in both molecule A and molecule B (Table 2), and thus cannot be assigned based on electron density. All these four residues are located either at loop region or at the surface of the molecules. However, these four residues are highly conserved among various RIPs (Fig. 4), and are thus tentatively assigned according to the sequences of their homologous proteins. Residues (29, 97, 217, 218, 225 and 228) do not have enough electron density of side chains in both molecules and are not conserved in RIP sequences, and thus cannot be assigned in this study. They are currently tentatively assigned as Ala or Gly. An inherent problem of X-ray sequencing is that some res- idue pairs (Glu/Gln, Asp/Asn, and Val/Thr) can not be dis- tinguished completely based purely on electron density. This is due to the facts that the numbers of electrons in outer shell of carbon, nitrogen, and oxygen are not too much different, and thus cannot be distinguished by X-ray diffraction of protein crystals at a resolution that is usually far less than atomic resolution. In order to solve this prob- lem, we used the information of 1) chemical environment evaluation of amino-acids (hydrophobic interactions, hydrogen bonds and salt bridges); 2) sequence compari- son with other RIPs. http://www.biomedcentral.com/1472-6807/7/29 BMC Structural Biology 2007, 7:29 where carbohydrate moieties were clearly identified from the electron density. Fig. 5a shows that Asp65A has a chemical environment perfect for an Asp, but less possible for an Asn because only an Asp, but not an Asn, can form salt bridge with Arg46. Moreover, Asp65 is quite con- served (Fig. 4) among all RIPs. Four additional residue pairs (Asp87, Glu159, Glu167 and Glu188) were found to form salt bridges with other residues, leading to their con- vincing assignment. Inspection of hydrophobic environ- ment can also assist to break down Val/Thr ambiguity. Fig. 5b shows an example where the electron density votes for either a Val or a Thr, but the perfect hydrophobic environ- ment makes it less possible for a Thr at this position. This evaluation of chemical environment of residues has increased the assigned residues from 160 (66%) to 208 (86%, see Table 2). It is not surprising that there are still a few residues (marked by asterisks in Table 2) that cannot be distinguished based on their chemical environment. Fig. 5c shows that Gln220 of molecule A has no interac- tion with any other residues except for a hydrogen bond with a water molecule (S327). The Gln220 in the mole- cule B is not observed in the electron density, so we can- not conclude A220 as a Glu or Gln. It was reported [3] that the side chain of Ser can distribute among two conforma- tions and may thus look like a Thr residue, albeit at weaker electron density. In our case, no such ambiguity was found. Superposition of molecule A (cyan) and B (magenta) of luffac- ulin 1 Figure 2 Superposition of molecule A (cyan) and B (magenta) of luffaculin 1. The slight deviation is indicated in a dashed circle. Residues not present in the molecule B are outlined in the boxes. Superposition of molecule A (cyan) and B (magenta) of luffac- ulin 1 Figure 2 Superposition of molecule A (cyan) and B (magenta) of luffaculin 1. The slight deviation is indicated in a dashed circle. Residues not present in the molecule B are outlined in the boxes. Superposition of molecule A (cyan) and B (magenta) of luffac- ulin 1 Figure 2 Superposition of molecule A (cyan) and B (magenta) of luffaculin 1. The slight deviation is indicated in a dashed circle. X-ray sequence of luffaculin 1 The reliability of this X-ray sequence assignment is summarized in Table 2. Overall str Figure 1 p g Overall structure of luffaculin 1 in stereo representa- tion. The two domains at the N- and C-terminal were colored in cyan and violet, respectively. Stick representations are residues Tyr70, Tyr110, Glu159 and Arg162 in the active site, and the two N-acetylglucosamines that are each cova- lently linked to Asn 77 and 84, respectively. All figures except for Fig. 4 were prepared using Pymol [46]. Based on the electron density, majority of the residues can be assigned unambiguously. Some examples are illus- Page 4 of 10 (page number not for citation purposes) Page 4 of 10 (page number not for citation purposes) http://www.biomedcentral.com/1472-6807/7/29 The prese Figure 3 Th The prese Figure 3 p y q g The presence of two molecules in the asymmetric unit facilitates sequence identification. (a) shows residues 3 and 64 in molecule A and B of luffaculin 1 contoured at 2σ and these residues are different from luffin a. (b) shows residues 46, 94 and 129 with clear electron density in molecule A and B contoured at 2σ. (c) shows residue 185 with weak electron density in molecule B can be clearly recognized as Ile in the electron density of molecule A. level as observed. This indicates that the function and the enzymatic mechanisms of luffaculin 1 are probably the same as other RIPs [35]. sequence of PAP-Saci was obtained from the exceptional quality of the electron density at 1.7 Å resolution, com- bined with the known sequence of the two PAP-S iso- forms. The authors claimed that almost all amino-acid side chains were identified with a high degree of certainty (with the exception of Asp/Asn and Glu/Gln ambiguities). The X-ray sequence of trichomaglin was obtained by com- bining those derived from electron density at 2.2 Å resolu- tion with the partial sequence information from mass spectroscopic analysis and the experimentally determined N-terminal sequence. 60% of the X-ray sequence was thus demonstrated to be highly reliable. In this paper we got the X-ray sequence of luffaculin 1 based on the high reso- lution (1.4 Å) electron density, cross-validated by the sec- ond molecule in the asymmetric unit of the crystals, and combined with the evaluation of the chemical environ- ment of selected residues. The active site is located in a cleft between the N-terminal domain and the C-terminal domain, which serves as the substrate-binding and catalysis site in RIPs (Fig. 1). Fig. 6 shows the conformations of luffaculin 1 catalytic residues, superimposed with those of other RIPs. The side chains of the active site residues have roughly the same position as the corresponding residues of other RIPs' structures [15,36], whereas Tyr70 shows relatively higher mobility in all analyzed RIPs. It has been reported that this residue interacts with the targeted adenine [37], and together with a second tyrosine (Tyr110 in luffaculin 1), forms an aro- matic stack of π electron system. The conformational flex- ibility of Tyr70 side chain may promote the substrate recognition and the formation of the aromatic stack. http://www.biomedcentral.com/1472-6807/7/29 Information of the glycosylation on Asn residues also facilitates to break down Asp/Asn ambi- guity as were the cases for residues Asn77 and Asn84, In summary, the evaluation of chemical environment greatly facilitates to break down the ambiguity (Table 2): 32 out of a total of 36 Val/Thr pairs and 16 out of a total of 38 Asp/Asn and Glu/Gln pairs were assigned by this method. By using electron density and evaluation of chemical environment, 86% of residues were assigned with confidence. Assignment based on sequence compar- ison, although not absolutely reliable, further increases the number of the identified residues to 227 (94% of a total of 241 residues). X-ray sequencing method has been successfully used to determine the amino-acid sequence of PAP-Saci [3], a Pokeweed antiviral protein, and trichomaglin [4]. The Page 5 of 10 (page number not for citation purposes) Page 5 of 10 (page number not for citation purposes) http://www.biomedcentral.com/1472-6807/7/29 BMC Structural Biology 2007, 7:29 The presence of two molecules in the asymmetric unit facilitates sequence identification Figure 3 The presence of two molecules in the asymmetric unit facilitates sequence identification. (a) shows residues 3 and 64 in molecule A and B of luffaculin 1 contoured at 2σ and these residues are different from luffin a. (b) shows residues 46, 94 and 129 with clear electron density in molecule A and B contoured at 2σ. (c) shows residue 185 with weak electron density in molecule B can be clearly recognized as Ile in the electron density of molecule A. Comparison with other RIPs and the active site 190 . . . . . . .200 . . . . . . . 210 . . . . . . .220 . . . . . . . 230 . . . . . . . 240 CC_A 1 0 Env HH H L HH L H H H L HH HHHHH HH H HH CC_B 1 0 Seq Ident Seq# 1 . . . . . . . . 10 . . . . . . . .20 . . . . . . . . 30 . . . . . . . .40 . . . . . . . . 50 . . . . . . . .60 . . . . . . . .70 . . . . . . . .80 . . . . . CC_A 0 1 1 CC_B Env H L H H HH L L HH L L HH L H H HHH H H L 0 Seq Ident Seq# . . . 90 . . . . . . . . 100 . . . . . . .110 . . . . . . . 120 . . . . . . .130 . . . . . . .140 . . . . . . .150 . . . . . . .160 . . . . . . .170 Seq# . . . . . . . 180 . . . . . . . 190 . . . . . . .200 . . . . . . . 210 . . . . . . .220 . . . . . . . 230 . . . . . . . 240 CC_A 1 0 Env HH H L HH L H H H L HH HHHHH HH H HH CC_B 1 0 Seq Ident CC_A CC_B 1 0 Env L H H HHL L H LHL L H H HH L L HL LH HHH H L L 0 1 Seq Ident X-ray sequence of luffaculin 1 and multiple sequence alignments Figure 4 X-ray sequence of luffaculin 1 and multiple sequence alignments. These alignments are of luffaculin 1 (LF1), luffin a (LUA), luffin b (LUB), α-momorcharin (MOM), trichosanthin (TCS) and bryodin (BRY). Highlighted residues are the active site residues. Panel Seq Ident indicates the sequence homology calculated by program clustalx 1.83. Comparison with other RIPs and the active site Fig. 4 shows the structure-based alignment of luffaculin 1 with selected type 1 RIPs, including luffaculin 1, luffin a, luffin b, α-momorcharin, trichosanthin and bryodin. The amino-acid sequence of luffaculin 1 shows high degree of sequence identities to other RIPs: 94% for luffin a, 83% for luffin b, 73% for α-momorcharin, 64% for bryodin, and 63% for trichosanthin, respectively. The active site is the most conserved region at both sequence and structure The final model of luffaculin 1 shows a common "RIP fold". The superposition of Cα atoms of luffaculin 1 to tri- chosanthin, α-momorcharin and β-luffin gave rmsd (root mean square deviation) values of 0.527, 0.492 and 0.359 Å, respectively. Despite this overall structural similarity of luffaculin 1 to other RIPs (Fig. 7), some noticeable differ- ences exist in surface exposed loop regions particularly those between β3-strand and α2-helix, and between α8- Page 6 of 10 (page number not for citation purposes) Page 6 of 10 (page number not for citation purposes) BMC Structural Biology 2007, 7:29 http://www.biomedcentral.com/1472-6807/7/29 ay sequence of luffaculin 1 and multiple sequence alignments ure 4 ay sequence of luffaculin 1 and multiple sequence alignments. These alignments are of luffaculin 1 (LF1), luffin a A), luffin b (LUB), α-momorcharin (MOM), trichosanthin (TCS) and bryodin (BRY). Highlighted residues are the active site dues. Panel Seq Ident indicates the sequence homology calculated by program clustalx 1.83. Panel CC_A and CC_B are the space fit (RS fit, calculated by program O ranging from 0 to 1) of the residues between current luffaculin 1 and composite t map of luffaculin 1. Panel Env represents the evaluation of chemical environment on residue pairs (Glu/Gln, Asp/Asn, Va ) that cannot be distinguished by X-ray crystallography. H/L stands for high/low confidence in breaking down this ambiguity ed on its chemical environment (hydrophobic interactions, hydrogen bonds and salt bridges). CC_A 0 1 1 CC_B Env H L H H HH L L HH L L HH L H H HHH H H L 0 Seq Ident Seq# . . . 90 . . . . . . . . 100 . . . . . . .110 . . . . . . . 120 . . . . . . .130 . . . . . . .140 . . . . . . .150 . . . . . . .160 . . . . . . Comparison with other RIPs and the active site .170 CC_A CC_B 1 0 Env L H H HHL L H LHL L H H HH L L HL LH HHH H L L 0 1 Seq Ident Seq# . . . . . . . 180 . . . . . . . 190 . . . . . . .200 . . . . . . . 210 . . . . . . .220 . . . . . . . 230 . . . . . . . 240 CC_A 1 0 Env HH H L HH L H H H L HH HHHHH HH H HH CC_B 1 0 Seq Ident Seq# 1 . . . . . . . . 10 . . . . . . . .20 . . . . . . . . 30 . . . . . . . .40 . . . . . . . . 50 . . . . . . . .60 . . . . . . . .70 . . . . . . . .80 . . . . . ay sequence of luffaculin 1 and multiple sequence alignments ure 4 ay sequence of luffaculin 1 and multiple sequence alignments. These alignments are of luffaculin 1 (LF1), luffin A), luffin b (LUB), α-momorcharin (MOM), trichosanthin (TCS) and bryodin (BRY). Highlighted residues are the active dues. Panel Seq Ident indicates the sequence homology calculated by program clustalx 1.83. Panel CC_A and CC_B are space fit (RS fit calculated by program O ranging from 0 to 1) of the residues between current luffaculin 1 and compo CC_A 0 1 1 CC_B Env H L H H HH L L HH L L HH L H H HHH H H L 0 Seq Ident Seq# . . . 90 . . . . . . . . 100 . . . . . . .110 . . . . . . . 120 . . . . . . .130 . . . . . . .140 . . . . . . .150 . . . . . . .160 . . . . . . .170 CC_A CC_B 1 0 Env L H H HHL L H LHL L H H HH L L HL LH HHH H L L 0 1 Seq Ident Seq# . . . . . . . 180 . . . . . . . Purification and crystallization Luffaculin 1 was extracted and purified by extraction with acetate buffer, ammonium sulfate fractional precipitation and cation exchange chromatography [25]. It was eluted as a single symmetrical peak in a cation exchange column (Mono S, Amersham Pharmacia Biotech) and gave a sin- gle band with an apparent molecular weight of about 28 kDa by reducing SDS-PAGE. The purified luffaculin 1 was thoroughly dialyzed against deionized water and lyophi- lized. For crystallization, lyophilized powder of luffaculin 1 was dissolved to a concentration of 15.8 mg/mL and then crystallized by hanging drop vapor diffusion method [38] at room temperature by mixing 2 μL protein (15.8 mg/mL) with an equal volume of reservoir solution (28% (w/v) PEG 6000, 0.1 M citrate buffer pH4.5, containing 0.02% (w/v) sodium azide) and equilibrating against 800 μL of the same reservoir solution. The crystal was briefly dipped into a cryoprotectant, 20% of glycerol (final con- centration) in the reservoir solution, before data collec- tion. Structural superposition of the active site residues Figure 6 Structural superposition of the active site residues. Trichosanthin, α-momorcharin, β-luffin and luffaculin 1 are colored as green, cyan, magenta and yellow, respectively. Conclusion h We present the 1.4 Å resolution crystal structure of luffac- ulin 1 and its X-ray sequence. This sequence was derived based on the high resolution electron density, validated against the second molecule present in the crystals, the evaluation of the chemical environment of selected resi- dues, and the sequence comparison with other homo- logues. A total of 86% (without using sequence comparison) or 94% (with sequence comparison) of luf- faculin 1 residues can be assigned with confidence by this approach. The luffaculin 1 is quite similar to luffin a at The electr Figure 5 y p , g The electron density of Asp65, Val212 and Gln220 of molecule A. This map (2Fo-Fc composite omit map) is con- toured at 1σ. Hydrogen bonds are denoted by dashed lines with numbers denoting the respective distances in the unit of Å. (a) shows that Asp65 of molecule A has hydrogen bond interaction with Thr67 and salt bridge with Arg46 besides interaction with water molecules S244 and S265. (b) shows that Val212 of molecule A resides in a hydrophobic environment. (c) shows that Gln220 of molecule A has no interaction with other residues except the hydrogen bond with the symmetry related water mol- ecule S327. and α9-helix. The largest deviation occurs at the β8-strand of the N-terminal region (box C in Fig. 7). both sequence and structural levels, suggesting its func- tions as an RIP. Comparison with other RIPs and the active site Panel CC_A and CC_B are the real space fit (RS fit, calculated by program O ranging from 0 to 1) of the residues between current luffaculin 1 and composite omit map of luffaculin 1. Panel Env represents the evaluation of chemical environment on residue pairs (Glu/Gln, Asp/Asn, Val/ Thr) that cannot be distinguished by X-ray crystallography. H/L stands for high/low confidence in breaking down this ambiguity based on its chemical environment (hydrophobic interactions, hydrogen bonds and salt bridges). Page 7 of 10 (page number not for citation purposes) Page 7 of 10 (page number not for citation purposes) http://www.biomedcentral.com/1472-6807/7/29 BMC Structural Biology 2007, 7:29 The electron density of Asp65, Val212 and Gln220 of molecule A Figure 5 The electron density of Asp65, Val212 and Gln220 of molecule A. This map (2Fo-Fc composite omit map) is con- toured at 1σ. Hydrogen bonds are denoted by dashed lines with numbers denoting the respective distances in the unit of Å. (a) shows that Asp65 of molecule A has hydrogen bond interaction with Thr67 and salt bridge with Arg46 besides interaction with water molecules S244 and S265. (b) shows that Val212 of molecule A resides in a hydrophobic environment. (c) shows that Gln220 of molecule A has no interaction with other residues except the hydrogen bond with the symmetry related water mol- ecule S327. Page 8 of 10 (page number not for citation purposes) Additional File 1 The electron density of one PEG1 and two PEG2. These maps (2Fo-Fc composite omit maps) is contoured at 1σ. The format of the additional file is JPEG and the file can be viewed through ACD systems. Click here for file [http://www.biomedcentral.com/content/supplementary/1472- 6807-7-29-S1.jpeg] [http://www.biomedcentral.com/content/supplementary/1472- 6807-7-29-S1.jpeg] Authors' contributions XMH refined the structure and drafted the manuscript. MHC obtained the initial crystallization conditions for luffaculin 1. EJM and LQC carried out the data collection and processing. MHC and MDH are advisors of XMH. MDH provided constructive advices and worked on the manuscript. JMX tested pharmacological activities of luf- faculin 1. All authors read and approved the final manu- script. and the value of Vm is 2.49 Å Da-1 corresponding to a sol- vent content of 48%. The statistics for the data set are sum- marized in Table 1. The final merged data set of 94795 unique reflections has high quality with an Rmerge of 0.03 and an averaged signal to noise ratio of 21.8. http://www.biomedcentral.com/1472-6807/7/29 The final model has R factor and Rfree of 0.213 and 0.232, respec- tively, containing 492 water molecules, one PEG1 (tetrae- thylene glycol), two PEG2 (diethylene glycol) and four N- acetylglucosamines (NAG) in the asymmetric unit. Data collection and model refinement statistics are listed in Table 1. Superposit momorcha low) Figure 7 Superposition of Cα atoms of trichosanthin (green), α- momorcharin (cyan), β-luffin (magenta) and luffaculin 1 (yel- low) Figure 7 Superposition of Cα atoms of trichosanthin (green), α-momorcharin (cyan), β-luffin (magenta) and luffac- ulin 1 (yellow). Loop deviations between β3-strand and α2- helix and between α8- and α9-helix are outlined in the boxes A and B, respectively. The largest deviation region at the β8- strand of the N-terminal region is outlined in the box C. p p (g ) ( y ) β ( g ) (y ) g Superposition of Cα atoms of trichosanthin (green), α-momorcharin (cyan), β-luffin (magenta) and luffac- ulin 1 (yellow). Loop deviations between β3-strand and α2- helix and between α8- and α9-helix are outlined in the boxes A and B, respectively. The largest deviation region at the β8- strand of the N-terminal region is outlined in the box C. Additional material The structure of luffaculin 1 was solved by the molecular replacement method (AMORE [41,42]) using a homology model built based on the sequence of luffin a [43,44]. Luf- fin a and luffaculin 1 were both purified from the same genus but different species in the Cucurbitaceae family, and were expected to share high sequence homology and structure similarity. Rotational solutions in the resolution range of 8-4 Å showed two clear peaks with the high cor- relation coefficients of 0.223 and 0.199, respectively, and the corresponding third highest value was 0.071. Then, the translation vector of the first solution was fixed because of the space group P1 of luffaculin 1 and the translation search was performed for the second solution, giving a higher correlation coefficient of 0.429. The refine- ment was performed with the program CNS [33] in a res- olution range from 50-1.4 Å. A total of 5% of the data was Data collection and processing Diffraction data of the crystals were collected using syn- chrotron radiation (APS SER-CAT beamline 22ID) at low temperature (100K) to improve the diffraction quality and to decrease the radiation decay. A 1.4 Å resolution data set was obtained and the diffraction data were proc- essed with the program package HKL2000 [39]. The crys- tals belong to space group P1, with unit-cell parameters a = 39.135 Å, b = 46.813 Å, c = 83.571 Å, α = 89.068°, β = 80.009°, γ = 72.143°. Matthews coefficient calculations [40] show two molecules present in the asymmetric unit Structural Figure 6 Structural Figure 6 p p g Structural superposition of the active site residues. Trichosanthin, α-momorcharin, β-luffin and luffaculin 1 are colored as green, cyan, magenta and yellow, respectively. Page 8 of 10 (page number not for citation purposes) Page 8 of 10 (page number not for citation purposes) http://www.biomedcentral.com/1472-6807/7/29 http://www.biomedcentral.com/1472-6807/7/29 BMC Structural Biology 2007, 7:29 Superposition of Cα atoms of trichosanthin (green), α- momorcharin (cyan), β-luffin (magenta) and luffaculin 1 (yel- low) Figure 7 Superposition of Cα atoms of trichosanthin (green), α-momorcharin (cyan), β-luffin (magenta) and luffac- ulin 1 (yellow). Loop deviations between β3-strand and α2- helix and between α8- and α9-helix are outlined in the boxes A and B, respectively. The largest deviation region at the β8- strand of the N-terminal region is outlined in the box C. randomly selected for Rfree calculation throughout the whole refinement. After a starting cycle of rigid body refinement, the R factor was 0.3360 and Rfree was 0.3518. Simulated annealing and restrained individual B-factor refinement were then performed. The sigma A weighted 2Fo-Fc and Fo-Fc electron density maps were used to guide the model building process. The model was exam- ined and manually rebuilt with the graphic program O [34]. In the final stage of the refinement, water molecules were added by CNS at locations where electron density was stronger than 3.0σ in sigma A-weighted Fo-Fc maps and had reasonable hydrogen-bond interaction with the protein, and then were inspected with program O. The carbohydrates and the polyethylene glycol (PEG) mole- cules [see Additional file 1] were visible at this stage in 2Fo-Fc and Fo-Fc electron density maps and were built in. Luffaculin 1 was crystallized from the solution containing PEG6000. It is inevitable that there are some low molecu- lar weight polyethylene glycol molecules existed in the PEG6000 and these small molecules most likely penetrate into crystals. Such cases can also be found in the PDB data bank, for example, in PDB entry 2FD6 [45]. The final model has R factor and Rfree of 0.213 and 0.232, respec- tively, containing 492 water molecules, one PEG1 (tetrae- thylene glycol), two PEG2 (diethylene glycol) and four N- acetylglucosamines (NAG) in the asymmetric unit. Data collection and model refinement statistics are listed in Table 1. Luffaculin 1 was crystallized from the solution containing PEG6000. 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Use of the Advanced Photon Source was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract No.W-31-109-Eng-38. We thank USA are gratefully acknowledged. Use of the Advanced Photon Source was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract No.W-31-109-Eng-38. We thank Yujun Wang of University of Alabamaba in Huntsville as well as the staffs of the APS SER-CAT beamline 22ID for help with data collection. The coor- dinates of luffaculin 1 have been deposited in PDB (code 2OQA). 22. Wang JH, Nie HL, Tam SC, Huang H, Zheng YT: Anti-HIV-1 prop- erty of trichosanthin correlates with its ribosome inactivat- ing activity. FEBS Lett 2002, 531(2):295-298. g y ( ) 23. Parikh BA, Tumer NE: Antiviral activity of ribosome inactivat- ing proteins in medicine. Mini Rev Med Chem 2004, 4(5):523-543. Yujun Wang of University of Alabamaba in Huntsville as well as the staffs of the APS SER-CAT beamline 22ID for help with data collection. The coor- dinates of luffaculin 1 have been deposited in PDB (code 2OQA). g p ( ) 24. Wang P, Tumer NE: Virus resistance mediated by ribosome inactivating proteins. Adv Virus Res 2000, 55:325-355. 25. Lin JK, Chen MH, Xie JM, Zhao R, Ye XM, Shi XL, Wang ZR: Purifi- cation and Characterization of Two Luffaculins, Ribosome- inactivating Proteins from Seeds of Luffa acutangula. Chinese Journal of Biochemistry and Molecular Biology 2002, 18(5):609-613. Acknowledgements g Financial support from the National Science Foundation of China (No. 39970872, 30625011), the Natural Science Foundation of Fujian Province (C97052), Special Fund of Fujian Development and Reform Commission and State Key Laboratory of Structural Chemistry, and NSF-EPSCoR of Page 9 of 10 (page number not for citation purposes) References Au TK, Collins RA, Lam TL, Ng TB, Fong WP, Wan DC: The plant ribosome inactivating proteins luffin and saporin are potent inhibitors of HIV-1 integrase. FEBS Lett 2000, 471(2-3):169-172. p ( ( ) 46. DeLano WL: The PyMOL Molecular Graphics System. 2002 [http://www.pymol.org]. Page 10 of 10 (page number not for citation purposes) Page 10 of 10 (page number not for citation purposes)
https://openalex.org/W4376645984	https://jurnalilmiahcitrabakti.ac.id/jil/index.php/jil/article/download/1691/555	English	null	STUDENTS ENGLISH PROFICIENCY AT LEMBAGA TAHFIZ QURAN	Jurnal ilmiah pendidikan citra bakti	2,023	cc-by-sa	3,395	Jurnal Ilmiah Pendidikan Citra Bakti \|\| 366 Coresponding author: Via Novia Sofiana (vnsofianaa19@gmail.com) STUDENTS ENGLISH PROFICIENCY AT LEMBAGA TAHFIZ QURAN The Qur'anic scholar who believes that the Qur'an is a noble text while also critically examining the Qur'anic text in order to discover its meaning. English proficiency show a strong positive correlation with overall academic accomplishment. Keywords: english profiency, students, tahfiz quran, grammar Article history Received: April 4, 2023 Accepted: May, 15 2023 Published: May, 15 2023 STUDENTS ENGLISH PROFICIENCY AT LEMBAGA TAHFIZ QURAN Egista Dwi Eriani1) dan Via Novia Sofiana2) Pendidikan Bahasa Inggris, Sekolah Tinggi Keguruan Ilmu Pendidikan Situs Banten 1) egiestariani@gmail.com, 2) vnsofianaa19@gmail.com Article history Abstract Received: April 4, 2023 Accepted: May, 15 2023 Published: May, 15 2023 Students English Profiency At Lembaga Tahfiz Quran. The purpose of this study is to identify the dominant skill among students who memorize the Quran in tahfiz utrujjah. The findings demonstrate that. According to the aforementioned findings, students' use of English in educational settings where the Quran is taught is mostly through watching and listening. Better than being forced is doing what you can because you begin to love and feel pleased first. Additionally, pupils at the tahfiz utrujjah institution were successful in liking the language and being content with it without being coerced and with the support of their own initiative. Tahfiz Quran Utrrujjah Institution is one of them. One of the non- formal educational facilities, Tahfiz Quran, in the city of Serang- Banten, is open to both children and adults. Students are undoubtedly excellent resources for learning the Quran. The Qur'anic scholar who believes that the Qur'an is a noble text while also critically examining the Qur'anic text in order to discover its meaning. English proficiency show a strong positive correlation with overall academic accomplishment. Keywords: english profiency, students, tahfiz quran, grammar Coresponding author: Via Novia Sofiana (vnsofianaa19@gmail.com) Article history Abstract Received: April 4, 2023 Accepted: May, 15 2023 Published: May, 15 2023 Students English Profiency At Lembaga Tahfiz Quran. The purpose of this study is to identify the dominant skill among students who memorize the Quran in tahfiz utrujjah. The findings demonstrate that. According to the aforementioned findings, students' use of English in educational settings where the Quran is taught is mostly through watching and listening. Better than being forced is doing what you can because you begin to love and feel pleased first. Additionally, pupils at the tahfiz utrujjah institution were successful in liking the language and being content with it without being coerced and with the support of their own initiative. Tahfiz Quran Utrrujjah Institution is one of them. One of the non- formal educational facilities, Tahfiz Quran, in the city of Serang- Banten, is open to both children and adults. Students are undoubtedly excellent resources for learning the Quran. Abstract Students English Profiency At Lembaga Tahfiz Quran. The purpose of this study is to identify the dominant skill among students who memorize the Quran in tahfiz utrujjah. The findings demonstrate that. According to the aforementioned findings, students' use of English in educational settings where the Quran is taught is mostly through watching and listening. Better than being forced is doing what you can because you begin to love and feel pleased first. Additionally, pupils at the tahfiz utrujjah institution were successful in liking the language and being content with it without being coerced and with the support of their own initiative. Tahfiz Quran Utrrujjah Institution is one of them. One of the non- formal educational facilities, Tahfiz Quran, in the city of Serang- Banten, is open to both children and adults. Students are undoubtedly excellent resources for learning the Quran. The Qur'anic scholar who believes that the Qur'an is a noble text while also critically examining the Qur'anic text in order to discover its meaning. English proficiency show a strong positive correlation with overall academic accomplishment. Keywords: english profiency, students, tahfiz quran, grammar Coresponding author: Via Novia Sofiana (vnsofianaa19@gmail.com) Jurnal Ilmiah Pendidikan Citra Bakti \|\| 366 DOI: https://doi.org/10.38048/jipcb.v10i2.1691 DOI: https://doi.org/10.38048/jipcb.v10i2.1691 Volume 10, Nomor 2 Tahun 2023 Volume 10, Nomor 2 Tahun 2023 p-ISSN 2355-5106 \|\| e-ISSN 2620-6641 Background The Qur'anic scholar who believes that the Qur'an is a noble text while also critically examining the Qur'anic text in order to discover its meaning. This group will continue to investigate the meaning of the Qur'an and compare approaches for interpreting the Qur'an in modern science such as literature, linguistics, philosophy, sociology, and others.(Cahyani et al., 2020) The Qur'an is Allah SWT's revelation to the Prophet Muhammad SAW to serve as a guidance for human life. A hafiz's obligation as a Muslim is to understand, interpret, and practice the Qur'an in everyday life, in addition to memorizing and being able to read it without glancing at the manuscripts. It is also the teacher's duty and responsibility to guide students through the process of memorizing the Qur'an accurately and adequately. One factor that can influence the quality of students' memorization is the teacher's teaching competence and motivation when the memorization process occurs. Other considerations include facility support, the climate, and student morale. With the practice of memorizing the Qur'an, this Tahfidz al-Qur'an institution has a noble purpose, one of which is to safeguard its purity and sanctity from falsification, against all types of both content and editorial by infidels. The most important thing is to keep the Qur'an authentic because it is the basis of all existent knowledge. As a result, a teacher in this educational process must be genuinely competent and grasp what will be taught to his students in order to develop quality generations of Qur'an memorizers. The Tahfidz al-Qur'an institution exists as a non-formal educational institution and serves as a forum for collaboration between a group of individuals, including leaders, staff, teachers, and students, in order to accomplish the planned and predetermined goals of educational institutions. (Noor, 2022). Tahfiz Quran Utrrujjah Institution is one of them. One of the non-formal educational facilities, Tahfiz Quran, in the city of Serang-Banten, is open to both children and adults. Students are undoubtedly excellent resources for learning the Quran. English is also quite popular among utrujjah students, and the goal of this study is to establish the level of language in the institution. Grammar is typically regarded as one of the language skills that students studying English must master, along with vocabulary and the sound system(Taqiyyah & Aswir, 2022). DOI: https://doi.org/10.38048/jipcb.v10i2.1691 p-ISSN 2355-5106 \|\| e-ISSN 2620-6641 Volume 10, Nomor 2 Tahun 2023 DOI: https://doi.org/10.38048/jipcb.v10i2.1691 Background One common issue with reading comprehension is that many students are accustomed to understanding a passage at the sentence level rather than at the level of the discourse. Because of this weakness, some students tend to read all texts at the same pace because they feel compelled to comprehend every single sentence in a text, even though doing so is not necessary to achieve their reading objectives. Additionally, the learner makes extensive use of dictionaries to learn the definitions of some unfamiliar words (Alzu’bi, 2013). DOI: https://doi.org/10.38048/jipcb.v10i2.1691 Jurnal Ilmiah Pendidikan Citra Bakti \|\| 367 p-ISSN 2355-5106 \|\| e-ISSN 2620-6641 Volume 10, Nomor 2 Tahun 2023 The most crucial language in the world is thought to be English. The majority of international communication takes place in this language (Kunasaraphan, 2015). It is the official language of the United Nations, as well as the language of business, government, and entertainment. It is frequently taught as a foreign language (EFL) in a large number of nations in order to fulfill educational requirements, gain access to better career opportunities, improve English language profiency, and for a variety of other reasons (Fernandez-Malpartida, 2021). English has become a global language as a result of the globalization process. Learning English is now required for everyone in order to compete in the global workforce. Language skill development is a crucial concern for language teachers and learners, particularly in Indonesia. (Melvina & Julia, 2021). Both primary and acute care facilities en-counter language obstacles when providing care. Patients with limited English proficien-cy are less likely to report having a regular source of care, continuity of care, or receiv-ing screening services in primary care settings (Shi et al., 2009). Reading will help to devel-op and feel human thinking power. Particu-larly in this age of globalization, knowledge will also increase and raise the standard of human resources. Without needing to physi-cally observe something, we can learn a vari-ety of things via reading (Sulfemi & Luthfianti, 2019). Levels of English profi-ciency show a strong positive correlation with overall academic accomplishment. Ad-ditionally, the researchers suggested that ini-tiatives to enhance academic success be fo-cused on enhancing the English language curriculum (Kadwa & Alshenqeeti, 2020). The capacity to teach in English is a skill that is typically seen as influenced by the teacher's language competency. Competency in English language teaching draws on content or subject matter knowledge, teaching abilities, and the ability to do so. Background The more one is familiar with a language, the more qualified one is to teach it, according to popular opinion (Richards, 2017). Learning strategies are one of the variables that affect the effectiveness of language acquisition; with the aid of these techniques, students can not only achieve academic success but also enhance their language proficiency. The purpose of this study is to ascertain whether test results for English proficiency correlate with cognitive reading techniques.(Gustanti & Ayu, 2021). It is crucial to understand and research the language abilities in tahfiz institutions in order to determine how much language development occurs in quran memorization. Jurnal Ilmiah Pendidikan Citra Bakti \|\| 369 Method The descriptive quantitative approach was employed to conduct this study by distributing questionnaires. A descriptive research is one that provides the most thorough picture or description of the situation while ignoring the behavior of the selected object. The perspective of Suharsimi Arikunto (2006: 224) depends on his point of view, and this DOI: https://doi.org/10.38048/jipcb.v10i2.1691 Jurnal Ilmiah Pendidikan Citra Bakti \|\| 368 Jurnal Ilmiah Pendidikan Citra Bakti \|\| 368 p-ISSN 2355-5106 \|\| e-ISSN 2620-6641 Volume 10, Nomor 2 Tahun 2023 questionnaire—or these questionnaires—can be identified based on their type being distinguished by (Kurniawati, 2015). How to react in a questionnaire—or these questionnaires—can be identified based on their type being distinguished by (Kurniawati, 2015). How to react in a 1. An open form that enables respondents to react with phrases of their own. 2. Closed questionnaire; respondents only need to select one response because the questions' answers are already known. For instance, the respondent may choose one response from a column (assessment 1-10) if one is provided.a. In light of the response provided a) A direct questionnaire in which respondents provide personal data b) An indirect questionnaire, in which respondents react to inquiries concerning different people or things. Consider the perception of literacy in a place or a particular item. 3. According to shape a) A closed questionnaire, often known as a multiple-choice questionnaire. b) The oral questionnaire is the same as the open questionnaire. c) Since a check list is only a list, the respondent only needs to tick the appropriate column. d) A rating scale, often known as a scaled scale, consists of a sentence and 3. According to shape a) A closed questionnaire, often known as a multiple-choice questionnaire. b) The oral questionnaire is the same as the open questionnaire. 3. According to shape a) A closed questionnaire, often known as a multiple-choice questionnaire. a) A closed questionnaire, often known as a multiple-choice questionnaire. b) The oral questionnaire is the same as the open questionnaire. c) Since a check list is only a list, the respondent only needs to tick the appropriate column. d) A rating scale, often known as a scaled scale, consists of a sentence and columns that each signify a level of agreement, from strongly agree to strongly disagree. Method Additionally, there are many benefits to using questionnaires, such as the following: a) Researchers are not required to be present; a) Researchers are not required to be present; b) A large number of respondents can be surveyed at once; c) Respondents can answer the survey at their own pace and in their free time; and d) The survey can be made anonymous so that respondents can respond honestly and without hesitation. e) Standards may be included in the design so that responses from all respondents are possible (Raharja et al., 2018). To obtain data regarding the topic under study, the researcher uses observational methods. a survey that was handed out to Utrujjah Students with the instrument The university used a tool in the form of questions to gauge the development of language, and up to 5 responders were chosen to represent all pupils. DOI: https://doi.org/10.38048/jipcb.v10i2.1691 Jurnal Ilmiah Pendidikan Citra Bakti \|\| 369 DOI: https://doi.org/10.38048/jipcb.v10i2.1691 Jurnal Ilmiah Pendidikan Citra Bakti \|\| 369 p-ISSN 2355-5106 \|\| e-ISSN 2620-6641 Volume 10, Nomor 2 Tahun 2023 Results and Discussion The outcomes are displayed in a graphic that shows how speaking and English language proficiency have developed in tahfiz utrujjah institutions, respectively. It was just created five years ago, but Utrujjah is also a tahfiz institution. Because of its young age, it has generated a large number of students who are productively memorizing the Qur'an, which is advantageous to all circles. There were a total of 5 respondents in the class of students at the tahfiz institution because, in contrast to the previous semester, this semester there are more parents and children present than students. To make it easier for responders to complete the survey, we've included some multilingual questions in the diagrams below. Jurnal Ilmiah Pendidikan Citra Bakti \|\| 370 DOI: https://doi.org/10.38048/jipcb.v10i2.1691 Table 0.4 Table 0.4 In this investigation, we were able to gather reliable data, which we then packed into the diagram above. Only women and mukim as for males are allowed to study the Quran in the tahfiz quran utrujjah institution; only parents and children are allowed. In this investigation, we were able to gather reliable data, which we then packed into the diagram above. Only women and mukim as for males are allowed to study the Quran in the tahfiz quran utrujjah institution; only parents and children are allowed. DOI: https://doi.org/10.38048/jipcb.v10i2.1691 Table 0.1 Table 0.1 Table 0.2 Table 0.3 Table 0.2 Table 0.2 Table 0.3 DOI: https://doi.org/10.38048/jipcb.v10i2.1691 Jurnal Ilmiah Pendidikan Citra Bakti \|\| 370 DOI: https://doi.org/10.38048/jipcb.v10i2.1691 p-ISSN 2355-5106 \|\| e-ISSN 2620-6641 Volume 10, Nomor 2 Tahun 2023 Table 0.4 In this investigation, we were able to gather reliable data, which we then packed into the diagram above. Only women and mukim as for males are allowed to study the Quran in the tahfiz quran utrujjah institution; only parents and children are allowed. Discussion The description of the data collection that follows. Table 0.1's findings showed that speaking and listening skills are more difficult for 60% of college students than for the remaining 40%. It has been demonstrated that many Indonesians struggle with these issues and that they tend to be more severe than other people. Foreign students may experience difficulties comprehending lectures and engaging with others, especially those from non-English speaking nations (Allen et al., 2011). According to table 0.2's findings, the majority of students memorizing the Qur'an at utrujjah tahfiz institutions choose to watch movies 60% of the time, write in English 20% of the time, and sing or sing songs in English for the remaining 20% of the time. This demonstrates, in our opinion, the lack of interest in reading English-language publications among the Quran memorizationists there. As a result, they feel more at ease observing, writing, and singing to further develop their language skills in a straightforward, independent manner. The benefits of watching and listening to inspiring movies are enormous(Kosimov, 2021). The definition's explanation does demonstrate that developing students' hearing and viewing skills is one technique to help them learn the Qur'an, even though reading is not the main form of instruction in tahfiz schools. After learning about their English-speaking abilities, the results demonstrate. In the basic phase, their English proficiency was 80% and in the intermediate phase, 20%. Because of their habits of memorization of the Quran and their objectives to memorize it, this is more important to them than learning to speak English, but this does not excuse them from wanting to proceed slowly to study English so that their knowledge is balanced. The findings demonstrate that people continue to like watching and listening as much as they do speaking it, with the same results showing no signs of changing. 60% favor listening, while 40% like watching. Some learning-related factors. DOI: https://doi.org/10.38048/jipcb.v10i2.1691 Jurnal Ilmiah Pendidikan Citra Bakti \|\| 371 DOI: https://doi.org/10.38048/jipcb.v10i2.1691 Jurnal Ilmiah Pendidikan Citra Bakti \|\| 371 p-ISSN 2355-5106 \|\| e-ISSN 2620-6641 Volume 10, Nomor 2 Tahun 2023 Conclussion The reach of this study is limited due to a shortage of respondents for researchers. According to the aforementioned findings, students' use of English in educational settings where the Quran is taught is mostly through watching and listening. However, since they emphasize their objectives to finish their memorization and both of them are making solid progress in the right direction, it cannot be ruled out that it will help their English language and process independently improvemay have an impact on the variation in students' levels of English proficiency. The motivation is one of the elements. (Septiyana & Aminatun, 2021). Better than being forced is doing what you can because you begin to love and feel pleased first. 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https://openalex.org/W2537754613	https://nottingham-repository.worktribe.com/preview/822932/gough%20et%20al%20301V%20assay.pdf	English	null	A sensitive 301V BSE serial PMCA assay	F1000Research	2,016	cc-by	5,400	METHOD ARTICLE A sensitive 301V BSE serial PMCA assay [version 1; referees: awaiting peer review] Kevin C. Gough , Keith Bishop , Robert A. Somerville , Nora Hunter , Ben C. Maddison2 1 2 3 3 2 School of Veterinary Medicine and Science, The University of Nottingham, Loughborough, UK ADAS-UK Biotechnology group, School of Veterinary Medicine and Science, The University of Nottingham, Loughborough, UK The Roslin Institute, The University of Edinburgh, Easter Bush, UK 1 2 3 School of Veterinary Medicine and Science, The University of Nottingham, Loughborough, UK ADAS-UK Biotechnology group, School of Veterinary Medicine and Science, The University of N The Roslin Institute, The University of Edinburgh, Easter Bush, UK 1 2 3 Open Peer Review Discuss this article (0) Comments Referee Status: AWAITING PEER REVIEW 18 Oct 2016, :2529 (doi: ) First published: 5 10.12688/f1000research.9735.1 18 Oct 2016, :2529 (doi: ) Latest published: 5 10.12688/f1000research.9735.1 v1 F1000Research 2016, 5:2529 Last updated: 18 OCT 2016 Abstract The prion strain 301V, is a mouse passaged form of bovine spongiform encephalopathy (BSE). It has been used as a model of BSE for more than 20 years, in particular in the investigation of tissue distribution of infectivity, the molecular phenotype and transmission properties of BSE, strain typing assays and prion inactivation studies. Most 301V experiments have required murine bioassay as a method for the quantitation of infectivity. To date this model strain has not been studied with the protein misfolding cyclic amplification assay (PMCA) which detects prion-associated PrP protein. The detection of BSE PrP by PMCA can be more sensitive than mouse bioassay and is carried out in a much shorter time frame of days as opposed to months/years. Here, we describe the development of a new highly sensitive and specific PMCA assay for murine 301V and assess the sensitivity of the assay in direct comparison with murine bioassay of the same material. This assay detected, in a few in vitro days, 301V at a brain dilution of at least 1x10 , compared to bioassay of the same material in VM mice that could detect down to a 1x10 dilution and took >180 days. The 301V PMCA may therefore offer a faster and more sensitive alternative to live animal bioassay when studying the BSE agent in VM mice. Sc Sc -9 -8 Ben C. Maddison ( ) Corresponding author: ben.maddison@adas.co.uk Gough KC, Bishop K, Somerville RA How to cite this article: et al. A sensitive 301V BSE serial PMCA assay [version 1; referees: awaiting 2016, :2529 (doi: ) peer review] F1000Research 5 10.12688/f1000research.9735.1 © 2016 Gough KC . This is an open access article distributed under the terms of the , which Copyright: et al Creative Commons Attribution Licence permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Data associated with the article are available under the terms of the (CC0 1.0 Public domain dedication). Creative Commons Zero "No rights reserved" data waiver This work was funded by DEFRA under project SE1433 (Robert Somerville) and by BBSRC Institute Strategic Grant Grant information: BB/J004332/1 (The Roslin Institute). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: No competing interests were disclosed. 18 Oct 2016, :2529 (doi: ) First published: 5 10.12688/f1000research.9735.1 Ben C. Ben C. Maddison ( ) Corresponding author: ben.maddison@adas.co.uk Gough KC, Bishop K, Somerville RA How to cite this article: et al. A sensitive 301V BSE serial PMCA assay [version 1; referees: awaiting 2016, :2529 (doi: ) peer review] F1000Research 5 10.12688/f1000research.9735.1 © 2016 Gough KC . This is an open access article distributed under the terms of the , which Copyright: et al Creative Commons Attribution Licence permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Data associated with the article are available under the terms of the (CC0 1.0 Public domain dedication). Creative Commons Zero "No rights reserved" data waiver This work was funded by DEFRA under project SE1433 (Robert Somerville) and by BBSRC Institute Strategic Grant Grant information: BB/J004332/1 (The Roslin Institute). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: No competing interests were disclosed. 18 Oct 2016, :2529 (doi: ) First published: 5 10.12688/f1000research.9735.1 Introduction In the last 15 years or so prion research has been revolutionised by the demonstration of in vitro assays that are thought to replicate the molecular events occurring in vivo during prion infection and the conversion of PrPC to the disease isomer PrPSc. First reported by Saborio and colleagues in 200110 the protein misfolding cyclic amplification (PMCA) assay is able to replicate prions in vitro within a source of PrPC (generally produced from a healthy brain homogenate) during cycles of PrPC to PrPSc seeded conversion fol- lowed by sonication with high frequency sound waves that break up aggregates of PrPSc to form new seeds or sites of nucleation. The products of this sensitive in vitro assay retain the biochemical characteristics of the prion seed and are infectious11. The sensitivity of the PMCA assay was improved by including the dilution of the reaction into fresh PrPC substrate after an optimal period of ampli- fication. This modification, known as serial PMCA (sPMCA)12 has been widely adopted by the research community and has been applied to several rodent prion strains13, scrapie in sheep14,15, BSE in cattle16, and CWD of cervids17. sPMCA can achieve levels of sensitivity significantly beyond that of animal bioassay18 and these experiments take days or weeks to perform compared to the months to years of animal bioassay, and at a fraction of the cost. As such, amplification of prions by sPMCA can be used as a surrogate for measuring infectivity in vivo. To date, the 301V strain of BSE has not been used in sPMCA based studies. Here, we describe a high sensitivity 301V sPMCA that can, over a period of 5 days detect higher dilutions of infectivity than are attained by a 170–200 day bioassay within the VM mouse line. The transmissible spongiform encephalopathies (TSE or prion diseases) form a group of infectious and fatal neurodegenerative diseases affecting several species of mammals for which there is no available treatment or cure. The cause is thought to be a novel infec- tious agent (the prion), itself a misfolded isomer (PrPSc) of a benign cell associated protein known PrP or PrPC. This group of diseases include scrapie in sheep and goats, Creutzfeldt Jakob disease (CJD) in humans and bovine spongiform encephalopathy (BSE) in cattle. Introduction The UK BSE epizootic of the mid-1980s to early 1990s was the result of recycling BSE through the cattle food chain via a high protein feed additive known as meat and bone meal. It is thought that over 460,000 BSE infected UK cattle entered the human food chain before a ban on feeding specified risk materials to cattle came into force1. As a consequence of this, a new human disease referred to as vCJD, the human form of BSE, began to present in a number of young adults from the mid-1990s. This demonstration of the zoonotic potential of prion diseases generated an acceler- ated program of research into these diseases and much of this has required animal models. The murine passaged BSE strain known as 301V was first described by Bruce and colleagues2 during trans- mission studies of cattle BSE to wild type mice. BSE 301V is the product of serial passage within the VM mouse line and this combination of 301V/VM has been well characterised and used in numerous studies, including those aimed at understanding the fundamental brain pathology during neuropathogenesis3. In addi- tion, the 301V/VM model has also been important in experiments analysing the effectiveness of various decontamination measures for BSE infectivity. For example, Taylor et al.4 demonstrated the effectiveness of formic acid in inactivating both 301V and scrapie in the context of occupational exposure to histological samples. This BSE model has also been used to show the lack of sufficient inactivation of BSE prions during historical rendering processes which resulted in the BSE outbreak in UK cattle5. For develop- ment of safe procedures in the context of human health, 301V has been used to model vCJD in the fractionation of plasma and the safe manufacture of blood products6, and additionally has been used to estimate BSE infectivity that is likely to remain after proc- esses in the derivation of bone gelatine from bovine products7. A further study by McLeod et al.8 screened a number of different proteases for their ability to reduce the infectious titre of 301V as a novel method for the decontamination of sensitive surgical instru- ments. More recently, the availability of transgenic mice express- ing the bovine PRNP transgene have become available, and with this their high susceptibility to bovine BSE prions has comple- mented the use of 301V in these types of experiments. Abstract Maddison ( ) Corresponding author: ben.maddison@adas.co.uk Gough KC, Bishop K, Somerville RA How to cite this article: et al. A sensitive 301V BSE serial PMCA assay [version 1; referees: awaiting 2016, :2529 (doi: ) peer review] F1000Research 5 10.12688/f1000research.9735.1 © 2016 Gough KC . This is an open access article distributed under the terms of the , which Copyright: et al Creative Commons Attribution Licence permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Data associated with the article are available under the terms of the (CC0 1.0 Public domain dedication). Creative Commons Zero "No rights reserved" data waiver This work was funded by DEFRA under project SE1433 (Robert Somerville) and by BBSRC Institute Strategic Grant Grant information: BB/J004332/1 (The Roslin Institute). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: No competing interests were disclosed. 18 Oct 2016, :2529 (doi: ) First published: 5 10.12688/f1000research.9735.1 Page 1 of 5 F1000Research 2016, 5:2529 Last updated: 18 OCT 2016 F1000Research 2016, 5:2529 Last updated: 18 OCT 2016 Introduction Unspiked PMCA samples were always negative (a total of 15 replicates are shown). Western blots were probed with the anti-PrP antibody SHa31, M, molecular mass markers at 41, 30 and 22 kDa. gure 1. sPMCA amplification of 301V BSE. A. Reactions were seeded with 10 µls of 10-4 to 10-13 dilution of 301V brain (as indicated). nspiked samples had 10 µl VM brain substrate only. Sc, scrapie positive brain sample was used as a blotting control. B. The assessment reproducibility of the 301V sPMCA using three separate batches of VM substrate, each were seeded with 301V brain dilutions 10-7 to 10-11 s indicated). Unspiked PMCA samples were always negative (a total of 15 replicates are shown). Western blots were probed with the Figure 1. sPMCA amplification of 301V BSE. A. Reactions were seeded with 10 µls of 10-4 to 10-13 dilution of 301V brain (as indicated). Unspiked samples had 10 µl VM brain substrate only. Sc, scrapie positive brain sample was used as a blotting control. B. The assessment of reproducibility of the 301V sPMCA using three separate batches of VM substrate, each were seeded with 301V brain dilutions 10-7 to 10-11 (as indicated). Unspiked PMCA samples were always negative (a total of 15 replicates are shown). Western blots were probed with the anti-PrP antibody SHa31, M, molecular mass markers at 41, 30 and 22 kDa. Figure 1. sPMCA amplification of 301V BSE. A. Reactions were seeded with 10 µls of 10-4 to 10-13 dilution of 301V brain (as indicated). Unspiked samples had 10 µl VM brain substrate only. Sc, scrapie positive brain sample was used as a blotting control. B. The assessment of reproducibility of the 301V sPMCA using three separate batches of VM substrate, each were seeded with 301V brain dilutions 10-7 to 10-11 (as indicated). Unspiked PMCA samples were always negative (a total of 15 replicates are shown). Western blots were probed with the anti-PrP antibody SHa31, M, molecular mass markers at 41, 30 and 22 kDa. total assay time taking little over a week. Whilst we report sensi- tivity of the assay at 5 days of amplification, it is very likely that much higher levels of sensitivity could be attained with additional rounds of amplification. Introduction A study published by Giles et al.9 directly compared the effectiveness of decontamination of both bovine BSE and 301V in transgenic and VM mice, respectively. 301V was more sensitive to both heat and chemical denaturation than cattle BSE, suggesting that the physical properties of the 301V BSE strain have diverged slightly from those of cattle BSE. Despite the more recent availability of these transgenic rodent strains the 301V/VM infection system remains a useful, well characterised model for BSE in TSE research and allows direct comparison with numerous previous studies Results and discussion Dataset 1. Raw uncropped images of the Western blots shown in Figure 1 http://dx.doi.org/10.5256/f1000research.9735.d138638 Dataset 1. Raw uncropped images of the Western blots shown in Figure 1 A pool of 301V mouse brain homogenate was used to assess the efficacy of a new sPMCA assay alongside conventional 301V/VM bioassay. For the in vitro assay we used a 5 day amplification method and a murine VM substrate. This 301V sPMCA assay demonstrated assay sensitivity to 1×10-9 dilution of brain homoge- nate (Figure 1A). The batch-to-batch variability test of a further 3 substrate preparations detected 301V to at least the same level(Figure 1B). The observed variations in sensitivity within these limiting dilution experiments (between 10-9 and 10-11) is likely a reflection of differences in individual substrate preparations that will be seen when making small volume preparations from limited numbers of brains. The same 301V sample was also analysed in a VM mouse bioassay by limiting dilution and this bioassay detected infectiv- ity in 1 out of 12 mice at the 10-8 dilution of 301V brain material (Table 1), equating to a 301V titre of the original brain pool of 108.5 LD50/g (as determined by Karber methodology19). The 301V sPMCA assay can therefore detect PrPSc at a level at least tenfold more sensitive than the VM mouse bioassay, in a Page 2 of 5 Page 2 of 5 F1000Research 2016, 5:2529 Last updated: 18 OCT 2016 Figure 1. sPMCA amplification of 301V BSE. A. Reactions were seeded with 10 µls of 10-4 to 10-13 dilution of 301V brain (as indicated). Unspiked samples had 10 µl VM brain substrate only. Sc, scrapie positive brain sample was used as a blotting control. B. The assessment of reproducibility of the 301V sPMCA using three separate batches of VM substrate, each were seeded with 301V brain dilutions 10-7 to 10-11 (as indicated). Introduction Dilu- tions of 301V brain homogenate are recorded as the dilution of brain spike before addition to the amplification reaction, ie 10-1 is 10 µl of a 10 % (w/v) preparation of brain, 10-2 is 10 µl of a 1 in 10 dilution of the 10-1 preparation of brain etc. All sPMCA was carried out at 37°C in a Misonix S3000 microplate horn. prion model is with cervid CWD17. In this instance, CWD amplifi- cation within cervid CNS tissue substrate is notoriously inefficient, and transgenic mice have been used as an animal bioassay model for infectivity studies and also to provide substrate to facilitate effi- cient in vitro amplification by sPMCA. were diluted 1 in 10 into fresh VM brain substrate and sonicated for a further 24 hour round of repeated sonication and incubation retaining the same three Teflon beads throughout the 5 rounds of sPMCA. Amplifications were carried out for a total of 5 days. Dilu- tions of 301V brain homogenate are recorded as the dilution of brain spike before addition to the amplification reaction, ie 10-1 is 10 µl of a 10 % (w/v) preparation of brain, 10-2 is 10 µl of a 1 in 10 dilution of the 10-1 preparation of brain etc. All sPMCA was carried out at 37°C in a Misonix S3000 microplate horn. In summary, we have developed a reliable in vitro method (sPMCA) for the detection of PrPSc resulting from infection with 301V (mouse passaged BSE). The assay is at least as sensitive as mouse bioassay and can derive data on the presence of PrPSc in a fraction of the time. This will be useful in studies such as those looking at BSE decontamination where the screening of large numbers of samples is required. Western blotting sPMCA reaction products (10 µl) were digested using a final concentration of 50 µg/ml proteinase K (Sigma-Aldrich), for 90 minutes at 40°C. Samples were then boiled for 5 minutes in 1X LDS buffer (Invitrogen) and electrophoresed through a NuPAGE SDS-PAGE gel system (Invitrogen) using 12% (w/v) acryla- mide gels. Molecular mass markers (prestained Seeblue plus2, Invitrogen LC5925) were run alongside samples. As a blotting control an aliquot of proteinase K digested (50 µg/ml proteinase K (Sigma-Aldrich), 60 minutes at 40°C) scrapie positive ovine brain (equivalent to 2 µl of a 10% w/v brain homogenate) was also loaded onto each SDS-PAGE gel. Introduction Proteins were transferred to PVDF (Roche) membrane by electroblotting, and the mem- branes were then blocked for 1 hour with 3 % (w/v) skimmed milk. Western blots were probed with the anti-PrP mouse monoclonal antibody SHa31 (SpiBio A03213) diluted to 1/80,000 and a poly- clonal goat anti-mouse immunoglobulins Horse Radish Peroxi- dase conjugate (Dako P04477), diluted to 1:20,000, as previously described21. Blots were imaged after the addition of EZ-ECL HRP substrate (Geneflow) using an ICCD225 photon counting camera system and IFS32 image software (Photek Ltd). sPMCA VM brains from healthy animals were supplied frozen, before preparation of the 10 % (w/v) homogenate substrate. Preparation of 10 % brain homogenates as substrates for sPMCA has been previously described21. Here, we included the sPMCA addi- tive digitonin22 (Sigma-Aldrich) which was added to reactions at 50 μg/ml. sPMCA reactions were assembled in 200 µl thin wall PCR tubes, and comprised 90 µl brain homogenate substrate with digitonin, three 2.4 mm Teflon beads (Precision plastic ball co. Ltd) and 10 µl of 301V sample to be amplified (10-4 to 10-13 dilution of 301V brain). Unspiked negative control samples were set up substi- tuting the 301V seed with 10 µl VM brain substrate only. Reaction tubes were placed in a Misonix S3000 sonicating water bath set on a program of 10 seconds sonication every 30 mins, for 24 hours at a power setting of 190–200 W at 37°C. Every 24 hours, samples Materials and methods All use of animals, the collection of animal tissues and the use of such tissue was carried out in accordance with the Animal (Scientific Procedures) Act (ASPA) 1986, under licences from the UK Government Home Office (Project licence 60/2544). All animal experiments were subject to review and approval (01-124) by The Roslin Institute Ethical Review Committee and euthanasia methods were approved by the UK Home Office. p g No competing interests were disclosed. p g No competing interests were disclosed. Author contributions Author contributions RAS and NH conceived the study and provided both the 301V and VM biological material, BCM and KCG devised and directed the experiments, KB carried out the experiments. BCM wrote the manuscript, all authors proof read the manuscript before submission. Introduction The highest dilution of 301V infectivity that could be detected within the VM bioassay was a 1×10-8 dilu- tion of brain at 184 days post inoculation, or 26 times longer than the sPMCA assay. Maintaining animals within bioassay, including their category 3 containment make these kind of titration experi- ments very costly and time consuming to carry out. That, coupled with the ethical implications of use of animals means sPMCA could be the method of choice unless there is a good scientific reason for requiring to demonstrate infectivity (the ability to cause disease), or a requirement to monitor strain phenotype, as opposed to the surrogate marker of disease, PrPSc protein. A useful way of incorporating these two assays into future studies, could be to assess 301V seeding activity within a wide range of samples to identify those that contain PrPSc. Bioassay could then be used on a lim- ited number of sPMCA-positive samples to confirm the presence of BSE infectivity. Another example of the application of sPMCA that has been routinely used for the detection of prions in a rodent Table 1. Titration of 301V infected mouse brain. Number of clinical and pathology positive mice/total injected. Mouse numbers exclude intercurrent deaths i.e. animals dying earlier than the 1st clinical case (there was one intercurrent death of a mouse receiving 10-7 brain dilution, and two receiving the 10-4 dilution). Total number of challenged mice were 6 per group for 10-4 and 10-5, 12 per group for 10-6 to 10-9. 301V Brain Dilution No. mice positive Incubation period (days) SD 10-4 4/4 142 10 10-5 5/6 149 14 10-6 10/12 170 12 10-7 4/11 191 18 10-8 1/12 184 - 10-9 0/12 - - Page 3 of 5 Page 3 of 5 F1000Research 2016, 5:2529 Last updated: 18 OCT 2016 F1000Research 2016, 5:2529 Last updated: 18 OCT 2016 were diluted 1 in 10 into fresh VM brain substrate and sonicated for a further 24 hour round of repeated sonication and incubation retaining the same three Teflon beads throughout the 5 rounds of sPMCA. Amplifications were carried out for a total of 5 days. Bioassay A serial dilution of pooled murine VM brains that were taken from 301V challenged animals was made as previously described20. A dilution series of this brain homogenate from 10-1 to 10-10 was made up in saline and used to inoculate groups of VM mice, bred in house at The Roslin Institute and of mixed sex, 6 weeks old (groups of 6 mice at 10-4 and 10-5, 12 mice from 10-6 to 10-10) with 20 µl of each dilution intracerebrally, as previously described20. Animals were observed daily for signs of ill health and euthanised by cervical dislocation when clinical signs of neurological disease or any intercurrent illness were observed. After euthanasia, brain tissue was confirmed as 301V positive or negative by detection of brain tissue vacuolation by light microscopy after Haematoxylin and Eosin staining20. This analysis was carried out blinded to the identity of the tissue in each case. Data availability Data availability F1000Research: Dataset 1. Raw uncropped images of the Western blots shown in Figure 1. 10.5256/f1000research.9735.d13863823 F1000Research: Dataset 1. Raw uncropped images of the Western blots shown in Figure 1. 10.5256/f1000research.9735.d13863823 ; ( ) PubMed Abstract \| Publisher Full Text 13. Yoshioka M, Imamura M, Okada H, et al.: Sc237 hamster PrPSc and Sc237- derived mouse PrPSc generated by interspecies in vitro amplification exhibit distinct pathological and biochemical properties in tga20 transgenic mice. Microbiol Immunol. 2011; 55(5): 331–40. PubMed Abstract \| Publisher Full Text 2. Bruce M, Chree A, McConnell I, et al.: Transmission of bovine spongiform encephalopathy and scrapie to mice: strain variation and the species barrier. Philos Trans R Soc Lond B Biol Sci. 1994; 343(1306): 405–411. PubMed Abstract \| Publisher Full Text 3. Williams A, Lucassen PJ, Ritchie D, et al.: PrP deposition, microglial activation, and neuronal apoptosis in murine scrapie. Exp Neurol. 1997; 144(2): 433–438. PubMed Abstract \| Publisher Full Text 3. Williams A, Lucassen PJ, Ritchie D, et al.: PrP deposition, microglial activation, and neuronal apoptosis in murine scrapie. Exp Neurol. 1997; 144(2): 433–438. PubMed Abstract \| Publisher Full Text 14. Thorne L, Terry LA: In vitro amplification of PrPSc derived from the brain and blood of sheep infected with scrapie. J Gen Virol. 2008; 89(Pt 12): 3177–84. PubMed Abstract \| Publisher Full Text 4. Taylor DM, Brown JM, Fernie K, et al.: The effect of formic acid on BSE and scrapie infectivity in fixed and unfixed brain-tissue. Vet Microbiol. 1997; 58(2–4): 167–174. \| 15. Thorne L, Holder T, Ramsay A, et al.: In vitro amplification of ovine prions from scrapie-infected sheep from Great Britain reveals distinct patterns of propagation. BMC Vet Res. 2012; 8: 223. PubMed Abstract \| Publisher Full Text \| Free Full Text ( ) PubMed Abstract \| Publisher Full Text 16. Franz M, Eiden M, Balkema-Buschmann A, et al.: Detection of PrPSc in peripheral tissues of clinically affected cattle after oral challenge with bovine spongiform encephalopathy. J Gen Virol. 2012; 93(Pt 12): 2740–8. PubMed Abstract \| Publisher Full Text 5. Taylor DM, Fernie K, McConnell I, et al.: Solvent extraction as an adjunct to rendering: the effect on BSE and scrapie agents of hot solvents followed by dry heat and steam. Vet Rec. 1998; 143(1): 6–9. PubMed Abstract \| Publisher Full Text 17. Kurt TD, Perrott MR, Wilusz CJ, et al.: Efficient in vitro amplification of chronic wasting disease PrPRES. J Virol. 2007; 81(17): 9605–8. PubMed Abstract \| Publisher Full Text \| Free Full Text 6. Reichl HE, Foster PR, Welch AG, et al.: Studies on the removal of a bovine spongiform encephalopathy-derived agent by processes used in the manufacture of human immunoglobulin. Vox Sang. 2002; 83(2): 137–145. PubMed Abstract \| Publisher Full Text 18. Saá P, Castilla J, Soto C: Ultra-efficient replication of infectious prions by automated protein misfolding cyclic amplification. J Biol Chem. 2006; 281(46): 35245–52. PubMed Abstract \| Publisher Full Text 7. Grobben AH, Steele PJ, Somerville RA, et al.: Inactivation of the bovine- spongiform-encephalopathy (BSE) agent by the acid and alkaline processes used in the manufacture of bone gelatine. Biotechnol Appl Biochem. 2004; 39(Pt 3): 329–338. 19. Kärber G: Beitrag zur kollektiven Behandlung pharmakologischer Reihenversuche. Archives of Experimental Pathology and Pharmacology. 1931; 162(4): 480–483. Publisher Full Text 1. Valleron AJ, Boelle PY, Will R, et al.: Estimation of epidemic size and incubation time based on age characteristics of vCJD in the United Kingdom. Science. 2001; 294(5547): 1726–1728. PubMed Abstract \| Publisher Full Text 39(Pt 3): 329 338. PubMed Abstract \| Publisher Full Text 8. Mcleod AH, Murdoch H, Dickinson J, et al.: Proteolytic inactivation of the bovine spongiform encephalopathy agent. Biochem Biophys Res Commun. 2004; 317(4): 1165–1170. 20. Grobben AH, Steele PJ, Somerville RA, et al.: Inactivation of transmissible spongiform encephalopathy agents during the manufacture of dicalcium phosphate from bone. Vet Rec. 2006; 158(11): 361–366. PubMed Abstract \| Publisher Full Text References 2005; 11(9): 982–5. PubMed Abstract \| Publisher Full Text 13. Yoshioka M, Imamura M, Okada H, et al.: Sc237 hamster PrPSc and Sc237- derived mouse PrPSc generated by interspecies in vitro amplification exhibit distinct pathological and biochemical properties in tga20 transgenic mice. Microbiol Immunol. 2011; 55(5): 331–40. PubMed Abstract \| Publisher Full Text 14. Thorne L, Terry LA: In vitro amplification of PrPSc derived from the brain and blood of sheep infected with scrapie. J Gen Virol. 2008; 89(Pt 12): 3177–84. PubMed Abstract \| Publisher Full Text 15. Thorne L, Holder T, Ramsay A, et al.: In vitro amplification of ovine prions from scrapie-infected sheep from Great Britain reveals distinct patterns of propagation. BMC Vet Res. 2012; 8: 223. PubMed Abstract \| Publisher Full Text \| Free Full Text 16. Franz M, Eiden M, Balkema-Buschmann A, et al.: Detection of PrPSc in peripheral tissues of clinically affected cattle after oral challenge with bovine spongiform encephalopathy. J Gen Virol. 2012; 93(Pt 12): 2740–8. PubMed Abstract \| Publisher Full Text 17. Kurt TD, Perrott MR, Wilusz CJ, et al.: Efficient in vitro amplification of chronic wasting disease PrPRES. J Virol. 2007; 81(17): 9605–8. PubMed Abstract \| Publisher Full Text \| Free Full Text 18. Saá P, Castilla J, Soto C: Ultra-efficient replication of infectious prions by automated protein misfolding cyclic amplification. J Biol Chem. 2006; 281(46): 35245–52. PubMed Abstract \| Publisher Full Text 19. Kärber G: Beitrag zur kollektiven Behandlung pharmakologischer Reihenversuche. Archives of Experimental Pathology and Pharmacology. 1931; 162(4): 480–483. Publisher Full Text 20. Grobben AH, Steele PJ, Somerville RA, et al.: Inactivation of transmissible spongiform encephalopathy agents during the manufacture of dicalcium phosphate from bone. Vet Rec. 2006; 158(11): 361–366. PubMed Abstract \| Publisher Full Text 21. Maddison BC, Baker CA, Terry LA, et al.: Environmental sources of scrapie prions. J Virol. 2010; 84(21): 11560–2. PubMed Abstract \| Publisher Full Text \| Free Full Text 22. Gonzalez-Montalban N, Makarava N, Ostapchenko VG, et al.: Highly efficient protein misfolding cyclic amplification. PLoS Pathogens. 2011; 7(2): e1001277. PubMed Abstract \| Publisher Full Text \| Free Full Text 23. Gough K, Bishop K, Somerville R, et al.: Dataset 1 in: A sensitive 301V BSE serial PMCA assay. F1000Research. 2016. Data Source PubMed Abstract \| Publisher Full Text \| Free Full Text 10. Saborio GP, Permanne B, Soto C: Sensitive detection of pathological prion protein by cyclic amplification of protein misfolding. Nature. 2001; 411(6839): 810–3. PubMed Abstract \| Publisher Full Text 22. Gonzalez-Montalban N, Makarava N, Ostapchenko VG, et al.: Highly efficient protein misfolding cyclic amplification. PLoS Pathogens. 2011; 7(2): e1001277. PubMed Abstract \| Publisher Full Text \| Free Full Text Grant information This work was funded by DEFRA under project SE1433 (Robert Somerville) and by BBSRC Institute Strategic Grant BB/J004332/1 (The Roslin Institute). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Page 4 of 5 Page 4 of 5 F1000Research 2016, 5:2529 Last updated: 18 OCT 2016 References ( ) PubMed Abstract \| Publisher Full Text 9. Giles K, Glidden DV, Beckworth R, et al.: Resistance of bovine spongiform encephalopathy (BSE) prions to inactivation. PLoS Pathogens. 2008; 4(11): e1000206. PubMed Abstract \| Publisher Full Text \| Free Full Text 21. Maddison BC, Baker CA, Terry LA, et al.: Environmental sources of scrapie prions. J Virol. 2010; 84(21): 11560–2. PubMed Abstract \| Publisher Full Text \| Free Full Text 12. Castilla J, Saá P, Soto C: Detection of prions in blood. Nat Med. 2005; 11(9): 982–5. PubMed Abstract \| Publisher Full Text References 1. Valleron AJ, Boelle PY, Will R, et al.: Estimation of epidemic size and incubation time based on age characteristics of vCJD in the United Kingdom. Science. 2001; 294(5547): 1726–1728. PubMed Abstract \| Publisher Full Text 2. Bruce M, Chree A, McConnell I, et al.: Transmission of bovine spongiform encephalopathy and scrapie to mice: strain variation and the species barrier. Philos Trans R Soc Lond B Biol Sci. 1994; 343(1306): 405–411. PubMed Abstract \| Publisher Full Text 3. Williams A, Lucassen PJ, Ritchie D, et al.: PrP deposition, microglial activation, and neuronal apoptosis in murine scrapie. Exp Neurol. 1997; 144(2): 433–438. PubMed Abstract \| Publisher Full Text 4. Taylor DM, Brown JM, Fernie K, et al.: The effect of formic acid on BSE and scrapie infectivity in fixed and unfixed brain-tissue. Vet Microbiol. 1997; 58(2–4): 167–174. PubMed Abstract \| Publisher Full Text 5. Taylor DM, Fernie K, McConnell I, et al.: Solvent extraction as an adjunct to rendering: the effect on BSE and scrapie agents of hot solvents followed by dry heat and steam. Vet Rec. 1998; 143(1): 6–9. PubMed Abstract \| Publisher Full Text 6. Reichl HE, Foster PR, Welch AG, et al.: Studies on the removal of a bovine spongiform encephalopathy-derived agent by processes used in the manufacture of human immunoglobulin. Vox Sang. 2002; 83(2): 137–145. PubMed Abstract \| Publisher Full Text 7. Grobben AH, Steele PJ, Somerville RA, et al.: Inactivation of the bovine- spongiform-encephalopathy (BSE) agent by the acid and alkaline processes used in the manufacture of bone gelatine. 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https://openalex.org/W2093422711	https://journals.vilniustech.lt/index.php/TEDE/article/download/6458/5598	English	null	ROBUSTNESS OF THE MULTI‐OBJECTIVE MOORA METHOD WITH A TEST FOR THE FACILITIES SECTOR	Technological and economic development of economy	2,009	cc-by	12,538	Technological and economic development OF ECONOMY Baltic Journal on Sustainability 2009 15(2): 352–375 Technological and economic development OF ECONOMY Baltic Journal on Sustainability 2009 15(2): 352–375 Technological and economic development OF ECONOMY Baltic Journal on Sustainability 2009 15(2): 352–375 15(2): 352–375 Received 21 January 2009; accepted 4 May 2009 Abstract. The definition of robustness in econometrics, the error term in a linear equation, was not only broadened, but in addition moved to the meaning of common language: from a cardinal to a qualitative one. These interpretations were tested by an application on the Facilities Sector in Lithuania. The application is multi-objective: like costs, experience and effectiveness at the side of the contractors; quality, duration of the work and cost price at the side of the owners. These objectives having all different units the dimensionless ratios of the MOORA method avoids the difficulties of normalization. In a first part of MOORA these ratios were aggregated and in a second one they were used as distances to a reference point. The results of both parts control each other, a test on robust- ness. Additionally, MOORA shows a robust domination on all other methods of multi-objective optimization. For the Facilities Sector in Lithuania, both parts of MOORA resulted in a comparable ranking. In this way a double check was made on the robustness of the results. Keywords: MOORA, robustness, multi-objective methods, ameliorated nominal group technique, Delphi. Reference to this paper should be made as follows: Brauers, W. K.; Zavadskas, E. K. 2009. Robust- ness of the multi-objective moora method with a test for the facilities sector, Technological and Economic Development of Economy 15(2): 352–375. Willem K. Brauers1, Edmundas K. Zavadskas2 1 Faculty of Applied Economics and Institute for Development Policy and Management, University of Antwerp 2 Department of Construction Technology and Management, Vilnius Gediminas Technical University, e-mail: edmundas.zavadskas@adm.vgtu.lt 2 Department of Construction Technology and Management, Vilnius Gediminas Technical University, e-mail: edmundas.zavadskas@adm.vgtu.lt Received 21 January 2009; accepted 4 May 2009 1. Definition of robustness By 1953, which is quite recent for statistics1, robust became a statistical term as “strong, 1 As well known, statistics already existed in Roman times with the census of population. doi: 10.3846/1392-8619.2009.15.352-375 Technological and Economic Development of Economy, 2009, 15(2): 352–375 353 healthy, sufficiently tough to withstand life’s adversities” (Stigler 1973:872). Nevertheless, already in 1969 the statistician Huber considered robustness as purely cardinal as a compro- mise between a normal distribution and its light deviations2. More recently the statisticians Casella and Berger call a robust alternative the Median Absolute Deviation for a sample X1, …..Xn (2002:509). The error term in a linear equation is the starting point for the definition of robustness in econometrics (Darnell 1997: 355). In addition, robustness is not only linked to error terms or random variables but also to residual terms, slack and dummy variables, outliers etc. Darnell concludes: “given the somewhat arbitrary ad hoc nature of the robust estimators ……these approaches have had limited application in econometrics” (1997: 356). Kennedy recognizes the existence of robust estimators “an estimator whose properties while not quite best”, he continues “the topic of robustness has become quite popular recently in econometrics, as researchers have become aware of the extreme sensitivity of some of their estimation pro- cedures”(1998: 298). Other well-known textbooks on econometrics do not even mention the name of robustness, like Thomas (1985), Intriligator (1978), Madansky (1976), Walters (1973), Wonnacott, R., Wonnacott, T. (1970) and Johnston (1963). More specificity is found by authors who consider robustness in forms of the error distribution (Rhodes and Fomby 1988), whereas Mills (1992) presents a Bayesian prediction test which is robust to certain forms of non-normality in the error distribution. Moreover, from the beginning Bayesian Analysis has to be characterized as cardinal, nevertheless with a high grade of arbitrariness. This arbitrariness could be softened by considerations on robustness3. Anyway, cardinal numbers form also the basis of robustness in the Poisson distribution, the t statistic and in sampling (Särndal, Swensson and Wretman 1992). However, even in econometrics, we observe a move to a more vague and qualitative defi- nition of robustness, namely to the meaning of common language4: from a cardinal towards a nominal scale: the most robust one, more robust than…, as robust as……, robust, weak robust, less robust than…, not robust etc., comparable to so many other nominal scales in multi-objective analysis (for instance, mentioned by Brauers 2004: 97–99). 2 At a later time, namely in 1981, Huber wrote a more complete book on Robust Statistics. In 1994 at the occasion of Huber’s birthday his colleagues edited a book on Robust Statistics (editor: Rieder). 3 A good overview of this problem of robustness and Bayesian Analysis is brought by Ruggeri, 2008. 4 Webster’s new Universal Unabridged Dictionary: robust: strong; stronger, strongest. 1. Definition of robustness A debate between Frisch (1933) and Tinbergen (1930) ensued as whether or not Tinbergen had estimated structural form representations robust to changes in policy regimes or reduced form representations not robust to shifting policy regimes (Heckman 1992: 878). Kreps (1990) maintains that more robustness is more important for bargaining theory than for auction theory as more information is available in the latter case than in the former. He esteems that robust predictions are crucial although the meaning given to robustness may depend on the context. (also Vincke 1999: 186(2)). Edin and Ohlson (1991) examine that institutional ar- rangements in the political process affect budget deficits. Sensitivity Analysis indicates that the results are robust. Admati and Pfleiderer (1994) speak of robustness in financial contracting. 354 W. K. Brauers, E. K. Zavadskas. Robustness of the multi-objective moora method with... Another qualitative approach of robustness is related to subjective probability by Machina and Schmeidler (1992). Dasgupta and Maskin (2008) maintain that the simple majority rule is the most robust voting rule. Finally, the context will determine robustness in benchmark- ing, in scenario writing and in simulation (Brauers et al. 2009). The remark that significance of robustness depends on the context can be specified in different ways. First, robustness can be considered as cardinal or as a nominal scale. Second, if robustness is indicated as vague or arbitrary, is it also not the case with inference statistics (Hoel 1971, 2 versus Hays, 1974: 47 and Casella et al. 2002, VII), probability theory (Hays 1974: 47) and statistical specification (Intriligator 1978: 2 and Matyas, Sevestre 1992, chap- ter 9 versus Thomas 1985: 71 and Wonnacott 1970: 312)? Third, robustness is characterized by completeness being present in the statistical population, when defined as covering events and opinions which are present, as well as in the statistical universe with events and opinions not only present but also possible (Brauers et al. 2009). 2.2. All objectivesh 2.2. All objectives The method of multiple objectives in which all non-correlated objectives are considered is more robust than this one in which only a limited number of objectives is considered. 2. Conditions of robustness in multi-objective methods The most robust multi-objective method has to satisfy the following conditions: 2.4. Non-subjective The method of multiple objectives which is non-subjective is more robust than this one which uses subjective approaches. Is this condition not purely theoretical? The difficulty lies in the many facets of multi-objective optimization in which excluding subjectivity seems to be impossible. Indeed, it concerns the subjectivity in the choice of the objectives, in the normalization procedure and in the importance given to an objective. 2.3. All interrelations between objectives and alternativesh The method of multiple objectives in which all interrelations between objectives and alterna- tives is taken into consideration at the same time is more robust than this one in which the interrelations are only examined two by two. 2.1. All stakeholders The method of multiple objectives in which all stakeholders are included is more robust than this one in which only one decision-maker or different decision-makers defending only their own objectives are involved. All stakeholders mean everybody interested in a certain issue. Consequently, the method of multiple objectives has to take into consideration consumer sovereignty too. The method taking into consideration consumer sovereignty is more robust than this one which does not respect consumer sovereignty. 5 The pioneer of Rank Correlation, Kendall (1948: 1), pretends the contrary but without any proof. Therefore, the introduction of the notion of Correlation is of no use for multiple objectives (Brauers 2007: 450–451). 2.6. Last available datah The method of multiple objectives which uses the last recent available data as a base in the response matrix is more robust than this one based on earlier data. 2.5. Cardinal The method of multiple objectives based on cardinal numbers is more robust than this one based on ordinal numbers: “an ordinal number is one that indicates order or position in a series, like first, second, etc.” (Kendall, Gibbons 1990: 1). i The robustness of cardinality is based first on the saying of Arrow (1974): “Obviously, a cardinal utility implies an ordinal preference but not vice versa” and, second, on the fact that the four essential operations of arithmetic: adding, subtracting, multiplication and division are only reserved for cardinal numbers5. a. Non-subjectivity in the choice of the objectives A creative and prospective thinking of all stakeholders interested in a certain issue has to generate a complete set of objectives. The Ameliorated Nominal Group Technique can assist for that purpose (see Appendix A). Non-subjectivity of the retained objectives results Technological and Economic Development of Economy, 2009, 15(2): 352–375 355 from unanimity or at least a convergence in the opinions of all the stakeholders concerned. Not only the Ameliorated Nominal Group Technique but in addition the Delphi Technique can bring support here (see Appendix B). Delphi will also assist in giving importance to the objectives, as explained underneath. h c. Non-subjectivity in the attribution of importance to an objective Convergence in opinion between all stakeholders to give more importance to an objective results again from a Delphi exercise. Therefore its dimensionless numbers are multiplied by a Significance Coefficient. The Attribution of Sub-Objectives represents another solution. The Attribution Method is more refined than the Coefficient Method, as the attribution method succeeds in characterizing an objective better. For instance, instead of giving a significance coefficient of 3 to pollution abatement, the objective “pollution abatement” is divided into 3 sub-objectives: the Greenhouse Effect, Energy Consumption and Other Pollution, each with their own characteristics. b. Non-subjectivity in the normalization proceduref Normalization affords a subjective solution for comparing the different units of the dif- ferent objectives. Consequently, the method of multiple objectives which uses non-subjective dimensionless measures, meaning that normalization is not needed, like in the ratio systems approach, is more robust than this one which uses subjective weights (already introduced by Churchman et al. in 1954, 1957) or subjective non-additive scores like in the traditional Reference Point Theory (Brauers 2004: 158–159). h y c. Non-subjectivity in the attribution of importance to an objective 7 Instead of a normal increase in productivity growth, a decrease remains possible. At that moment the interval becomes [–1, 1]. Take the example of productivity, which has to increase (positive). Consequently, we look for a maximization of productivity, e.g. in European and American countries. What if the opposite does occur? For instance, take the original transition from the USSR to Russia. Contrary to the other European countries, productivity decreased. It means that in formula (2) the numerator for Russia was negative with the whole ratio becoming negative. Consequently, the inter- val changes to: [–1, +1] instead of [0, 1]. 6 Brauers and Zavadskas (2006) prove that the most robust choice for this denominator is the square root of the sum of squares of each alternative per objective. 3. The moora method atrix of responses of all alternative solutions on all objectives: xij (1) d starts with a matrix of responses of all alternative solutions on all objectives: The method starts with a matrix of responses of all alternative solutions on all object The method starts with a matrix of responses of all alternative solutions on all objectives: xij , (1) with: xij as the response of alternative j on objective or attribute i; i = 1,2, …, n as the objec- (1) xij , with: xij as the response of alternative j on objective or attribute i; i = 1,2, …, n as the objec- tive or the attribute; j = 1,2, …, m as the alternatives. In order to define objectives better, we have to focus on the notion of attribute. Keeney and Raiffa (1993: 32) present the example of the objective “reduce sulfur dioxide emissions” to be measured by the attribute “tons of sulfur dioxide emitted per year”. An objective and a correspondent attribute always go together. Consequently, when the text mentions “objective”, the correspondent attribute is meant as well.h The MOORA method consists of 2 parts: the ratio system and the reference point ap- proach. 2.7. Different methods Once the previous 6 conditions are fulfilled, the use of 2 different methods of multi-objective optimization is more robust than the use of a single method; the use of 3 methods is more robust than the use of 2, etc. The Multi-Objective Optimization by Ratio Analysis Method (MOORA) satisfies the first 6 conditions if Non-Subjectivity in the choice of the objectives and Non-Subjectivity in the at- The Multi-Objective Optimization by Ratio Analysis Method (MOORA) satisfies the first 6 conditions if Non-Subjectivity in the choice of the objectives and Non-Subjectivity in the at- 5 The pioneer of Rank Correlation, Kendall (1948: 1), pretends the contrary but without any proof. Therefore, the introduction of the notion of Correlation is of no use for multiple objectives (Brauers 2007: 450–451). W. K. Brauers, E. K. Zavadskas. Robustness of the multi-objective moora method with... 356 tribution of importance to an objective are solved. Therefore the Ameliorated Nominal Group Technique but also the Delphi Technique can bring support. In addition, MOORA satisfies partially the seventh condition by using 2 different methods of Multi-Objective Optimization. MOORA is the most robust method as no other method satisfies the 7 conditions better. 8 Brauers 2008(b) proves that the Min-Max metric is the most robust choice between all the possible metrics of reference point theory. 3.1. The ratio system as a part of MOORA We go for a ratio system in which each response of an alternative on an objective is compared to a denominator, which is representative for all alternatives concerning that objective6: x x x ij ij ij j m * = =∑ 2 1 , (2) (2) with: xij = response of alternative j on objective i; j = 1,2, ..., m; m the number of alterna- tives; i = 1,2, … n; n the number of objectives; xij a dimensionless number representing the response of alternative j on objective i. with: xij = response of alternative j on objective i; j = 1,2, ..., m; m the number of alterna- tives; i = 1,2, … n; n the number of objectives; xij a dimensionless number representing the response of alternative j on objective i. Dimensionless Numbers, having no specific unit of measurement, are obtained for instance by multiplication or division. The normalized responses of the alternatives on the objectives belong to the interval [0; 1]. However, sometimes the interval could be [–1; 1]. Indeed, for Technological and Economic Development of Economy, 2009, 15(2): 352–375 357 instance, in the case of productivity growth some sectors, regions or countries may show a decrease instead of an increase in productivity, i.e. a negative dimensionless number7. For optimization, these responses are added in case of maximization and subtracted in case of minimization: y x x j ij i i g ij i g i n * * * = − = = = + = ∑ ∑ 1 1 , (3) (3) with: i = 1, 2, …, g as the objectives to be maximized; i = g + 1, g + 2, …, n as the objectives to be minimized; y j * a dimensionless number representing the response of alternative j with respect to all objectives; y j * can be positive or negative depending of the totals of its maxima and minima. An ordinal ranking of the yij * shows the final preference. Indeed, cardinal scales can be compared in an ordinal ranking after Arrow (1974: 256): “Obviously, a cardinal utility implies an ordinal preference but not vice versa”. 3.2. The reference point approach as a part of MOORA Reference Point Theory will go out from the ratios found in formula (2), whereas a Maximal Objective Reference Point is also considered. The Maximal Objective Reference Point ap- proach is called realistic and non-subjective as the co-ordinates (ri), which are selected for the reference point, are realized in one of the candidate alternatives. In the example, A (10;100), B (100;20) and C (50;50), the maximal objective reference point Rm results in: (100;100). The Maximal Objective Vector is self-evident, if the alternatives are well defined, as for projects in Project Analysis and Project Planning. Given the dimensionless number representing the normalized response of alternative j on objective i, namely xij * of formula (2) and in this way arriving to: ( r x i ij − ), (4) (4) with: i = 1,2, …, n as the attributes; j = 1,2, …, m as the alternatives; ri = the ith co-ordinate of the reference point; xij a dimensionless number representing the response of alternative j on objective i, then this matrix is subject to the Min-Max Metric of Tchebycheff (Karlin and Studden 1966)8: j i i ij Min r x ( ) ( ) − ( ) * max , (5) (5) r x i ij − * means the absolute value if xij * is larger than ri, for instance, by minimization. Concerning the use of the maximal objective reference point approach as a part of MOORA, some reserves can be made in connection with consumer sovereignty. Consumer sovereignty is measured with the community indifference locus map of the consumers (Brau- ers 2008b: 92–94). Given its definition, the maximal objective reference point can be pushed r x i ij − * means the absolute value if xij * is larger than ri, for instance, by minimization. 8 Brauers 2008(b) proves that the Min-Max metric is the most robust choice between all the possible metrics of reference point theory. W. K. Brauers, E. K. Zavadskas. Robustness of the multi-objective moora method with... 358 in the non-allowed non-convex zone of the highest community indifference locus and will try to pull the highest ranked alternatives in the non-allowed non-convex zone too (Brauers, Zavadskas 2006: 460–461). 3.2. The reference point approach as a part of MOORA Therefore an aspiration objective vector can be preferred, which moderates the aspirations by choosing smaller co-ordinates than in the maximal objec- tive vector and consequently can be situated in the convex zone of the highest community indifference locus. Indeed stakeholders may be more moderate in their expectations. The co-ordinates qi of an aspiration objective vector are formed as: q r i i ≤ (ri – qi) being a subjective element we do not like to introduce subjectivity in that way again. Instead, a test shows that the min-max metric of Tchebycheff, even by using the maximal objective reference point, delivers points inside the convex zone of the highest community indifference locus (Brauers 2008b: 98–103). 4. Application of the moora method for evaluating contractor’s alternatives in the facilities sector in Lithuania The facilities sector (Real Estate) in Lithuania provides the following services – acquisition, leasing and renting of existing buildings; – management of buildings, which is a multifunctional service. This means that all su- pervision, maintenance and repairing is included in the sector.h – management of buildings, which is a multifunctional service. This means that all su- pervision, maintenance and repairing is included in the sector. The facilities sector is only a very small sector in Lithuania, composed of a small number of small firms, which even perform other tasks outside facilities management, such as waste management. The largest firm in the sector counts only 179 employees. Official statistics are not separately available for the facilities sector (Brauers, Lepkova 2003). In theory the facilities sector could include the entire management of corporate real estate. This means the effective management, which is called the fifth resource. Indeed, in the report of “the Industrial Development Research Foundation of the United States”, the corporate real estate assets are indicated as a fifth resource, after the resources of people, technology, information and capital (McGregor; Shiem-Shim Then 1999). h An application in facility management, namely on the choice of a contractor for the maintenance of private dwellings tests the definitions of robustness. The problem is multi- objective: like cost of building management, territory cleaning, experience, effectiveness, and size at the side of the contractors; quality of the construction, duration of the work and cost price at the side of the owners. Objectives are determined on basis of the opportunities of the contractors and of the wishes of the customers, here the owners of dwellings. The 15 largest maintenance contractors for private dwellings of Vilnius, capital of Lithua- nia, agreed to fix and estimate their main objectives, namely 9 objectives, as given in Table 1. The full names of the contractors are not provided for the sake of confidentiality9. Table 1. Main attributes and objectives of contractors in Vilnius (2004) No. 3.3. The importance given to an objective The normalized responses of the alternatives on the objectives belong to the interval [0; 1] (see formula 2). Nevertheless, it may turn out to be necessary to stress that some objectives are more important than others. In order to give more importance to an objective its normalized responses on an alternative could be multiplied with a Significance Coefficient: y s x s x j i i i g ij i i g i n ij * * * = − = = = + = ∑ ∑ 1 1 , (6) (6) with: i = 1, 2, …, g as the objectives to be maximized; i = g+1, g+2, …, n as the objectives to be minimized; si= the significance coefficient of objective i; y j a dimensionless number representing the response of alternative j with respect to all objectives with significance coefficients. fi The Attribution of Sub-Objectives represents another solution. Take the example of the purchase of fighter planes (Brauers 2002). For economics, the objectives concerning the fighter planes are threefold: price, employment and balance of payments, but there is also military effectiveness. In order to give more importance to military defense, effectiveness is broken down in, for instance, the maximum speed, the power of the engines and the maximum range of the plane. Anyway, the Attribution Method is more refined than that a significance coefficient method could be as the attribution method succeeds in characterizing an objective better. For instance, for employment two sub-objectives replace a significance coefficient of two and in this way characterize the direct and indirect side of employment. Of course, at that moment the problem raises of the subjective choice of objectives in general, or could we call it robustness of a choice? The Ameliorated Nominal Group Tech- nique will gather all stakeholders interested in the issue to determine the objectives in a non-subjective and anonymous way (see Appendix A). The importance given to an objective results from convergence in the stakeholders’ opinions, which will happen with the assistance in a robust way of the Delphi Technique (see Appendix B). 359 Technological and Economic Development of Economy, 2009, 15(2): 352–375 9 Dr. Tatjana Vilutienė took care of the field work. Previously, she already participated in a similar studies (Zavadskas; Vilutienė 2006, 2004; Vilutienė, Zavadskas 2003). ji y (Zavadskas; Vilutienė 2006, 2004; Vilutienė, Zavadskas 2003). 4. Application of the moora method for evaluating contractor’s alternatives in the facilities sector in Lithuania Attributes Units of measurement Max Min 1 Cost of building management Lt/m2 Min X1 2 Cost of common assets management Lt/m2 Min X2 3 Hvac system maintenance cost (mean) Lt/m2 Min X3 4 Courtyard territory cleaning (in summer) Lt/m2 Min X4 5 Total service cost Lt/m2 Min X5 6 Length of time in maintenance business (experience) Years Max X6 7 Market share for each contractor % Max X7 8 Number of projects per executive Units/person Max X8 9 Evaluation of management cost (cmin/cp) – Max X9 * Lt means Litas, the official currency of Lithuania Table 1. Main attributes and objectives of contractors in Vilnius (2004) 9 Dr. Tatjana Vilutienė took care of the field work. Previously, she already participated in a similar studies (Zavadskas; Vilutienė 2006, 2004; Vilutienė, Zavadskas 2003). 360 W. K. Brauers, E. K. Zavadskas. Robustness of the multi-objective moora method with... The management cost (cmin/cp) evaluates the cost competitiveness of a contractor. Cmin represents the minimal cost in all offerings, cp is the cost offered by the contractor considered. Consequently: The management cost (cmin/cp) evaluates the cost competitiveness of a contractor. Cmin represents the minimal cost in all offerings, cp is the cost offered by the contractor considered. Consequently: cmin/cp ≤ 1. The nature of the construction industry involves that the total number of the minima is mostly larger than the total number of the maxima. Contractor’s rating is performed accord- ing to the attributes (Table 2). The nature of the construction industry involves that the total number of the minima is mostly larger than the total number of the maxima. Contractor’s rating is performed accord- ing to the attributes (Table 2). Table 2. Initial decision making matrix of 15 contractors in Vilnius (2004) Table 2. 10 These additional objectives were: quality standard of management services, quality of maintenance of common property, work organization, the efficiency of information use, certification of company, range of services, reliability of company, company reputation, staff qualification and past experience, communication skills, and geographical market restrictions. 4. Application of the moora method for evaluating contractor’s alternatives in the facilities sector in Lithuania Initial decision making matrix of 15 contractors in Vilnius (2004) Alternatives ↓ x1 x2 x3 x4 x5 x6 x7 x8 x9 a1 0.064 0.11 0.18 0.31 0.67 12 11.75 4.6 0.83 a2 0.06 0.14 0.37 0.12 0.5 3 0.39 0.33 0.885 a3 0.057 0.11 0.18 0.15 0.69 12 5.25 1.47 0.935 a4 0.058 0.12 0.09 0.15 0.57 12 7.09 2.78 0.912 a5 0.058 0.1 0.18 0.2 0.45 12 5.56 1.39 0.912 a6 0.071 0.3 0.18 0.26 0.82 13 26.62 5.67 0.746 a7 0.11 0.14 0.18 0.12 0.55 5 2.82 1.2 0.483 a8 0.058 0.18 0.37 0.19 0.61 11 9.48 3.03 0.916 a9 0.053 0.14 0.16 0.23 0.8 11 2.23 0.76 1 a10 0.071 0.26 0.29 0.23 0.73 11 13.47 9.05 0.746 a11 0.12 0.2 0.09 0.2 0.81 4 4.7 1.5 0.443 a12 0.071 0.28 0.18 0.28 0.73 12 2.35 0.86 0.746 a13 0.078 0.2 0.18 0.3 0.76 8 5.6 3.25 0.681 a14 0.056 0.14 0.18 0.12 0.5 11 2.66 1.7 0.948 a15 0.12 0.14 0.09 0.21 0.56 3 0.04 0.03 0.531 From information of the dwelling owners association, a panel of 30 owners of dwellings chosen at random agreed with these 9 objectives, but they increased the objectives with 11 other ones10. However, these additional objectives were only expressed in qualitative points, showed some overlapping and after their rating represented only 25.9% importance of the total. If these opinions are only taken as indicative, these qualitative objectives can be dropped. Is the owner’s information perhaps useful to allot significance coefficients? Therefore the sample is not enough representative. Indeed, a significance coefficient of importance was not possible to give to the 9 objectives as 30 interviews even chosen at random mean a confidence level of only: 361 cal and Economic Development of Economy, 2009, 15(2): 352–375 Technological and Economic Development of Economy, 2009, 15(2): 352–375 361 Standard error = = = pq n 25 30 0 09 . , which means 9% under or 9% above the real percentage. pq 25 Standard error = = = pq n 25 30 0 09 . , which means 9% under or 9% above the real percentage. pq 25 Standard error = = = pq n 25 30 0 09 . , which means 9% under or 9% above the real percentage. pq 25 : pq n = = 25 100 0 05 . Both parts of MOORA rank more or less in the same way the first 4 positions after the quality of the contractors. In this way a double check is made on the robustness of the results, as shown in Table 4. Contractor 6 P Contractor 10 P Contractor 1 P Contractor 4 (P preferred to Contractor 6 P Contractor 10 P Contractor 1 P Contractor 4 (P preferred to) In other words, 4 contractors are classified in a robust order, whereas contractor A15 is the very last one. The other 10 contractors are ranked low, but it is unclear in what position.if Contractor 6 is ranked first for size and experience and second for effectiveness. Contractor 10 is ranked first for effectiveness and second for size. Contractor 1 together with contractor 4 is second ranked for experience. All these strong contractors are not so good in efficiency (costs), which seems rather unusual. On the other side, the size of the enterprise seems to be very important. In this way the comments that from the beginning no small firms were considered are without any value. 4. Application of the moora method for evaluating contractor’s alternatives in the facilities sector in Lithuania , Economics generally accept 100 interviews with a standard error of: pq n = = 25 100 0 05 . , which means 5% under or 5% above the real percentage (p = expected probability; q = 1 – p; in a symmetric distribution: p = q).ifi Instead of attributing significance coefficients the contractors and the small group of own- ers preferred the attribution of sub-objectives. Indeed, 5 objectives concern minimization of costs (efficiency). Even the last maximization forms a cost consideration. Furthermore, one objective is related to experience, as measured by length of time in maintenance business, one to size as measured by marked share and finally one objective measures effectiveness as expressed by the number of projects per executive. Table 3 presents the results of the calculation process of MOORA. Appendix C gives the details of this calculation. Table 3. Ranking of the 15 contractors by the 2 parts of MOORA Contractors MOORA Square Root Part MOORA Reference Point Part a1 4 3 a2 12 14 a3 6 8 a4 3 5 a5 5 7 a6 1 1 a7 11 10 a8 8 4 a9 9 13 a10 2 2 a11 14 9 a12 13 12 a13 10 6 a14 7 11 a15 15 15 Table 3. Ranking of the 15 contractors by the 2 parts of MOORA Both parts of MOORA rank more or less in the same way the first 4 positions after the quality of the contractors. In this way a double check is made on the robustness of the results, as shown in Table 4. W. K. Brauers, E. K. Zavadskas. Robustness of the multi-objective moora method with... 362 Table 4. No discussion on the ranking of the first four positions of the 15 contractors after the 2 parts of MOORA Contractors MOORA Square Root Part MOORA Reference Point Part A6 1 1 A10 2 2 A1 4 3 A4 3 5 A2, A3, A5; A7-A9; A11-A14 Not very clear Not very clear A15 15 15 According to the results of table 4 we can find the priority between the contractors: Table 4. 4. Application of the moora method for evaluating contractor’s alternatives in the facilities sector in Lithuania No discussion on the ranking of the first four positions of the 15 contractors after the 2 parts of MOORA Contractors MOORA Square Root Part MOORA Reference Point Part A6 1 1 A10 2 2 A1 4 3 A4 3 5 A2, A3, A5; A7-A9; A11-A14 Not very clear Not very clear A15 15 15 According to the results of table 4, we can find the priority between the contractors: Table 4. No discussion on the ranking of the first four positions of the 15 contractors after the 2 parts of MOORA According to the results of table 4, we can find the priority between the contractors: According to the results of table 4, we can find the priority between the contractors: 5. Conclusions The remark that significance of robustness depends on the context is specified in different ways. First, robustness can be considered either as cardinal robust or as a nominal scale. Sec- ond, if robustness is indicated as vague or arbitrary, perhaps it is also the case with inference statistics, probability theory and statistical specification. Third, robustness is characterized by completeness being present in the statistical population covering events and opinions which are present, as well as in the statistical universe with events and opinions not only present but also possible. Concerning the most robust method of multi-objective optimization the following condi- tions are to be satisfied: 1) the method of multiple objectives in which all stakeholders are involved is more robust than one in which only one decision-maker or different decision-makers defending only a limited number of objectives are involved. All stakeholders mean everybody interested in a certain issue. Consequently, the method of multiple objectives which takes into considera- tion consumer sovereignty is more robust than this one which does not respect consumer sovereignty. Consumer sovereignty is measured with community indifference loci. 5. Conclusions Solutions have to deliver points inside the convex zone of the highest community indifference locus; 363 Technological and Economic Development of Economy, 2009, 15(2): 352–375 2) the method of multiple objectives in which all non-correlated objectives are considered is more robust than this one, in which only a limited number of objectives is considered; 2) the method of multiple objectives in which all non-correlated objectives are considered is more robust than this one, in which only a limited number of objectives is considered; 3) the method of multiple objectives in which all interrelations between objectives and alternatives are taken into consideration, at the same time is more robust than this one in which the interrelations are only examined two by two; 4) the method of multiple objectives based on cardinal numbers is more robust than this one based on ordinal numbers.hi 4) the method of multiple objectives based on cardinal numbers is more robust than this one based on ordinal numbers.hi The robustness of cardinality is based first on the saying of Arrow: “obviously, a cardinal utility implies an ordinal preference but not vice versa” and second on the fact that the four essential operations of arithmetic: adding, subtracting, multiplication and division are only reserved for cardinal numbers; 5) the method of multiple objectives which is non-subjective is more robust than this one using subjective estimations. Consequently, a method of multiple objectives which uses non-subjective dimensionless measures is more robust than this one which for normalization uses subjective weights or subjective non-additive scores like in the traditional Reference Point Theory. A creative and prospective thinking of all stakeholders interested in a certain issue has to generate a complete set of objectives. The Ameliorated Nominal Group Technique can assist for that purpose (see Appendix A). Non-subjectivity of the retained objectives results from unanimity or at least a convergence in the opinions of all the stakeholders concerned. Not only the Ameliorated Nominal Group Technique but in addition the Delphi Technique can bring support here. 5. Conclusions Delphi will also assist in giving importance to the objectives (see Appendix B); 6) the method of multiple objectives which uses the last recent available data as a base in the response matrix is more robust than this one based on earlier data;if 6) the method of multiple objectives which uses the last recent available data as a base in the response matrix is more robust than this one based on earlier data;if 7) once the previous 6 conditions are fulfilled the use of 2 different methods of multi- objective optimization is more robust than the use of a single method; the use of 3 methods is more robust than the use of 2, etc. 7) once the previous 6 conditions are fulfilled the use of 2 different methods of multi- objective optimization is more robust than the use of a single method; the use of 3 methods is more robust than the use of 2, etc.hi The Multi-Objective Optimization by Ratio Analysis Method (MOORA) satisfies the 6 conditions with the assistance of the Ameliorated Nominal Group Technique and the Delphi Method. In addition, MOORA satisfies partially the seventh condition by using 2 different methods of Multi-Objective Optimization. MOORA is the most robust method as no other method satisfies the 7 conditions better. The MOORA method, based on dimensionless measures, consists of 2 parts: the aggrega- tion of dimensionless ratios and these ratios used as distances to a reference point. The 2 parts of MOORA control each other. Does the application of MOORA for the contractor’s alterna- tives in the facilities sector of Lithuania respond to the seven conditions of robustness? 1) First condition of robustness 3) Third condition of robustness All interrelations between objectives and alternatives were involved at the same time under the form of a matrix of responses considered as a whole and as a starting point for the application of MOORA. 4) Fourth condition of robustness MOORA is based on cardinal numbers; 5) Fifth condition of robustnessh 5) Fifth condition of robustnessh The use of dimensionless measures is a more robust method than subjective methods of normalization. In the application MOORA’s dimensionless ratios satisfied this condition. 6) Sixth condition of robustnessh The last available data were used. 7) Seventh condition of robustnessi 1) First condition of robustness All stakeholders, on the one side the contractors, on the other side the owners of private dwellings were involved. Indeed, MOORA was applied for the choice between the 15 main contractors of dwellings to satisfy the wishes of the owners of dwellings in the city of Vilnius, capital of Lithuania. Consequently, this condition also respects consumer sovereignty. 364 W. K. Brauers, E. K. Zavadskas. Robustness of the multi-objective moora method with... 2) Second condition of robustness All objectives were taken into consideration, though a part at the side of the owners was ignored as: – the objectives were only qualitative – there was overlapping with the contractor’s objectives – they were only a less significant part of the questioning. 3) Third condition of robustness 1. The Original Nominal Group Technique The nominal group technique consists of a sequence of steps, each of which has been designed to achieve a specific purpose. The nominal group technique consists of a sequence of steps, each of which has been designed to achieve a specific purpose. 1) The steering group or the panel leader carefully phrases as a question the problem to be researched. Much of the success of the technique hinges around a well-phrased question. Otherwise the exercise can easily yield a collection of truisms and obvious statements. A successful question is quite specific and refers to real problems. The question has to have a singular meaning and a quantitative form as much as possible. 2) The steering group or the panel leader explains the technique to the audience. This group of participants is asked to generate and write down ideas about the problem under examination. These ideas too have to have a singular meaning and a quantitative form as much as possible. Participants do not discuss their ideas with each other at this stage. This stage lasts between five and twenty minutes. 3) Each person in round-robin fashion produces one idea from his own list and eventually gives further details. Other rounds are organized until all ideas are recorded. 4) The steering group or the panel leader will discuss with the participants the overlapping of the ideas and the final wording of the ideas. 4) The steering group or the panel leader will discuss with the participants the overlapping of the ideas and the final wording of the ideas. 5) The nominal voting consists of the selection of priorities, rating by each participant separately, while the outcome is the totality of the individual votes. A usual procedure con- sists of the choice by each participant of the n best ideas from his point of view, with the best idea receiving n points and the lowest one point. All the points of the group are added up. A ranking is the democratic result for the whole group. The Original Nominal Group Technique can be characterized as weak robust as the participants expressed too much their personal feeling. For that reason amelioration was proposed. 2. The Ameliorated Nominal Group Technique 7) Seventh condition of robustnessi All the previous 6 conditions are fulfilled and also the 7th condition as 2 different methods of Multi-Objective Optimization are used. No other Multi-Objective Optimization Method exists which uses more than 2 Multi-Objective Optimization Methods and fulfill the previ- ous six conditions. The MOORA method came to the following results: 3 contractors take the first 3 posi- tions. A fourth one has still to be mentioned with its favourable ranking position, whereas one contractor is classified the very last one. The other 10 contractors are ranked low, but it is unclear in what position. The best contractors are not the best in efficiency (costs), which seems rather unusual. On the other side, the size of the enterprise seems to be very impor- tant. In this way the comments that from the beginning no small firms were considered are without any value. Even more, for contractors and their clients the firm quality as measured by size, expe- rience and effectiveness seems to dominate the cost price of maintenance for a dwelling. A new research based on newer data, larger samples and a larger number of quantifiable objectives may verify this unexpected outcome and will rather increase the robustness of the outcome. Is the robustness of the conclusion acceptable: quality chosen before cost price? Prob- ably after the well-known bad quality of the Soviet time, Lithuania was an integral part of the Soviet Union until 1990, the owners of dwellings prefer good quality for an acceptable price. Otherwise, it is possible that robustness of the research was rather weak. Indeed, the research dates from 2004. Data from 2008 would have a better robustness and the period 2004–2008 is even still better. Given that the robustness of the MOORA method itself was effectively proven, a new research based on the latest data, larger samples and a larger number of quantifiable objectives may increase the application robustness. Technological and Economic Development of Economy, 2009, 15(2): 352–375 365 The assistance by the ameliorated nominal group technique With experts representing all stakeholders for a certain issue the relation with robustness seems rather fuzzy, unless an Ameliorated Nominal Group Technique is used. Even better if with the produced information one can deduct complete scenario writing.h The ameliorated approach of the nominal group technique, which is explained here, was ameliorated by Brauers (1987, 2004: 44–64), but the Nominal Group Technique was first elaborated by Van de Ven and Delbecq (1971). 1. The Original Nominal Group Technique E = R x P. E = R x P. Once again, the effectiveness rates of the group are ordered by ranking. One may conclude that the Ameliorated Nominal Group Technique is more robust than the Original Nominal Group Technique. 3. Examples of the Ameliorated Nominal Group Technique 1) Which events will influence the most the economic aspect of the developing countries in the next 10 years to come (1987–1996)? 23 post-graduate students, non-experts, from French speaking Africa participated (Brauers 2004: 49–51). Robustness of this example? Robustness of this example? An Evaluation ex-post showed an astonishing approximation of realization. 2) Which events (maximum 5 per participant) will influence the most the economic aspect of Lithuania in the next 10 years to come (2002–2011)? 18 post-graduate students, non-experts, from Lithuania participated (Brauers, Lepkova 2002). Robustness of this example? Robustness of this example? A partial Evaluation ex-post showed a sufficient result. al Evaluation ex-post showed a sufficient result. A partial Evaluation ex-post showed a sufficient result. 3) Which events (economic, technical, political, social, medical and other events) will influence the most the business outlook of the Facilities Sector (Corporate Real Estate) of Lithuania in the next 10 years to come (2003–2012)? 15 experts as Lithuanian delegates from the facilities sector, the ministerial departments involved and from the academic world participated. In this way an attempt was made to involve all stakeholders (Brauers, Lepkova 2003). 3) Which events (economic, technical, political, social, medical and other events) will influence the most the business outlook of the Facilities Sector (Corporate Real Estate) of Lithuania in the next 10 years to come (2003–2012)? 15 experts as Lithuanian delegates from the facilities sector, the ministerial departments involved and from the academic world participated. In this way an attempt was made to involve all stakeholders (Brauers, Lepkova 2003). 22 events were recorded. Each participant could select 5 events with as points: 5 + 4 + 3 + 2 + 1 = 15. With 15 participants assisting the total arrived at 225 and, as all participants used their privileges, the total of 225 was maintained. The usefulness of the introduction of median probabilities was demonstrated by the fact that the total of 225 was reduced to 145.21. Robustness of this example? A partial evaluation ex-post showed a sufficient result. A partial evaluation ex-post showed a sufficient result. 2. The Ameliorated Nominal Group Technique As there was too much wishful thinking even between experts, better results were obtained if the group was also questioned about the probability of occurrence of the event. In this way the experts became more critical even about their own ideas. The probability of the group is found as the median of the individual probabilities. 366 W. K. Brauers, E. K. Zavadskas. Robustness of the multi-objective moora method with... Finally, the group rating (R) is multiplied with the group probability (P) in order to obtain the effectiveness rate of the event (E): E = R x P. The results of this nominal group technique inspired the panel leader to deduce 3 different scenarios for the future of the facilities sector in Lithuania: the fifth resource scenario, the status quo scenario and the cut throat competitive scenario (Brauers, Lepkova 2003: 7). The fifth resource scenario Foreign direct investments will come to Lithuania. They may find industrial zones with ready premises. However, they want more and they like to decide themselves on location. This is the moment that the fifth resource beside the resources of people, technology, information and capital, comes fully alive, namely the management of the corporate real estate assets (McGregor et al. 1999). New companies will respond to this demand. These new companies of real estate assets management will look after space (location), design of buildings, construc- Technological and Economic Development of Economy, 2009, 15(2): 352–375 367 tion, reparations, maintenance, waste management and eventually demolition. They will look after the direct investments, so to say “from the cradle until the grave”. This has to happen in the most effective way, i.e., with an optimal multiple objective utility. In this way, the foreign firms can look for an optimal multiple objective utility for people, technology, information and capital, but also for their corporate real assets. Synergy effects will play fully. The status quo scenario h In this scenario the situation in the facilities sector does not change. A set of small firms will remain operative in the facilities sector of Lithuania. Some small firms will disappear by competition, aging, disagreement, etc., but new ones will take their places. The cut-throat competitive scenario In the cut-throat competitive scenario the consequences of the productivity effect of the European Union will fully play. The productivity in the new member countries of the European Union will rise in the internationally traded sectors. The result is an increase in wages. However, after the “Balassa-Samuelson Effect” the more national services have to raise their wages too, without an increase in productivity of the same size. This increase in wages will have an inflationary effect in the country (Balassa 1964; Balazs et al. 2002; Samuelson 1994, 1964). The facilities sector in Lithuania will fully undergo this influence. It will remain only a nationally traded sector. If it increases its prices, together with the other not internationally traded services, inflation will go up in Lithuania and ipso facto the cost of living will rise. E = R x P. l Instead of increase in prices, diminution of quality of its services forms another alterna- tive for the facilities sector. At that moment cut-throat competition between the facilities management companies will occur. In this struggle for life only the fittest will survive. It is also the moment that mala fide companies will appear, which will exploit the customers as much as possible. Robustness of these scenarios? Robustness of these scenarios is rather weak; the set of scenarios is not complete. Perhaps still many other scenarios are possible. Appendix B The assistance by the Delphi technique The assistance by the Delphi technique In questionnaires, referenda and Delphi the face-to-face dialogue is absent in order to decrease subjective influence. Questionnaires fail if broad issues are involved. Indeed the steering group may influence the opinions by its phrasing of the questions. In addition, the steering group may make a too subjective summary when analyzing the questionnaires. With question- naires, it is difficult to reach consensus. Opinions can be too divergent for a consensus to be reached, which is certainly the case with broad problems. Could the mean average be useful for this purpose? Mueller et al. (1970: 140) remark that: “since it reflects every value in the array, it will be affected by the extremely high or low values that are always found in a skewed distribution and therefore it will lose its typicality and perhaps mislead the reader”. At that moment manipulation is possible. Suppose, for instance, in a jury a jury member can influence the voting by giving very high points to his protégé (skewed to the right) and W. K. Brauers, E. K. Zavadskas. Robustness of the multi-objective moora method with... 368 very low points to the other candidates (skewed to the left). These excesses can be left out too, but in a subjective way. Who can judge what is excessive and what not? Here the median is helpful. The median is defined as the middle measurement after the measurements have been arranged in order of magnitude. To measure skewness beside the median one could find two other values, one dividing the histogram at a point such that one fourth of the area to the left of it (first quartile) and the other such that one fourth of the area is to the right of it (third quartile). In fact the median itself is the second quartile. In this way one may speak of skewness to the left and skewness to the right. A task could be to find ways and means to decrease skewness by trying to bring the quartiles nearer to each other. This point is taken up later. Referenda, as a direct vote of the population, may fail too, if broad issues are involved. Not only they will show the same disadvantages as the questionnaires, but they can also create opposition from a large part (eventually until 49.99%) of the population against the outcome. The assistance by the Delphi technique All depends if the referendum is simply indicative or really directive, like in direct democracy. In indirect democracy, democracy through representatives, majority voting would be the most robust voting rule (Dasgupta and Maskin, 2008).h Delphi tries to improve either the committee or the questionnaire approach. The Delphi method is a method for obtaining and processing judgmental data. It consists of a sequenced program of interrogation (in session or by mail) interspersed with feedback of persons in- terested in the issue, while everything is conducted through a steering group. We advocate most this method as it also takes care of: – Quantitative treatment – Expert knowledge – Anonymity – Convergence. Dalkey and Helmer (1963) used Delphi in its present form for the first time around 1953. The essential features of it are: 1. A group of especially knowledgeable individuals (experts) 2. Inputs with a singular meaning and quantitative as much as possibleh 3. The opinions about the inputs are evaluated with statistical indexes 4. Feedback of the statistical indexes with request for re-estimation, also after considera- tion of reasons for extreme positions 5. The sources of each input are treated anonymouslyh 6. Two developments: meeting and questionnaires. The organization of a meeting produces quicker results. However, the meeting has to be organized in such a way that commu- nication between the panel members is impossible. Therefore, a central computer with desk terminals, television screen and computer controlled feedback is advisable.i As an example of Delphi, a music competition ended with 12 finalists (Brauers 2008a). Beside the personal preferences of the jury members, different music schools or tendencies exist. Total points and the medians were the same for the first four candidates but for the 5th and the 6th ranks, the laureates were reversed. However, the large diversion between the first and the third quartiles illustrated a possible frustration between the jury members for the laureates ranking 5 and 6 and the other finalists ranking 7, 8, 9, 10, 11 and 12. At that moment As an example of Delphi, a music competition ended with 12 finalists (Brauers 2008a). Beside the personal preferences of the jury members, different music schools or tendencies exist. Total points and the medians were the same for the first four candidates but for the 5th and the 6th ranks, the laureates were reversed. The assistance by the Delphi technique However, the large diversion between the first and the third quartiles illustrated a possible frustration between the jury members for the laureates ranking 5 and 6 and the other finalists ranking 7, 8, 9, 10, 11 and 12. At that moment 369 Technological and Economic Development of Economy, 2009, 15(2): 352–375 Delphi interferes. The voting is repeated several times. In the beginning skewness is still too large, but then a new round may help. Delphi experiences a better convergence in opinion as the medians and quartiles approach more and more to one another in different rounds until convergence as much as possible is reached and automatically robustness is increased. At that moment, the ranking of the finalists in the positions 5 till 12 may be entirely reversed, but the members of the jury, like the public and the press, will be more satisfied. Delphi interferes. The voting is repeated several times. In the beginning skewness is still too large, but then a new round may help. Delphi experiences a better convergence in opinion as the medians and quartiles approach more and more to one another in different rounds until convergence as much as possible is reached and automatically robustness is increased. At that moment, the ranking of the finalists in the positions 5 till 12 may be entirely reversed, but the members of the jury, like the public and the press, will be more satisfied. Appendix C MOORA: square root method (1a until 1c) and MOORA reference point theory (1d- MOORA: square root method (1a until 1c) and MOORA reference point theory (1d-1e) Table 1. MOORA applied to 9 objectives for Lithuanian contractors 1a. Matrix of responses of alternatives on objectives: (xij) 1 2 3 4 5 6 7 8 9 min. min. min. min. min. max. max. max. max. a1 0.064 0.11 0.18 0.31 0.67 12 11.75 4.6 0.83 a2 0.06 0.14 0.37 0.12 0.5 3 0.39 0.33 0.885 a3 0.057 0.11 0.18 0.15 0.69 12 5.25 1.47 0.935 a4 0.06 0.12 0.09 0.15 0.57 12 7.09 2.78 0.912 a5 0.058 0.1 0.18 0.2 0.45 12 5.56 1.39 0.912 a6 0.071 0.3 0.18 0.26 0.82 13 26.62 5.67 0.746 a7 0.11 0.14 0.18 0.12 0.55 5 2.82 1.2 0.483 a8 0.058 0.18 0.37 0.19 0.61 11 9.48 3.03 0.916 a9 0.053 0.14 0.16 0.23 0.8 11 2.23 0.76 1 a10 0.07 0.26 0.29 0.23 0.73 11 13.47 9.05 0.746 a11 0.12 0.2 0.09 0.2 0.81 4 4.7 1.5 0.443 a12 0.071 0.28 0.18 0.28 0.73 12 2.35 0.86 0.746 a13 0.078 0.2 0.18 0.3 0.76 8 5.6 3.25 0.681 a14 0.056 0.14 0.18 0.12 0.5 11 2.66 1.7 0.948 a15 0.12 0.14 0.09 0.21 0.56 3 0.04 0.03 0.531 1b. Sum of squares an d their square roots a1 0.004096 0.0121 0.0324 0.0961 0.4489 144 138.0625 21.16 0.6889 a2 0.0036 0.0196 0.1369 0.0144 0.25 9 0.1521 0.1089 0.783225 a3 0.003249 0.0121 0.0324 0.0225 0.4761 144 27.5625 2.1609 0.874225 a4 0.0036 0.0144 0.01 0.0225 0.3249 144 50.41 7.7284 0.81 a5 0.003364 0.01 0.0324 0.04 0.2025 144 30.9136 1.9321 0.81 a6 0.005041 0.09 0.0324 0.0676 0.6724 169 708.6244 32.1489 0.556516 a7 0.0121 0.0196 0.0324 0.0144 0.3025 25 7.9524 1.44 0.233289 a8 0.003364 0.0324 0.1369 0.0361 0.3721 121 89.8704 9.1809 0.839056 a9 0.002809 0.0196 0.0256 0.0529 0.64 121 4.9729 0.64 1 a10 0.0049 0.0676 0.0841 0.04 0.49 121 182.25 81.9025 0.5625 a11 0.0144 0.04 0.0081 0.04 0.6561 16 22.09 2.25 0.196249 a12 0.005041 0.0784 0.0324 0.0784 0.5329 144 5.5225 0.7396 0.556516 a13 0.006084 0.04 0.0324 0.09 0.5776 64 31.36 10.5625 0.463761 a14 0.003136 0.0196 0.0324 0.0144 0.25 121 7.0756 2.89 0.898704 a15 0.0144 0.0196 0.0081 0.0441 0.3136 9 0.0016 0.0009 0.281961 sum 0.089184 0.495000 0.66890 0.6734 6.5096 1496 1306.8205 174.8456 9.554902 root 0.2986369 0.70356236 0.81786307 0.82060953 2.55139178 38.6781592 36.1499723 13.2229195 3.09110045 Table 1. Appendix C MOORA applied to 9 objectives for Lithuanian contractors 1a. Matrix of responses of alternatives on objectives: (xij) Table 1. MOORA applied to 9 objectives for Lithuanian contractors 1a. Matrix of responses of alternatives on objectives: (xij) 1 2 3 4 5 6 7 8 9 min. min. min. min. min. max. max. max. max. a1 0.064 0.11 0.18 0.31 0.67 12 11.75 4.6 0.83 a2 0.06 0.14 0.37 0.12 0.5 3 0.39 0.33 0.885 a3 0.057 0.11 0.18 0.15 0.69 12 5.25 1.47 0.935 a4 0.06 0.12 0.09 0.15 0.57 12 7.09 2.78 0.912 a5 0.058 0.1 0.18 0.2 0.45 12 5.56 1.39 0.912 a6 0.071 0.3 0.18 0.26 0.82 13 26.62 5.67 0.746 a7 0.11 0.14 0.18 0.12 0.55 5 2.82 1.2 0.483 a8 0.058 0.18 0.37 0.19 0.61 11 9.48 3.03 0.916 a9 0.053 0.14 0.16 0.23 0.8 11 2.23 0.76 1 a10 0.07 0.26 0.29 0.23 0.73 11 13.47 9.05 0.746 a11 0.12 0.2 0.09 0.2 0.81 4 4.7 1.5 0.443 a12 0.071 0.28 0.18 0.28 0.73 12 2.35 0.86 0.746 a13 0.078 0.2 0.18 0.3 0.76 8 5.6 3.25 0.681 a14 0.056 0.14 0.18 0.12 0.5 11 2.66 1.7 0.948 a15 0.12 0.14 0.09 0.21 0.56 3 0.04 0.03 0.531 1b. Sum of squares an d their square roots 1b. Sum of squares an d their square roots a1 0.004096 0.0121 0.0324 0.0961 0.4489 144 138.0625 21.16 0.6889 a2 0.0036 0.0196 0.1369 0.0144 0.25 9 0.1521 0.1089 0.783225 a3 0.003249 0.0121 0.0324 0.0225 0.4761 144 27.5625 2.1609 0.874225 a4 0.0036 0.0144 0.01 0.0225 0.3249 144 50.41 7.7284 0.81 a5 0.003364 0.01 0.0324 0.04 0.2025 144 30.9136 1.9321 0.81 a6 0.005041 0.09 0.0324 0.0676 0.6724 169 708.6244 32.1489 0.556516 a7 0.0121 0.0196 0.0324 0.0144 0.3025 25 7.9524 1.44 0.233289 a8 0.003364 0.0324 0.1369 0.0361 0.3721 121 89.8704 9.1809 0.839056 a9 0.002809 0.0196 0.0256 0.0529 0.64 121 4.9729 0.64 1 a10 0.0049 0.0676 0.0841 0.04 0.49 121 182.25 81.9025 0.5625 a11 0.0144 0.04 0.0081 0.04 0.6561 16 22.09 2.25 0.196249 a12 0.005041 0.0784 0.0324 0.0784 0.5329 144 5.5225 0.7396 0.556516 a13 0.006084 0.04 0.0324 0.09 0.5776 64 31.36 10.5625 0.463761 a14 0.003136 0.0196 0.0324 0.0144 0.25 121 7.0756 2.89 0.898704 a15 0.0144 0.0196 0.0081 0.0441 0.3136 9 0.0016 0.0009 0.281961 sum 0.089184 0.495000 0.66890 0.6734 6.5096 1496 1306.8205 174.8456 9.554902 root 0.2986369 0.70356236 0.81786307 0.82060953 2.55139178 38.6781592 36.1499723 13.2229195 3.09110045 W. K. Brauers, E. K. Zavadskas. Appendix C Robustness of the multi-objective moora method with. 370 1c. Objectives divided by their square roots and MOORA (1) 1 2 3 4 5 6 7 8 9 Sum (2) 1.9335294 (3) Rank a1 0.21430707 0.15634719 0.22008574 0.37776797 0.26260177 0.31025261 0.325 0.348 0.269 0.0205713 1.9541007 4 a2 0.20091288 0.19898734 0.45239847 0.14623276 0.19597147 0.07756315 0.01078839 0.02495667 0.2863058 –0.7948889 1.1386405 12 a3 0.19086724 0.15634719 0.22008574 0.18279096 0.27044063 0.31025261 0.14522833 0.11117061 0.30248127 –0.1513989 1.7821305 6 a4 0.20091288 0.17056057 0.12226986 0.18279096 0.22340748 0.31025261 0.19640402 0.21024101 0.29115844 0.1081144 2.0416438 3 a5 0.19421578 0.14213381 0.22008574 0.24372127 0.17637432 0.31025261 0.15380371 0.10512051 0.29115844 –0.1161957 1.8173337 5 a6 0.23774691 0.42640143 0.22008574 0.31683766 0.32139321 0.336107 0.73637677 0.42880092 0.241338 0.2201577 2.1536871 1 a7 0.36834028 0.19898734 0.22008574 0.14623276 0.21556862 0.12927192 0.07800836 0.09075152 0.15625503 –0.6949279 1.2386015 11 a8 0.19421578 0.25584086 0.45239847 0.23153521 0.23908519 0.28439823 0.26224086 0.22914758 0.29633459 –0.3009542 1.6325752 8 a9 0.17747304 0.19898734 0.19563177 0.28027947 0.31355435 0.28439823 0.06168746 0.06050101 0.32350938 –0.4358299 1.4976995 9 a10 0.23439836 0.36954791 0.35458258 0.24372127 0.27436006 0.28439823 0.37344427 0.68441769 0.24263204 0.1082820 2.0418114 2 a11 0.40182576 0.28426762 0.1100429 0.2437213 0.3174738 0.1034175 0.1300139 0.1134394 0.1433147 –0.8671458 1.0663836 14 a12 0.23774691 0.39797467 0.2200857 0.3412098 0.2861183 0.3102526 0.0650070 0.0650386 0.2413380 –0.8014993 1.1320301 13 a13 0.26118674 0.28426762 0.2200857 0.3655819 0.2978766 0.2068351 0.1549102 0.2457854 0.2203099 –0.6011581 1.3323713 10 a14 0.18751869 0.19898734 0.2200857 0.1462328 0.1959715 0.2843982 0.0735824 0.1285646 0.3066869 –0.1555639 1.7779655 7 a15 0.40182576 0.19898734 0.1100429 0.2559073 0.2194880 0.0775632 0.0011065 0.0022688 0.1717835 –0.9335294 1.0000000 15 (1) The nature of the construction industry involves that the total number of the minima is mostly larger than the total number of the maxima with very often negative sums as a result, which is the case here. Therefore, to make the ranking more comprehensive, the supplement to make the smallest total, here a15, equal to one, is added to every total. (2) Due to the many minima the majority of the alternatives shows a negative total. On the contrary, alternatives A6, A10, A4 and A1 finally classified in good order, show positive totals. (3) Supplement to make all totals positive and the total for A15 equal to one. Technological and Economic Development of Economy, 2009, 15(2): 352–375 371 1d. Reference point theory with ratios: co-ordinates of the reference point equal to the maximal objective values 1 2 3 4 5 6 7 8 9 ri 0.1775 0.1421 0.1100 0.1462 0.1764 0.336107 0.73637677 0.68441769 0.32350938 1e. References Admati , A. R.; Pfleiderer, P. 1994. Robust financial contracting and the role of venture capitalists, Journal of Finance 49(2): 371–402. Arrow, K. J. 1974. General economic equilibrium: purpose, analytic techniques, collective choice, American Economic Review, June: 253–272. Balassa, B. 1964. The purchasing power parity doctrine: a reappraisal, Journal of Political Economy, 584–596. Balazs, E.; Drine, I.; Lommatzsch, K.; Rault, C. 2002. The Balassa-Samuelson Effect and Central and Eastern Europe: Myth or Reality? Working Paper 483, the William Davidson Institute, University of Michigan Business School. Brauers, W. K. 2008a. Group decision making with multi-objective optimization, Foundations of Comput- ing and Decision Science 33(2): 167–179. Poznan University of Technology. Brauers, W. K. 2008b. Multi-objective decision making by reference point theory for a wellbeing economy, Operations Research International Journal 8: 89–104. Brauers, W. K. 2007.What is meant by normalization in decision making? In W. K. Brauers, E. K. Zavadskas (Ed.). Special Issue: Normalization in Decision-Making Methods, International Journal of Management and Decision Making 8(5/6): 441–460. Genève, Inderscience Publishers. Brauers, W. K. 2004. Optimization Methods for a Stakeholder Society, a Revolution in Economic Think- ing by Multi-Objective Optimization: Nonconvex Optimization and its Applications. Boston, U.S.A.: Kluwer Academic Publishers. Brauers, W. K. 2002. The multiplicative representation for multiple objective optimization with an ap- plication for arms procurement, Naval Research Logistics 49: 327–340. Wiley. Brauers, W. K. 1987. Nominal Methods in Group Multiple Decision Making. Research Paper N°3, Institute For Developing Countries, University of Antwerp, Antwerpen, Be, Ruca. Brauers, W. K.; Zavadskas, E. K. 2009. Is Robustness Really Robust? Robustness from the Point of View of Statistics and Econometrics, in C. Zopounides (Ed.). Multi Criteria Decision Aid, Series “Advances in Operations Research”, Nova Publishers, Hauppauge, New York (in press). Brauers, W. K.; Zavadskas, E. K. 2006. The MOORA Method and its application to privatization in a transition economy, Control and Cybernetics 35(2): 443–468. Brauers, W. K.; Lepkova, N. 2003.The application of the nominal group technique to the business out- look of the facilities sector of Lithuania over the period 2003–2012, International Journal of Strategic Property Management 7(1): 1–9. Brauers, W. K.; Lepkova, N. 2002.The application of the nominal group technique to the economic outlook of Lithuania over the period 2002–2011, Technological and Economic Development of Economy [Ūkio technologinis ir ekonominis vystymas] 8(1): 19–24. Casella, G.; Berger, R. L. 2002. Statistical Inference, second edition. Pacific Grove, CA, US, Duxbury, Thomson Learning. Churchman, C. W.; Ackoff, R. Appendix C Robustness of the multi-objective moora method with. 372 Appendix C Reference point theory: deviations from the reference point 1 2 3 4 5 6 7 8 9 Max. Rank Min. a1 0.0368 0.0142 0.1100 0.2315 0.0862 0.02585438 0.41134195 0.33653687 0.0550 0.4113 3 a2 0.0234 0.0569 0.3424 0.0000 0.0196 0.25854384 0.72558838 0.65946102 0.0372 0.7256 14 a3 0.0134 0.0142 0.1100 0.0366 0.0941 0.02585438 0.59114845 0.57324708 0.0210 0.5911484 8 a4 0.023 0.028 0.012 0.037 0.047 0.02585438 0.53997275 0.47417667 0.03235094 0.5399728 5 a5 0.0167 0.0000 0.1100 0.0975 0.0000 0.02585438 0.58257306 0.57929718 0.03235094 0.5825731 7 a6 0.0603 0.2843 0.1100 0.1706 0.1450 0 0 0.25561677 0.08217138 0.2842676 1 a7 0.1909 0.0569 0.1100 0 0.0392 0.20683508 0.65836842 0.59366617 0.16725435 0.6583684 10 a8 0.017 0.114 0.342 0.085 0.063 0.05170877 0.47413591 0.45527011 0.02717479 0.4741359 4 a9 0.0000 0.0569 0.0856 0.1340 0.1372 0.05170877 0.67468931 0.62391668 0 0.6746893 13 a10 0.0569 0.2274 0.2445 0.0975 0.0980 0.05170877 0.3629325 0 0.08087735 0.3629325 2 a11 0.2244 0.1421 0.0000 0.0975 0.1411 0.2326895 0.6063628 0.5709783 0.1801947 0.6063628 9 a12 0.0603 0.2558 0.1100 0.1950 0.1097 0.0258544 0.6713698 0.6193791 0.0821714 0.6713698 12 a13 0.0837 0.1421 0.1100 0.2193 0.1215 0.1292719 0.5814666 0.4386323 0.1031995 0.5814666 6 a14 0.0100 0.0569 0.1100 0.0000 0.0196 0.0517088 0.6627944 0.5558530 0.0168225 0.6627944 11 a15 0.2244 0.0569 0.0000 0.1097 0.0431 0.2585438 0.7352703 0.6821489 0.1517259 0.7352703 15 1 2 3 4 5 6 7 8 9 Max. Rank Min. a1 0.0368 0.0142 0.1100 0.2315 0.0862 0.02585438 0.41134195 0.33653687 0.0550 0.4113 3 a2 0.0234 0.0569 0.3424 0.0000 0.0196 0.25854384 0.72558838 0.65946102 0.0372 0.7256 14 a3 0.0134 0.0142 0.1100 0.0366 0.0941 0.02585438 0.59114845 0.57324708 0.0210 0.5911484 8 a4 0.023 0.028 0.012 0.037 0.047 0.02585438 0.53997275 0.47417667 0.03235094 0.5399728 5 a5 0.0167 0.0000 0.1100 0.0975 0.0000 0.02585438 0.58257306 0.57929718 0.03235094 0.5825731 7 a6 0.0603 0.2843 0.1100 0.1706 0.1450 0 0 0.25561677 0.08217138 0.2842676 1 a7 0.1909 0.0569 0.1100 0 0.0392 0.20683508 0.65836842 0.59366617 0.16725435 0.6583684 10 a8 0.017 0.114 0.342 0.085 0.063 0.05170877 0.47413591 0.45527011 0.02717479 0.4741359 4 a9 0.0000 0.0569 0.0856 0.1340 0.1372 0.05170877 0.67468931 0.62391668 0 0.6746893 13 a10 0.0569 0.2274 0.2445 0.0975 0.0980 0.05170877 0.3629325 0 0.08087735 0.3629325 2 a11 0.2244 0.1421 0.0000 0.0975 0.1411 0.2326895 0.6063628 0.5709783 0.1801947 0.6063628 9 a12 0.0603 0.2558 0.1100 0.1950 0.1097 0.0258544 0.6713698 0.6193791 0.0821714 0.6713698 12 a13 0.0837 0.1421 0.1100 0.2193 0.1215 0.1292719 0.5814666 0.4386323 0.1031995 0.5814666 6 a14 0.0100 0.0569 0.1100 0.0000 0.0196 0.0517088 0.6627944 0.5558530 0.0168225 0.6627944 11 a15 0.2244 0.0569 0.0000 0.1097 0.0431 0.2585438 0.7352703 0.6821489 0.1517259 0.7352703 15 W. 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Zavadskas Santrauka Patikimumo apibrėžimas ekonometrikoje, kaip neteisingas terminas tiesinėje lygtyje, buvo ne tik papil- dytas, bet ir išreikštas įprasta kalba: nuo kiekybinio prie kokybinio. Šios interpretacijos buvo patikrintos taikant jas Lietuvos paslaugų sektoriuje. Taikymas yra daugiatikslis: iš rangovo pusės kaip išlaidos, patirtis, efektyvumas; kokybė, darbo trukmė, kaina iš užsakovo pusės. Minėtieji tikslai turi skirtingus matavimo vienetus. O jų santykiniai dydžiai neturi mato vienetų, todėl taikant MOORA metodą yra išvengiama sunkumų juos normalizuojant. Pirmoje MOORA metodo taikymo dalyje šie santykiai yra sujungiami, o antroje dalyje ieškoma atstumo iki geriausio sprendinio. Abiejų metodo dalių rezultatai pagrindžia sprendinio teisingumą. Tai rodo aiškų MOORA metodo pranašumą, palyginti su kitais daugiatikslio optimizavimo metodais. Taikant abi MOORA metodo dalis Lietuvos paslaugų sektoriui buvo sudarytas lyginamasis rangavimas, buvo atliktas dvigubas rezultatų patikimumo patikrinimas. Reikšminiai žodžiai: MOORA, patikimumas, daugiatiksliai metodai, patobulintas naminalių grupių metodas, Delphi. Willem K. Brauers was graduated as Ph.D. in economics (Un. of Leuven), Master of Arts (in economics) of Columbia Un. (New York), Master in Management and Financial Sciences, in Political and Diplomatic Sciences and Bachelor in Philosophy (Un. of Leuven). He is professor at the Faculty of Applied Economics and at the Institute for Development Policy and Management of the University of Antwerp. Previously, he was professor at the University of Leuven, the Belgian War College, the School of Military Admin- istrators, and the Antwerp Business School. He was a research fellow in several American institutions like Rand Corporation, the Pentagon, the Institute for the Future, the Futures Group and extraordinary 375 Technological and Economic Development of Economy, 2009, 15(2): 352–375 advisor to the Center for Economic Studies of the University of Leuven. He was consultant in the public sector, such as the Belgian Department of National Defense, the Department of Industry in Thailand, the project for the construction of a new port in Algeria (the port of Arzew) and in the private sector such as the international seaport of Antwerp and in electrical works. He was Chairman of the Board of Directors of SORCA Ltd. Brussels, Management Consultants for Developing Countries, linked to the world-wide group of ARCADIS. At the moment he is Chairman of the Board of Directors of MARESCO Ltd. Antwerp, Marketing Consultants and General Manager of CONSULTING, Systems Engineering Consultants. Brauers is member of many international scientific organizations. Santrauka His specialization covers: Optimizing Techniques with Several Objectives, Forecasting Techniques, Public Sector Economics such as for National Defense and for Regional Sub-optimization and Input-Output Techniques. His scientific publications consist of twelve books and hundreds of articles and reports. Edmundas Kazimieras Zavadskas. Academic Experience: Dr. 1973, Lecturer (1973–1974), Senior Lec- turer (1974–1978), Associate Professor (1978–1985), Senior Researcher (1985–1987), Dr. Sc. 1987, Pro- fessor (1988–), Head of Construction Technology Department (1987–1990), Rector of Vilnius Technical University (1990–1996), Rector of Vilnius Gediminas Technical University (VGTU) (1996–2002). First Vice Rector of VGTU and Head of Department of Construction Technology and Management (2002–) E. K. Zavadskas is participating in two Framework 5 programmes and two Framework 6. The spheres of his current research are internet based and e-business systems (property, construction and export), decision-making theory, decision support systems, etc. E. K. Zavadskas is the author of 527 research publications and 14 monographs.
https://openalex.org/W2565915370	http://old.scielo.br/pdf/abmvz/v68n6/0102-0935-abmvz-68-06-01465.pdf	Portuguese	null	Proteinograma sérico de éguas com placentite ascendente e seus respectivos neonatos: dados preliminares	Arquivo Brasileiro de Medicina Veterinária e Zootecnia/Arquivo brasileiro de medicina veterinária e zootecnia	2,016	cc-by	3,254	Recebido em 15 de outubro de 2015 Aceito em 29 de março de 2016 Autor para correspondência (corresponding author) E-mail: lucianaalins@yahoo.com.br C. Haetinger1, B.R. Curcio2, L.A. Lins2, V. Müler3, L.S. Feijó1, F.M. Pazinato1, L.A. Amaral1, C.E.W. Nogueira2 1Aluna de pós-graduação  Faculdade de Veterinária ˗ UFPel ˗ Pelotas, RS 2Faculdade de Veterinária ˗ UFPel ˗ Pelotas, RS 3 3Aluna de graduação  Faculdade de Veterinária ˗ UFPel ˗ Pelotas, RS RESUMO Este estudo teve por objetivo estabelecer o proteinograma sérico em éguas com placentite induzida e em seus respectivos neonatos. Foram coletadas amostras de sangue das éguas em oito momentos diferentes e dos potros em quatro momentos. Para obtenção da concentração das frações proteicas, utilizou-se eletroforese em gel de acrilaminada contendo dodecil sulfato de sódio (SDS-PAGE). No método utilizado, foram observadas 23 bandas proteicas, cujos pesos moleculares variaram de 16KDa a 245KDa, sendo possível a identificação das seguintes frações: 175KDa, 102KDa, 83KDa, 63KDa, 50KDa, 41KDa, 39KDa e 28KDa. De todas as bandas proteicas encontradas, somente as de 39KDa e 41KDa apresentaram alteração na cinética nos momentos avaliados. De acordo com a solução marcadora, pode-se sugerir que essas proteínas seriam α1-glicoproteína ácida (39KDa) e haptoglobina (41KDa). A concentração de imunoglobulinas nos potros apresentou aumento significativo a partir das 12 horas de nascimento. Não está elucidado se estes níveis refletem a persistência do processo inflamatório placentário ou se são alterações fisiológicas do periparto. Não foram observadas alterações na cinética das proteínas nos potros nas primeiras 48 horas. Palavras-chave: haptoglobina, α1-glicoproteína ácida, obstetrícia equina, proteínas de fase aguda Serum protein concentrationin mares with ascendingplacentitis and their newborns: preliminary data] C. Haetinger1, B.R. Curcio2, L.A. Lins2*, V. Müler3, L.S. Feijó1, F.M. Pazinato1, L.A. Amaral1, C.E.W. Nogueira2 http://dx.doi.org/10.1590/1678-4162-8818 http://dx.doi.org/10.1590/1678-4162-8818 Arq. Bras. Med. Vet. Zootec., v.68, n.6, p.1465-1469, 2016 INTRODUÇÃO A placentite ascendente na égua é uma das principais causas de parto prematuro, aborto e nascimento de potros comprometidos (LeBlanc, 2010), sendo o Streptococcus equi, subespécie zooepidemicus, o principal agente causador (Giles et al., 1993). A pesquisa para se encontrarem marcadores precoces da inflamação vem sendo foco nas medicinas humana e veterinária ao longo dos anos, utilizando-se principalmente a identificação bioquímica das proteínas de fase aguda (PFA) como marcador do grau da inflamação (Crisman et al., 2008). As PFAs são proteínas sanguíneas que alteram sua concentração em processos inflamatórios, infecciosos ou estresse (Murata et al., 2004). O emprego da técnica SDS-PAGE em medicina veterinária pode ser útil na avaliação da cinética das proteínas de fase aguda de resposta inflamatória, facilitando a definição do diagnóstico das doenças animais (Fagliari et al., 2003). O objetivo deste estudo é estabelecer o proteinograma sérico em éguas com placentite induzida e em seus respectivos neonatos. O estudo foi realizado com o plantel de éguas do Centro de Ensino e Experimentação em Equinocultura da Palma (CEEP), da Universidade Federal de Pelotas, no município de Capão do Leão, Rio Grande do Sul (31°48'08.2"S; 52°29'51.4"O), durante a temporada reprodutiva 2012-2013. Para o modelo experimental, foram utilizadas oito éguas mestiças, com média de 11±2 anos e média de peso de 437±22kg, e seus respectivos neonatos. Este estudo foi aprovado pelo Comitê de Ética e Experimentação Animal da Universidade Federal de Pelotas, sob nº 3072/2013. A assistência imediata ao neonato constou de limpeza das vias aéreas e ventilação com auxílio de ambu, além de oferta de colostro até uma hora apos o parto. Na ausência ou no retardo do reflexo de sucção, o colostro era fornecido via sonda nasogástrica. Os neonatos foram tratados com ampicilina (Cilinon®, ampicilina sódica, Ariston, Brasil), na dose 22mg/kg e intervalo de seis horas, e flunixinmeglumine (Desflan®, flunixinmeglumine, Ouro Fino Agronegócio, Brasil), na dose de 1,1mg/kg, a cada oito horas. As éguas foram submetidas à indução de placentite ascendente, por meio da infusão intracervical de Streptococcus equi, subespécie zooepidemicus, na concentração de 107UFC, entre os dias 290-300 de gestação, conforme protocolo descrito por Bailey et al. (2010). Todos os animais foram submetidos à coleta de sangue e o soro foi separado e armazenado a - 20oC. Haetinger et al. secreção vaginal mucopurulenta, sendo coletada para avaliação microbiológica e citológica, quando foi identificado crescimento de S. equizooepidemicus e presença de células inflamatórias por citologia, determinando o diagnóstico clínico de placentite. As éguas foram tratadas com sulfametoxazol e trimetoprim (Trissulfin®, sulfametoxazol e trimetoprim, Ouro Fino Agronegócio, Brasil), na dose de 30mg/kg, a cada 12 horas, além de flunixinmeglumine (Desflan®, flunixinmeglumine, Ouro Fino Agronegócio, Brasil), na dose 1,1mg/kg, a cada 24 horas, sendo iniciado a partir de 48 horas de indução. Todos os partos foram assistidos para avaliar a necessidade de intervenção. Após a expulsão da placenta, foram coletados fragmentos de sete pontos desta, conforme o método de Schlafer (2004), os quais foram encaminhados para avaliação histopatológica. Das oito éguas avaliadas, somente quatro apresentaram, no momento do parto, lesões histopatológicas de placentite aguda caracterizadas por infiltrados celulares mistos, predominantemente histiolinfocitários, com presença de alguns neutrófilos, conforme o padrão descrito por Hong et al. (1993) e Lins et al. (2012). Foi realizada a comparação do proteinograma sérico entre as éguas com lesões compatíveis com placentite aguda (n=4) contra as éguas que não apresentaram esse padrão de placenta (n=4). ABSTRACT The aim of this paper was to identify the serum acute phase protein concentration in mares with induced placentitis and their neonates. Blood samples were collected from the mares in 8 different moments, and from the foals, in 4 moments. To obtain the concentration of protein fractions acrilaminada gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE) was used. In the used method 23 protein bands whose molecular weights ranged from 16kDa to 245kDa were observed, it is possible to identify the following fractions: 175kDa, 102kDa, 83kDa, 63kDa, 50kDa, 41kDa, 39kDa and 28kDa. Of all the protein bands found only the 39KDa and 41KDa have changes in the kinetics in the evaluated times. According to the marker solution, we would suggest that these proteins are α1-acid glycoprotein (39kDa) and haptoglobin (41kDa). The concentration of immunoglobulins in foals increased significantly from 12 hours of birth. Keywords:Haptoglobin, α1-acid glycoprotein, equine obstetrics, acute phase proteins Haetinger et al. Arq. Bras. Med. Vet. Zootec., v.68, n.6, p.1465-1469, 2016 INTRODUÇÃO Foram coletadas amostras de sangue das éguas em oito momentos: 60 dias pré-parto (M1), 30 dias pré-parto (M2), no dia da indução (M3), 24 horas (M4) e 48 horas após a indução (M5), no dia que antecedeu o parto (M6), imediatamente após o parto (M7) e 24 horas após Todas as éguas desenvolveram sinais clínicos de placentite, como espessamento da junção uteroplacentária e descolamento placentário 24- 48h após a indução, avaliados por ultrassonografia. Também foi observada 1466 Arq. Bras. Med. Vet. Zootec., v.68, n.6, p.1465-1469, 2016 Proteinograma sérico... 39KDa e 28KDa. De todas as bandas proteicas encontradas, somente as de 39KDa e 41KDa apresentaram alteração na cinética nos momentos avaliados. De acordo com a solução marcadora (Sigma – Saint Louis, EUA), pode-se sugerir que essas proteínas seriam α1-glicoproteína ácida (39KDa) e haptoglobina (41KDa). Nas Tab. 1 e 2, são demonstrados os níveis dos valores das frações proteicas identificados nas éguas e nos potros, respectivamente. o parto (M8). Os neonatos foram submetidos a coletas de sangue em quatro momentos: imediatamente após o nascimento (C1), 12 horas (C2), 24 horas (C3) e 48 horas após o nascimento (C4). O processamento das amostras foi realizado no Laboratório de Apoio à Pesquisa do Departamento de Clínica e Cirurgia Veterinária da Unesp - Jaboticabal. A concentração plasmática de proteína total foi determinada pelo método colorimétrico, por reação com o biureto, utilizando-se kit comercial (Labtest®). Para obtenção da concentração das frações proteicas, utilizou-se eletroforese em gel de acrilaminada contendo dodecil sulfato de sódio (SDS-PAGE), conforme técnica descrita por Laemmli (1970). Após o fracionamento, o gel foi corado durante 10 minutos em solução de azul de coomassie e, em seguida, colocado em solução de ácido acético a 7% para retirar o excesso de corante, até que as frações proteicas se apresentassem nítidas. As concentrações dessas proteínas foram determinadas em densitômetro computadorizado (Shimadzu CS 9301 - Tokio, Japan). Como referência, utilizou-se uma solução marcadora (Sigma - Saint Louis, EUA) com pesos moleculares 29.000, 45.000, 66.000, 97.400, 116.000 e 205.000 dáltons (Da), além de proteínas purificadas – albumina, IgG, haptoglobina e transferrina. A concentração da fração proteica de 39Kda (α1- glicoproteína ácida) nas éguas apresentou crescimento gradativo a partir das 48 horas após a indução da placentite. Já a PFA 41KDa (haptoglobina) demonstrou curva de crescimento gradativo conforme aproximação do parto, não apresentando aumento significativo entre os momentos. INTRODUÇÃO Não foi observada diferença entre os grupos de éguas, de acordo com as lesões histopatológicas da placenta, para nenhuma das proteínas testadas nos diferentes momentos. Nas comparações realizadas entre as éguas e os potros, não houve correlação entre os valores das proteínas nas duas categorias e nos diferentes momentos. No presente estudo, a fração proteica de39KDa (α1-glicoproteína ácida) se destacou das demais identificadas na eletroforese, apresentando aumento na sua concentração a partir de 48 horas após a inoculação da bactéria, atingindo maiores concentrações no dia prévio ao parto e após o parto. De acordo com Eckersall e Bell (2010), os níveis das proteínas de fase aguda apresentam um pico em seu acréscimo a partir de 24-48 horas após o estímulo, o que ocorreu nos dados do presente estudo. No entanto, para os mesmos autores, os níveis devem rapidamente declinar quando na extinção do processo. Para se avaliar a influência dos diferentes momentos nas variações dos proteinogramas séricos, foi realizada análise de variância simples pelo teste One Way AOV, com a comparação entre médias por meio do teste LSD. Os dados que não apresentaram distribuição normal no teste de normalidade Shapiro-Wilk foram analisados pelo teste Kruskal-Wallis One-Way Nonparametric AOV. Para se avaliar a correlação entre as bandas proteicas identificadas em éguas e potros, foi realizado o teste de correlação de Pearson. As comparações foram realizadas entre todos os momentos avaliados das éguas, contra os dois momentos avaliados nos potros. Foi utilizado o software Statistix 9.0 para a análise. Não houve diferença entre os valores das proteínas nas éguas que apresentaram placentite no momento do parto para as que não demonstraram alteração placentária no histopatológico, o que pode sugerir que este acréscimo nas taxas das frações proteicas de 39KDa (α1-glicoproteína ácida) ocorre de forma fisiológica no periparto. No método utilizado, foram observadas 23 bandas proteicas, cujos pesos moleculares variaram de 16KDa a 245KDa, sendo possível a identificação das seguintes frações: 175KDa, 102KDa, 83KDa, 63KDa, 50KDa, 41KDa, Os níveis da fração proteica de 39KDa (haptoglobina) nas éguas demonstraram uma curva semelhante e, apesar de os aumentos não serem significativos, são também indicativos de 1467 Arq. Bras. Med. Vet. Zootec., v.68, n.6, p.1465-1469, 2016 Haetinger et al. que houve resposta dessa proteína ao estímulo, sem regressão após o tratamento. INTRODUÇÃO Por serem ambas PFA do tipo positivas, seus níveis elevados ou crescentes indicam presença e persistência do estímulo inflamatório ((Takiguchi et al., 1990; Kaneko et al., 1997). Canisso et al. (2014) demonstraram elevação dos níveis plasmáticos de haptoglobina em éguas com placentite induzida. Entretanto, não está esclarecido se esses níveis apresentam comportamento similar em placentite espontânea (Canisso et al., 2015). imunoglobulinas adquiridas por transferência passiva, através do colostro. Aqueles potros que apresentaram graus de imaturidade apresentaram esse mesmo acréscimo, o que se justifica em função da assistência neonatal com administração de colostro via sonda nasogástrica nos neonatos que não apresentaram reflexo de sucção. O colostro fornece uma fonte imediata de imunoglobulinas, e a falha na absorção ou na transferência passiva destes anticorpos representa um importante fator de risco para a infecção (Cohen, 1994). Em estudo de Bailey et al. (2010), mediante tratamento à base de sulfametoxazol e trimetoprim na dose de 30mg/kg, em modelo experimental de placentite induzida, foi possível prolongar a gestação de éguas com placentite ascendente. No entanto, para os mesmos autores, o protocolo terapêutico não foi eficaz em eliminar completamente a infecção bacteriana no útero ou em aumentar a viabilidade neonatal. Assim, os níveis crescentes das frações proteicas após a indução da placentite podem indicar a persistência do processo inflamatório. Nos potros, não foram observadas alterações na cinética das frações proteicas durante o período avaliado neste estudo, contemplando desde o nascimento até as primeiras 48 horas de vida. No presente estudo, foi estabelecido o proteinograma sérico em éguas com placentite induzida, em que ficou demonstrado um aumento crescente dos níveis das frações proteicas de 39Kda (α1-glicoproteína ácida) e 41Kda (haptoglobina) 48 horas após a indução de placentite, até o momento do parto. Na avaliação do proteinograma dos neonatos, não houve alteração na cinética das proteínas avaliadas durante o período. Os potros apresentaram aumento na concentração de IgG a partir das 12 horas de nascimento, que representa a concentração de Tabela 1. 1468 Arq. Bras. Med. Vet. Zootec., v.68, n.6, p.1465-1469, 2016 Arq. Bras. Med. Vet. Zootec., v.68, n.6, p.1465-1469, 2016 As letras maiúsculas (A e B) representam diferença estatística (P<0.05) entre os momentos/colunas. As letras maiúsculas (A e B) representam diferença estatística (P<0.05) entre os momentos/colunas. 1468 Arq. Bras. Med. Vet. Zootec., v.68, n.6, p.1465-1469, 20 INTRODUÇÃO Média e desvio-padrão das concentrações das frações proteicas (mg/dL) e da proteína total (g/dL) nas éguas com placentite ascendente nos momentos: 60 (M1) e 30 dias pré-parto (M2), no dia da indução da placentite (M3), 24 horas (M4) e 48 horas após a indução (M5), no dia que antecedeu o parto (M6), imediatamente após (M7) e 24 horas após o parto (M8) (M1) e 30 dias pré-parto (M2), no dia da indução da placentite (M3), 24 horas (M4) e 48 horas após a indução (M5), no dia que antecedeu o parto (M6), imediatamente após (M7) e 24 horas após o parto (M8) Proteínas/ Frações proteicas N M1 N M2 N M3 N M4 N M5 N M6 N M7 N M8 PT (g/dL) 8 8275,0± 361,54AB 7 7742,9± 472,08B 7 8172,9± 703,20AB 4 7985,7± 740,33AB 6 8233,3± 508,59AB 7 7771,4± 468,03B 8 8587,5± 768,00A 6 8083,3± 507,61AB 63KDa 8 4366,9± 1604,3A 7 4495,3± 425,51ABC 7 4602,0± 508,66ABC 4 4263,6± 604,80BC 6 4748,7± 598,41AC 7 4134,4± 276,69C 8 4916,9± 614,49A 6 4358,3± 393,61ABC 102KDa 8 79,12± 214,12 7 3,42± 2,22 7 5,85± 3,07 4 4,71± 3,59 6 3,83± 3,25 7 5,00± 4,93 8 6,37± 7,38 6 7,00±5,36 39KDa 7 10,42± 2,69C 7 10,57± 3,50C 7 11,00± 3,87C 4 10,57± 4,19C 6 12,00± 4,4272C 7 13,14± 5,39BC 8 17,37± 5,63AB 6 19,50± 7,71A 41KDa 8 58,37± 24,47C 7 69,00± 6,73BC 7 73,28± 19,61BC 4 78,28± 23,67BC 6 72,50± 28,17BC 7 79,71± 37,98BC 8 96,12± 31,85AB 6 116,50± 36,85A 175KDa 8 144,00± 107,44 7 158,29± 51,71 7 174,29± 50,43 4 187,00± 50,84 6 172,00± 58,89 7 172,14± 20,60 797,29± 95,61 8 145,25± 48,59 6 176,33± 50,87 28KDa 8 791,75± 206,54 7 801,29± 148,56 7 840,86± 97,65 4 887,57± 90,03 6 890,67± 133,92 7 8 866,38± 102,41 6 865,67± 132,13 50KDa 8 638,13± 236,58 7 693,43± 104,05 7 748,86± 143,57 4 841,29± 142,71 6 757,83± 145,88 7 825,29± 191,69 8 796,25± 305,71 6 715,67± 133,40 83KDa 8 1236,3± 1724,0AB 7 600,29± 104,17AB 7 587,71± 77,10B 4 606,57± 84,49AB 6 592,50± 71,74AB 7 579,71± 55,71B 8 689,63± 143,62A 6 598,83± 83,73AB As letras maiúsculas (A e B) representam diferença estatística (P<0.05) entre os momentos/colunas. As letras maiúsculas (A e B) representam diferença estatística (P<0.05) entre os momentos/colunas. 1468 Proteinograma sérico... Tabela 2. REFERÊNCIAS GILES, R.C.; DONAHUE, J.M.; HONG C.B. et al. Causes of abortion, stillbirth, and perinatal death in horses: 3,527 cases (1986-1991). J. Am. Vet. Med. Assoc., v.203, p.1170-1175, 1993. BAILEY, C.S.; MACPHERSON, M.L.; POZOR, M.A. et al. Treatment efficacy of trimethoprim- sulfamethoxazole, pentoxifylline and altrenogest in experimentally induced equine placentitis. Theriogenology, v.74, p.402-412, 2010. BAILEY, C.S.; MACPHERSON, M.L.; POZOR, M.A. et al. Treatment efficacy of trimethoprim- sulfamethoxazole, pentoxifylline and altrenogest in experimentally induced equine placentitis. Theriogenology, v.74, p.402-412, 2010. HONG, C.B.; DONAHUE, J.M.; GILES Jr., R.C. et al. Etiology and pathology of equine placentitis, J. Vet. Diag. Invest., v.5, p.56-63, 1993. CANISSO, I.F.; BALL, B.A.; CRAY, C. et al. Serum amyloid a and hatoglobin concentrations are increased in plasma of mares with ascending placentitis in the absence of changes in peripheral leukocyte counts or fibrinogen concentration. J. Am. Reprod. Immunol., v.72, p.376-385, 2014. KANEKO, J.J.; HARVEY, J.W.; BRUSS, M.L. Clinical biochemistry of domestic animals. 5.ed. San Diego: Academic Press, 1997. 932p. in plasma of mares with ascending placentitis in the absence of changes in peripheral leukocyte counts or fibrinogen concentration. J. Am. Reprod. Immunol., v.72, p.376-385, 2014. LAEMMLI, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, v.227, p.680-685, 1970. CANISSO, I.F.; BALL, B.A.; SCOGGIN , K.E. et al., Alpha-fetoprotein is present in the fetal fluids and is increased in plasma of mares with experimentally induced ascending placentitis. Anim. Reprod. Sci., 2015. Available in: <http://dx.doi.org/10.1016/j.anireprosci.2014.12.019>. Accessed in: 19 fev. 2016. 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Acute phase proteins: biomarkers of infection and inflammationin veterinary medicine - review. Vet. Arq. Bras. Med. Vet. Zootec., v.68, n.6, p.1465-1469, 2016 Ouro Fino Agronegócio, Fapergs, CNPQ, Capes. Ouro Fino Agronegócio, Fapergs, CNPQ, Capes. AGRADECIMENTOS FAGLIARI, J.J.; WEISS, D.J.; McCLENAHAN, D. et al. Serum protein concentrations in calves with experimentally induced pneumonic pasteurellosis. Arq. Bras. Med. Vet. Zootec., v.55, p.383-387, 2003. FAGLIARI, J.J.; WEISS, D.J.; McCLENAHAN, D. et al. Serum protein concentrations in calves with experimentally induced pneumonic pasteurellosis. Arq. Bras. Med. Vet. Zootec., v.55, p.383-387, 2003. INTRODUÇÃO Média e desvio-padrão das concentrações das frações proteicas (mg/dL) e da proteína total (g/dL) nos potros provenientes de éguas com placentite ascendente nos momentos: imediatamente após o nascimento (C1), 12 horas (C2), 24 horas (C3) e 48 horas após o nascimento (C4) Proteínas/Frações proteicas N C1 N C2 N C3 N C4 PT (g/dL) 7 4,3±0,4 8 4,9±0,4 7 5,4±0,4 4 5,4±0,6 63KDa 7 3311±244 8 3240±228 7 3288±244 4 3235±323 102KDa 7 11,6±3,5 8 11,1±3,2 7 11,3±3,5 4 11,2±4,6 39KDa 7 8,9±3,6 8 11,8±3,4 7 19,2±3,6 4 16,7±4,8 41KDa 7 64±19 8 60 ±18 7 76±19 4 50,5±25 175KDa 7 91±21 8 90±19 7 86±21 4 80±28 28KDa 7 3,3±113B 8 229±105A 7 373±113A 4 402±149A 50KDa 7 6,2±133B 8 291±125A 7 474±133A 4 541±177A IgG Total 7 34±240B 8 521±225A 7 847±240A 4 943±318A 83KDa 7 533±55 8 507±51 7 504±55 4 489±72 As letras maiúsculas (A e B) representam diferença estatística (P<0.05) entre os momentos/colunas. Tabela 2. Média e desvio-padrão das concentrações das frações proteicas (mg/dL) e da proteína total (g/dL) nos potros provenientes de éguas com placentite ascendente nos momentos: imediatamente após o nascimento (C1), 12 horas (C2), 24 horas (C3) e 48 horas após o nascimento (C4) REFERÊNCIAS J., v.185, p.23-27, 2010. TAKIGUCHI, M.; FUJINAGA, T.; NAIKI, M. et al. Isolation, characterization, and quantitative analysis of C-reative protein from horses. Am. J. Vet. Res., v.51, p.1215-1220, 1990. 1469 Arq. Bras. Med. Vet. Zootec., v.68, n.6, p.1465-1469, 2016
https://openalex.org/W4388129883	https://journal-ems.com/index.php/emsj/article/download/851/761	English	null	Religious Leadership and the Success of Sustainable Development Goals in Nigeria (2020-2023): A Critical Appraisal	European modern studies journal	2,023	cc-by	8,084	ISSN 2522-9400 European Modern Studies Journal Vol 7 No 4 available at www.journal-ems.com DOI: 10.59573/emsj.7(4).2023.4 Religious Leadership and the Success of Sustainable Development Goals in Nigeria (2020-2023): A Critical Appraisal ISSN 2522-9400 European Modern Studies Journal available at www.journal-ems.com Vol 7 No 4 Emeaku, A. O. Chidi, PhD Nigerian Institute of Social and Economic Research (NISER) P.M.B. 5, U. I. Post Office, Ibadan, Oyo State, Nigeria Emeaku, A. O. Chidi, PhD Nigerian Institute of Social and Economic Research (NISER) P.M.B. 5, U. I. Post Office, Ibadan, Oyo State, Nigeria Abstract. Sustainable Development Goals (SDGs) represent a call by the international community to produce and implement a set of universally accepted goals that meet the urgent economic, environmental and political challenges of our time by 2030. The paper examines Religious Leadership and Success of Sustainable Development Goals in Nigeria. The research method is descriptive, and secondary sources of data were utilized. Qualitative approach was adopted. It was discovered that few religious leaders had contributed to the success of SDGs, while the actions of greater number of them were diametrically opposed to the success of it. Federal government of Nigeria should sponsor an executive bill titled ‘Ethical Conduct and Limitations of Religious Leaders, 2024; to forestall their penchant for: child abuse, sexual molestation, ritualism, monetization of prayers; self-kidnap, and fake prophecies. Each State Government of the Federation should replicate Federal government’s policy implementation guidelines on the matter. These measures will act as a deterrence to other institutions in Nigeria. Key words: Millennium Development, Nigeria, Religious Leadership, Sustainable Goals Introduction Three specific needs were designed in consonance with the needs of each zone. Phase 1: 2016-2020, Phase 2: 2020-2026, Phase 3: 2026-2030. As an off-shoot of the Millennium Development Goals, signatories to the agreement were expected to mobilize other institutions and/or stakeholders, towards building on the achievements recorded (2000-2015) in order to meet the SDGs by 2030. In Nigeria, the key stakeholders comprised of Governments, Civil societies, Religious institutions, the Media, Education sector, Health sector, Labour leaders, Legislature, Judiciary, and Nigerian citizens at large. g In recognition of religious institutions roles in the society, Tomalin et al. (2019) posits: “Religion is a major socio-cultural, political, and economic factor in many official development assistance (ODA) in recipient countries, and after decades of being ignored by global development processes, greater portions of development aids are now channeled via faith- based organizations”. In this light, it is evident that religion is increasingly recognized as a human resource rather than just an obstacle to development (Tomalin et al., 2019). j p ( ) Moreover, the Quranic verse; Sura 39:6 reads: “He makes you in the wombs of your mothers in stages, one after another in three veils of darkness”. The provision of this verse might have informed the majority of Islamic scholars to believe that, the basic goal of development is to create an environment that enables people to enjoy moral, spiritual, and socio-economic wellbeing on the planet earth and thereafter (www.arabianjbmr.com>pdfs). In the same vein, Christian scholars believe that, religion informs social change which in-turn propels development that transcend the physical and material for the wellbeing of mankind. Hence, Christian religious leaders are instructed by Jesus Christ not to be selfish in the discharge of their roles in the society: Don’t do anything from selfish ambition or from a cheap desire to boast but, be humble towards one another; always considering others better than yourselves. And, look out for one another’s interest, not just for your own interest in (Philippians, 2:3-6, P. 246). Don’t do anything from selfish ambition or from a cheap desire to boast but, be humble towards one another; always considering others better than yourselves. And, look out for one another’s interest, not just for your own interest in (Philippians, 2:3-6, P. 246). Introduction In pursuant to a new set of universally accepted agenda for sustainable development, one hundred and ninety three (193) World leaders on 25 September, 2015 at the UN Headquarters in New York City, entered into an agreement to create a better World spanning from 2015 to 2030 with the aim of incorporating a Post-Millennium Development agenda of nipping the preponderance of global poverty and hunger, achieving universal economic development, social inclusion, including environmental sustainability in the bud by 2030. , g y y To this end, 17 achievable Developmental Goals were agreed upon: (i) End poverty in all its forms everywhere; (ii) End hunger, achieve food security and improve nutrition, and promote sustainable agriculture; (iii) Ensure healthy lives, and promote wellbeing for all at all ages; (iv) Ensure inclusive, and equitable quality education and promote life-long learning opportunities for all; (v) Achieve gender equality, and empower all women and girls; (vi) Ensure availability and sustainable management of water and sanitation for all; (vii) Ensure access to affordable, reliable, sustainable, and modern energy for all; (viii) Promote sustained, inclusive, and sustainable economic growth, full and productive employment and decent work for all; (ix) Build resilient infrastructure, promote inclusive and sustainable industrialization and foster innovation; (x) Reduce inequality within and among countries; (xi) Make cities and human settlements inclusive safe, resilient, and sustainable; (xii) Ensure sustainable consumption and production pattern; (xiii) Take urgent action to combat climate change and its impacts; (xiv) Conserve and sustainably use the oceans, seas, and Marin resources for sustainable development; (xv) Protect, restore and promote sustainable use of terrestrial ecosystem, sustainably manage forests, combat desertification, halt and reserve land degradation; (xvi) Promote peaceful and inclusive societies for sustainable development, provide access to justice for all and build effective, accountable and inclusive institutions at all levels; and (xvii) Strengthen the means of implementation, and revitalize the global partnership for sustainable development (UNDP, 2015). 47 European Modern Studies Journal, 2023, 7(4) European Modern Studies Journal journal-ems.com Nigeria is one amongst 193 countries of the world that entered into an agreement for the adoption of Sustainable Development Goals (SDGs) after the expiration of the Millennium Development Goals (MDGs) with limited scope, and the attendant failure of its developmental objectives. The Nigerian road map according to Adoke for implementing the Sustainable Development Goals is to focus on six thematic areas: Policies, data management, institutions, partnership, communications and finance. Conceptual Clarification /Analysis Conceptual Clarification /Analysis p y The concept of Sustainable Economic Development like every other concept suffers universally accepted definition. Edward Barbier (1987:1) clarifies the difficulties in defining the concept with precision. According to the scholar, the concept is difficult to define for one to grasp analytically. He contends that if one try to describe the environmental, economic and social features of an on-going process, the difficulty lies in arriving at a universally accepted definition that is also analytically proven. However, he described its characteristics capable of distinguishing the concept from other concept of development as follows: “First, it is one part of the total development of society, and can be distinguished and analyzed separately. Second, its quantitative dimension is associated with economic, or growth in real per capital output. Third, its Qualitative dimension is associated with technology and institutional change, or innovation broadly defined, and fourth, it should ideally be measured i.e. economic development is associated with direct readily measurable economic gain”. He maintains that the total development of society involves not only changes in economic activities but also political, social and cultural transformations (Barkier, 1987:1). In another perspective, Brundmam Report, cited in Jacumbus (2006:1) tries to define the concept as the development that meets the needs of the present without compromising the ability of future generation to meet their own needs. As par the origin of Sustainable Development Goals, Jacombus posits that, the concept emerged in the context of a growing awareness of an imminent ecological crisis seems to have been one the driving forces of world history in the period around the end of the 20th century (Jacombus, 2006:1). On the other hand, religion also suffers a universally accepted definition as a concept. It is better described than defined. However, it is a set of beliefs and practices in the worship of a supernatural being. For Emile Durkheim (1915), religious beliefs is a social glue that help to keep the society. Sigmund Freud (1927) sees religious beliefs as an illusion; a childlike yearning for a fatherly figure, and for Napoleon Bonaparte, religion is what keeps the poor from murdering the rich. For Ajaegbu (2012), religion is a system of social coherence commonly understood as a group of beliefs or attitude concerning an object, person, unseen or imaginary being or system of thought considered to be supernatural, sacred or divine (Ajaegbu, 2012). Religion is a belief in spirits. Introduction h 20 2 28 li i l d f h i d h In Matthew 20:25-28 religious leaders are further instructed thus: You know that the rulers of the heathen have power over them, and the leaders have complete authority. This, however, is not the way it shall be among you. If one of you wants to be great he must be the servant of the rest, and if one of you wants to be first, he must be your slave like the son of man who did not come to be served but to serve and give his life to redeem many people (Matthew 20:25-28, P. 29). Can we rightly say that religious leaders in Nigeria have lived up to the expectations of these biblical and /or Quranic injunctions? Nonetheless, the study cannot lay claim that all religious leaders’ conduct are inimical to the successful implementation of SDGs. In carrying out the study, we relied on descriptive method, and secondary sources of data were utilized. Qualitative approach was adopted, and a combination of theories for the legitimization of religious leaders’ role was also adopted. The main objective of the study is to examine religious leadership and success of the Sustainable Development Goals (SDGs) in Nigeria; with a view to establishing the conforming and non-conforming roles religious leaders have made and still making towards the success of the SDGs in Nigeria. 48 European Modern Studies Journal, 2023, 7(4) European Modern Studies Journal journal-ems.com Conceptual Clarification /Analysis to this study, the implication of Marx’s viewpoint above is that, religious leaders have no obligation in the developmental objectives of the Nigerian State than to serve the interest of only one economically and politically dominant class (Nigerian political class). In other words, our expectations from the religious leaders for the success of Sustainable Development Goals to Marx, is a misplaced priority. But, the writer is opposed to this submission as religious leaders are part and parcel of the society, and should contribute their own quota towards sustainable economic growth and development in their countries. Incidentally, there are three major religious institutions in Nigeria: Christianity, Islam and African Traditional religion. However, two dominant religious institutions are Christianity and Islam. These religious denominations constituted Christian and Islamic religious institutions in Nigeria. According to Pew Forum (2006); Odumosu & Simbine (2011) cited in Omobolaji (2012:231), Roman Catholic is the largest denomination that constitutes 28%. The Protestants comprised of Anglicans, Baptists, Presbyterians, Methodists and Lutherans 31%. The Evangelical and Pentecostal institutional churches make up the rest of the Christian population in Nigeria. The Muslim faith in Nigeria follows the broad line that exists within the Muslim faith worldwide; Sunni and Shi’a. Majority of Nigerian Muslims belong to the Malaki School of the Sunni branch of Islam. The different is that, what is refer to as the Shi’a movement in Nigeria is actually a blend of Shi’a and Sunni Islam introduced by Ibrahim Elzakzaky (Umar, 2001; Omobolaji, 2012:231). Interestingly, religious institutions are involved in various economic activities, and remains very important institutions that have mass audience to influence the policy direction of the Nigerian government. Observably, religious leaders are into real estate, some own hospitals, schools: nursery, primary, and secondary up to world class universities. To mention but a few; Al- Hikmah University at Ilorin, Kwara state belongs to a Muslim Cleric - Abdulraheed Oladimeji, Benson Idahosa University belongs to the Idahosa, and Babcock University is owned by the Seventh Day Adventist Church, and Ajayi Crowther University is being run by Anglican Communion, and Redeemers’ University belongs to Pastor Adegboye while, the Covenant University is for Bishop Oyedepo of the Winners’ Chapel, Bowen University is owned by a Baptist Convention, and Madonna University is owned by the Catholic Church (Enegho, 2017). Conceptual Clarification /Analysis Arising from the findings above, it is evident that a good number of religious leaders in Nigeria have contributed tremendously in education sector of the Nigerian economy; even though their schools and tuition fees are not within the reach of the Hoi pol-loi. Invariably, the high cost of tuition fees constitutes serious setback to the attainment of quality education for all as in Goal 4 of SDGs. Conceptual Clarification /Analysis Spirits were gods, animating powers, animal-spirit companions, all of which seemed to have a religious cast (Tylor, 1958). Alsatian (2006) cited in Casley (2008) provides two sided controversial discourses of religion. First, religion as a sanctimonious aspiration to peace and love that is often ignored in politics. Second, as the source of all terrorist extremism, particularly since the 9/11 attacks in the United States of America. The discussion on religion in the context of national and international affairs automatically raises the spectre of religious- based-conflicts. Alsatian cited in Smock (2011) contends that the contribution religious institutions can make to global peace-making as a flip aide to religious conflict is only beginning to be explored (Smock, 2011). g g p ( , ) Above all, Karl Marx perceived religion with precision: Above all, Karl Marx perceived religion with precision: The State promotes inverted belief in religion just to pacify the exploited and the oppressed class, and consolidate the power of the owners and controllers of means of production (who invariably constituted the ruling class (Aja, 1997:-28-30). Religion is the spiritual aroma of the world. Religious distress is at the same time the expression of real distress and the protest against real distress. Religion is the sigh of the oppressed creatures, the heart of the heartless world, just as it is the spirit of the spiritless situation. Religion is the opium of the people (Maynard, 1974:12; Aja, 1997:28). ) In point of analysis, Marx sees religion and its institutions as make-shift organizations only for the pacification of the downtrodden and helpless masses in the society. As it applies 49 European Modern Studies Journal, 2023, 7(4) European Modern Studies Journal journal-ems.com to this study, the implication of Marx’s viewpoint above is that, religious leaders have no obligation in the developmental objectives of the Nigerian State than to serve the interest of only one economically and politically dominant class (Nigerian political class). In other words, our expectations from the religious leaders for the success of Sustainable Development Goals to Marx, is a misplaced priority. But, the writer is opposed to this submission as religious leaders are part and parcel of the society, and should contribute their own quota towards sustainable economic growth and development in their countries. Structural Functionalism and Multi-Tracks Diplomacy Structural Functionalism and Multi-Tracks Diplomacy There are plurality of scholars that theorized on the justification for and against the role of religion in nation building. They provides useful insights into how the society is structured in conformity for solidarity and the survival of the state. In 19th century, functionalism as a sociological theory had its exponents as; Emile Durkheim, Talcott Parsons and Robert K. Merton. They unanimously came to a conclusion that society is a complex social system whose independent component parts work in unison to promote solidarity and stability needed for the survival of the society. The implication of this assumption is that institutions such as; the Family, Religious institutions, the Media, Civil society groups, Government, Education sector, Judiciary, Legislature, and the Executive arm etcetera across the globe are potential constituent parts of the society. However, they have divergent views on how those institutions functions for the survival of the whole system. 50 European Modern Studies Journal, 2023, 7(4) European Modern Studies Journal journal-ems.com Durkheim, from macro level of social structure perspectives, posits that society is a complex system of interrelated and interdependent parts that work together to maintain stability (Durkheim, 1893). Talcott parson (1930s) came up with “Functional prerequisites”: Adaptation, Goal Attainment, Integration and Pattern Maintenance (AGIP) as alternative sociological theory that, the society is like a system with component parts that must adapt to the prevailing environment, pursue developmental objective of the state in collaboration with other stakeholders for solidarity and survival of the state. He believed in functional indispensability of all the independent parts. However, the theory has been criticized by Robert Merton, a lecturer in Harvard University in America. He opposed the views of Talcott Parsons on functional indispensability of the component parts of a system. He argues that, there is no way the society will have a complete functional unity in a workable system. He contends that if religion, for instance, is removed as component part of the state, citizens will go back to what they were known for in the past. For him therefore, no component part of a system is indispensable hence, his functional alternative proposition. Herbert Spencer also opposed Parsons’ theory of functionalism by looking at social system as an organism/ human being that is naturally saddled with emotions, sentiments, feelings of self, and the consciousness that will surely bring about change in behavior in a social environment. Structural Functionalism and Multi-Tracks Diplomacy g Nevertheless, the theory provides useful insights into the conforming roles of social institutions, particularly what the religious institutions can contribute on peaceful co-existence, maintenance, and the survival of the state. Aside from its immense contribution towards character reformation, it also calls for cooperation and harmonious relationship based on mutual trust in the society. As corroborated by Parsons, the institution treats all social institutions in a state as equal partners in progress for the solidarity and survival of the state in general (Parsons, 1975). g ( ) On the other hand, the theory of Multi-Tracks Diplomacy propounded by McDonald and Diamond (1996) holds the need to evolve a comprehensive approach to peace building by engaging a variety of actors in which track seven represents religious institutions. The theory brings the relevance of faith-based institutions as indispensable partners in national development to the fore. The theory equally suggests that, Governments in Track 1 is no longer an effective institution for securing international co-operation for conflict resolution, and developmental programmes without other stake-holders such as ; the religious institutions. Interestingly, religious leaders have formidable mass audience at their disposal to influence peace processes globally. They are expected in SDG 16 to exhibit and dramatize nonviolent strategies: apology, dialogue, prayer and fasting, commendation, condemnation, mediation, conciliation, social justice, forgiveness and repentance, and by extension, show the fear of God as policy thrusts in them (Emeaku, 2019 Field work). The Conduct of Religious Leaders that Negates the Successful Implementation of SDGs in Nigeria The section finds it necessary to explore what obtained from religious leaders in other climes that fought the spread of COVID-19 to a standstill in 2020 as opposed to our religious leaders in Nigeria. For instance, the objective of Goal 3 of SD agenda is to “Ensure healthy lives, and promote wellbeing for all at all ages.” During the COVID-19 Pandemic, it was expected that given the mass audience at the disposal of religious leaders, they would assist in the awareness campaigns by Federal government to stemming the spread of the scourge as quickly as possible. Rather, we discovered that greater number of the leading religious institutions indulged in falsehood prophesy whereas in other saner climes, as in South Korea, the Meeting of the Shincheonji Church of Jesus; a secretive doomsday group according to Bremmer (2020) 51 European Modern Studies Journal, 2023, 7(4) European Modern Studies Journal journal-ems.com complied with all safety measures put in place by the Korean Government without rancour. In Saudi Arabia, one of the Islamic centre piece across the World, closed all worship centres, and limited social gatherings in which a religious pilgrimage was cancelled; arrivals and visits to Mecca and Medina for Umrah were outlawed. Also cancelled in Kuwait, Malaysia and Thailand in 2020 were Friday services and Call for prayers (Bremmer, 2020). Whereas in Nigeria, many people believed unscientifically that the climatic condition of Africa could not have allowed the virus to survive as it lasted. For others, the virus was for the rich and the aged persons. It was only few Nigerians that believed the existence of the virus. The most worrisome aspect of the whole issue was that the majority of those who did not believe in the existence of the virus were from the Mega religious institutions, especially the Pentecostals in the country. The congregants have had so much confidence and trust in whatever their spiritual leaders would tell them, and not what governments’ policy objectives require from the citizens as corresponding obligations. For instance, the Federal government of Nigeria through the Center for Disease Control (NCDC) issued guidelines on lockdown and social distancing measures to curtail the spread of the pandemic. In a swift reaction to these guidelines, Prophet Temitope Joshua of the Synagogue church of all nations falsely raised the hope of the unsuspecting congregants that, there is nothing to fear about Corona virus. The Conduct of Religious Leaders that Negates the Successful Implementation of SDGs in Nigeria He boasted that “by 27 March, 2020, whether we like it or not, no matter the medicine they might have produced to cure whatever, it will go the way it come” (The Punch, 17 July, 2020:3). Meanwhile, COVID-19 report from NCDC as at 23 August, 2020 showed confirmed cases of 50,964 patients; recovered figure was 37,304; death cases was 9, 92 persons while, death cases globally recorded 792,475 persons. In New Zealand, the virus was detected in February 28, and the Country in less than three weeks shut its borders to outside travellers, and a week later, shut down both essential businesses as well as coming up with total lockdown in which people could only communicate within homes (Bremmer, 2020). In South Africa, the Buddhist New year public water fights to mark the celebration was brought to a halt, the same in other Asian countries. It is important to note that these countries have functional institutions that can withstand the vagaries of diseases yet, their religious leaders complied with the safety measures adopted by their various Governments. In Nigeria, the case was / is too far from being the same even though, we borrowed religious doctrines from the above core Islamic countries. In Nigeria’s case, one of the safety guidelines by Federal government via NCDC was that the social distancing measures should be adhered to by all Nigerians. But, at the heat of the dreaded COVID-19 virus, the Muslim religious leaders came together for the burial of Abba Kyari in April 18, 2020 (BBC, 2020). The implication of this action is that religious leaders in Nigeria have the penchant for violating health policies that has direct impact on the lives of the masses they pretend to serve. The policy thrusts of SDG 8 & 16 are targeted on the betterment and sustainability of human life, and not to expose it to infections. Incidentally, when the entire world was passing through a delicate virus in 2020; COVI- 19 pandemic, and it was up to every country to halt its spread or risk the colossal loss of her citizens to the virus. So, when thousands of worshippers were falsely assured of their safety under the ravaging COVID-19 pandemic, the implication is the tendency to ignore all safety measures such as; social distancing and face masks, and the entire nation were at risk. European Modern Studies Journal European Modern Studies Journal European Modern Studies Journal journal-ems.com The implication of the Sheik Ismail’s position is that, the federal government lock-down, and Social distancing measures to curb the spread of the scourge were inconsequential to them. For him, the movement of the Almajirai children from one place to another in the North has been an age-long history, and as such, cannot be discontinued even when the increasing cases of the infected persons were being reported. Similarly, in Niger state, the Police command handed over 15 children rescued from one Umar Ahmed who runs an Almajirai school, and cases of brutal torture; chains were used to tie their legs, and scars all over their bodies were reported. This dehumanizing treatment by a cleric is in conflict with the objectives of Goals 3, 4, and 5 of Sustainable Development strategy of the federal government. Religious leaders are supposed to be educating their followers on the dangers of not adhering to those safety guidelines from the Nigerian Centre for Disease Control in order to help in the fight against the virus but, the reverse is the case (Punch, 27 July, 2020). ( y ) A cursory look at Goals 3: Good health and wellbeing for people shows the urgent need to reducing the alarming rate of premature death, and decline in life expectancy rate due to the preponderance of diseases such as; malaria parasite, tuberculosis, polio, HIV/AIDS, and the recently reported cases of Lassa fever with 72% in three states bordering Cameroun: Ondo (32), Edo (29), and Bauchi (11), according to World Health Organization (2023). In Goal 16: Peace, justice, and strong institutions are required by countries for the successful implementation of SDGs. This is because without peaceful environment, social justice and fairness, successful implementation of SDGs, driven by humans will be elusive. Furthermore, arising from our deductions from the objectives of the Goals above, it is evident that, the death sentence passed on a -22 year Yahaya Sharif on Monday 10, 2020 in Kano state of Nigeria by Muslim leaders was antithetical to the cardinal objectives of SDGs in Nigeria. This is because, for Nigeria to achieve success in Goals 3 & 16, human lives must have been protected in a peaceful and safe environment at all cost by 2030. In the same vein, Goal 10 is about how to reduce income inequality within Nigeria and not to aggravate it. The Conduct of Religious Leaders that Negates the Successful Implementation of SDGs in Nigeria For instance, Sheik Ismaila Alkashnawi had criticized the Northern Governors’ resolution to evacuate Almajirai on the street to curb the wide spread of Corona virus. He maintained that Almajirai had come to stay, and cannot be outlawed as the concept started from the time of the great prophet Mohammed. The cleric advised Federal and State governments to establish a body similar to the Universal Basic Education to coordinate Islammiyya and local Islamic schools, and he concluded by saying that, “any one supporting the evacuation of Almajirai is Anti- Islam” (The Punch, 27 July, 2020:2). 52 European Modern Studies Journal, 2023, 7(4) European Modern Studies Journal In another development, the general overseer of new Life Church of God, Pastor Adetokunbo and his gang on 29 July, 2020, were apprehended and paraded by the Police Public 53 European Modern Studies Journal, 2023, 7(4) European Modern Studies Journal journal-ems.com Relation officer, Frank Mba for the abduction of a dispatch rider who had come to the church to deliver an item for a church member, and was kidnapped and kept in an underground cabin inside the pastor’s church building in Ogijo in Ogun state, who demanded the sum of N20 million naira ransom. When they were caught, the pastor confessed that he embarked on the evil venture for the expansion of his church, charity works, and Ileya. p y y On 4 July 2020, Punch newspaper also reported the arrest of one Pastor Caleb Agu in collaboration with a community leader in Owerri, Imo state by the Anti-kidnapping unit and special Ant-robbery squad for the alleged killing of two NDLEA personnel and recovered the sum of N4.2 million naira ransom they collected from one of the victims, including AK-47 riffle , one riffle belonging to the slain NDLEA officer, 120 rounds of live ammunition , one Lexus Suv vehicle, and other exhibits (The Punch, 4 July, 2020:5). In another development, a religious leader in Kaduna, James Clement staged his own kidnap and demanded 5million ransom from his followers (Vanguard 29, 2020). In the same vein, a Methodist Pastor-- Adegoke at Shagamu branch Lagos was reported by the Sahara Reporters to have lodged himself in a hotel, and claimed that he had been kidnapped and demanded the sum of 3million ransom, and when it was uncovered by the Police, he claimed it was spiritual attack on him that was responsible (Sahara Reporters, 2020). In May, 2020, the President of love world incorporated otherwise known as the Christ Embassy kicked against the lockdown measure by the Federal Government, saying that churches should be opened for healing. In a broad cast at the pulpit posted on the social media on the August 1 2020, he criticized the new guidelines for the re-opening of worship centers this way: y I am appalled that some people have the audacity to tell us how many hours we can spend in church. How dare you! What in the world do you think you are? You cannot tell God‘s people how long they can spend in Church. European Modern Studies Journal If you built the building for them and you are saying you are not going to let them enter the building, may be because you built it. But, if you did not, you have no right to tell God’s people you are going to spend one hour or two hours in church. I tell you, the number of hours you tell them they have in the church is the number of hours you have left (The Punch, July 17, 2020:3). In the like manner, the Presiding Bishop of Living Faith Church worldwide: Winning Chapel International, Oyedepo Posits: The decision by the Federal Government to open market and keep churches closed was a demonic plot to silence the body of Christ. There is something wrong; for people to be allowed to be in the market for six hours and cannot be in the church for two hours, it is an upside down way of looking at things. Which one is more orderly? The market or the church? I can smell a rat. Behind all this, how do we stop the church from exploding? The voice of darkness is influencing people at various levels, targeting the church because the growth and expansion of the church is the greatest headache of the devil. But the gates of hell shall not prevail against it. The devil and all his agents shall pay for this. International, Oyedepo Posits: The decision by the Federal Government to open market and keep churches closed was a demonic plot to silence the body of Christ. There is something wrong; for people to be allowed to be in the market for six hours and cannot be in the church for two hours, it is an upside down way of looking at things. Which one is more orderly? The market or the church? I can smell a rat. Behind all this, how do we stop the church from exploding? The voice of darkness is influencing people at various levels, targeting the church because the growth and expansion of the church is the greatest headache of the devil. But the gates of hell shall not prevail against it. The devil and all his agents shall pay for this. Pastor Chris Okotie, the founder of the household of God’s Church international Ministries stated that COVID-19 is grand scheme by international forces to reduce the world population. European Modern Studies Journal Going by the incessant warnings and threats that those who don’t pay their tithes irrespective of the situation they found themselves during COVI-19 pandemic will not go to heaven. There is every tendency that some members who might have spent the supposedly tithe sum may steal or go a- borrowing for the fear of not missing heaven as if; the very key to heaven is in their pastors’ pocket. The Goal 10, otherwise known as shared prosperity is to complement Goal l1; the eradication of extreme poverty, and it was targeted poor countries of the world. How can the Nigerian Government succeed in realizing these objectives when religious leaders kept on impoverishing their members? In Goal 16 of the Sustainable development programme, various institutions are expected to promote peaceful co-existence; act and behave peacefully. Without peaceful environment, no meaningful development takes place (silent lege inter armo). But, on the contrary, actions and inactions of religious leaders in recent past had generated interreligious violence, and many lives were lost. Nigerians are known to be too religious, and if it comes to taking instructions from their religious leaders, we are the best in the world, and yet, at the slightest provocative statement from other religious denominations, interreligious conflict is bound to occur. For instance, the sporadic attacks on Christians in Kaduna state on the 16 May 2020 as reported by the Punch Newspaper alleged that, it was carried out by the armed Muslim bandits, and about 15000 Christians were displaced. It was also reported that the Christians in Southern Kaduna had resolved to defend themselves (The Punch, 16 May, 2020). The implication is that the crisis was not resolved amicably till date. To this end, making cities and settlements inclusive, safe, resilient, sustainable, and having access to justice for all in Goals; 11 and 16 will be difficult to be achieved by 2030. European Modern Studies Journal He condemned Christians listening to sermons on line rather than attending churches. He said it is unbiblical for not worshipping together as God commanded. However, some Nigerians have argued that Jesus Christ did not lay hands on every one he healed. But, spoke the word and they were healed (The Punch, July 17, 2020:3-4). Adegboye’s sermon on Tithing is most disappointing. He places much of his energy at the pulpit on Tithing and offering regardless of the state of extreme poverty which Goals 1 and 2 of Sustainable development intend to address in Nigeria. Many Nigerians are casual workers, 54 European Modern Studies Journal, 2023, 7(4) European Modern Studies Journal journal-ems.com and as such, they are grossly underpaid in their place of work, as at March, 2020 when the Federal government rolled out a set of safety guidelines to curtail the spread of COVID-19 virus, including : lockdowns; sit-at home orders; wearing of face masks; social distancing; closure of government schools; borders; restriction of social engagements, and economic activities at work places, including worshipping in churches and mosques, and as a result, many Nigerian have lost their sources of livelihood, living from hands to mouth. In the face of the hardship in the country, pastors were threatening to pass a curse on its members for not paying their tithes. When has tithing become more important than the gospel for survival and salvation? Adegboye warns the pastors and the congregants thus: g y g g I am going to give every one of you pastors and opportunity to repent tonight and then to go back immediately after this convention and restitute your ways with your congregation, make it clear to them. Anyone who is not paying his/her Tithe is not going to heaven, full-stop! Pay your Tithes to avoid a divine curse. When we say pay your Tithes it is because it attract a curse, particularly to your generation (BBC, 2018). This warning is more of a threat to the congregation, and someone may wonder if the Pastor has the key to heaven in his pocket. Many of the pastors have functional Universities, and cruises in their private jets while, greater number of their followers are passing through untold hardship due to Corona Virus pandemic. European Modern Studies Journal Another worrisome dimension in the conduct of some religious leaders in Northern part of the country is their look warm attitude in the cases of gruesome killings without recourse to due process in the law of the Federation. Nigeria is not being governed under theocratic laws but, a constitutional democracy in which fair hearing remains its core tenet. What our leaders must know, is that, an unlawful killing without thorough investigation and punitive measures being meted to the culprits, will beget many more killings in the future. For example, on the 12 may, 2022, a 200 level student of Shehu Shagari College of Education, Sokoto was gruesomely murdered, and burnt to ashes under the aegis of ‘blasphemy’ in a broad day light by her colleagues (The Guardia, 2022). And, as a follow-up, Usman Buda; a butcher was beaten to death for the same alleged blasphemy during an argument with a trader in the same state (The Punch, 6 June, 2023). ( ) Although, there were condemnations from Islamic leaders after the dastardly act in the case of Deborah, but, it’s over one year, and counting without any body being prosecuted. It is a known fact that, the culprits have leaders in various Mosques on whose instructions they always obeyed. The look warm attitude being exhibited by our religious leaders in the North is antithetical to the cardinal objectives of Goal 3, 11, and 16; which placed much premium on safety and sanctity of human life. False Prophecies g y g ( ) Most of the prophecies released concerning President Muhammadu Buhari, the Presidency, Nigeria and some popular figures in 2021 did not come to pass. Some of these prophecies include but not limited to the death of President Buhari, crises in the Presidency, Vice President, Yemi Osinbajo suddenly becoming President. Presidency, Nigeria and some popular figures in 2021 did not come to pass. Some of these prophecies include but not limited to the death of President Buhari, crises in the Presidency, Vice President, Yemi Osinbajo suddenly becoming President. On the 12th of June, 2021, the leader of INRI Evangelical Spiritual Church, Primate Ayodele came up with a Prophecy of a Clash between Buhari and Osinbajo as well as their Aides clashing against each other. He went further to claim that he saw a Presidential convoy attacked. The Primate concluded his unfounded prophecy thus: There will be threats in the Villa, I’m seeing confusion in the Villa, Buhari will lose one of his aides. There is going to be a clash between Buhari and Osinbajo and their aides, On the 12th of June, 2021, the leader of INRI Evangelical Spiritual Church, Primate Ayodele came up with a Prophecy of a Clash between Buhari and Osinbajo as well as their Aides clashing against each other. He went further to claim that he saw a Presidential convoy attacked. The Primate concluded his unfounded prophecy thus: There will be threats in the Villa, I’m seeing confusion in the Villa, Buhari will lose one of his aides. There is going to be a clash between Buhari and Osinbajo and their aides, mark my word (Daily Post Nigeria, 16 Jan., 2022) There will be threats in the Villa, I’m seeing confusion in the Villa, Buhari will lose one of his aides. There is going to be a clash between Buhari and Osinbajo and their aides, mark my word (Daily Post Nigeria, 16 Jan., 2022) 55 European Modern Studies Journal, 2023, 7(4) European Modern Studies Journal, 2023, 7(4) European Modern Studies Journal journal-ems.com Discussion This study cannot lay claim to the effect that all religious leaders’ conducts are inimical to the successful implementation of SDGs. However, the conduct of greater number of them were diametrically opposed to the developmental objectives of the Federal Government on SDGs within the period under review. For instance, in Goal 5: of the SDGs is to achieve gender equality and empower all women and girls. Whereas, the report cases of sexual molestation and rape by some religious leaders were, and still common place in Nigeria. The sexual exploitation of Women and Girls in the name of spiritual cleansing negates the objective of Goal 5. This is because, achieving gender equality begins from the elimination of practices that tend to retard Women and Girls’ freedom and safety. The implementation of Goal 5 would be seen as successful, if their priorities and needs are taken into account by all institutions, especially, the religious institutions that controls greater number of women and girls. Child marriage is another impeding factor to the success of SDGs in Nigeria, and some religious leaders, particularly, in Northern part of Nigeria, do not see child marriage as an act of child abuse with concomitant health implications. In the Northern part of the country untold number of girl below 18 years of age were married by religious clerics, and in most cases without their consent. Many observers attributes this early marriage to the rate of poverty and hunger ravaging many families in the North. In Goal 16: Peace, justice, and strong institutions are required by countries for the successful implementation of SDGs. This is because, without peaceful environment, social justice and fairness, successful implementation of SDGS, driven by humans will be elusive. Arising from our deductions from the objectives of the Goals above, it is evident that, the death sentence passed on a -22 year Yahaya Sharif on Monday 10, 2020 in Kano state of Nigeria by Muslim leaders was antithetical to the cardinal objectives of SDGs in Nigeria. This is because, for Nigeria to achieve success in Goals 3 & 16, human lives must have been protected in a peaceful and safe environment at all cost by 2030. Conclusion and Policy Recommendations Arising from the findings above, religious leaders in a generalized context have been paying lip service towards the eradication of poverty amongst Nigerians. Prophecies and predictions based on falsehood were the greatest obstacles to the realization of SDGs in Nigeria. Majority of the religious leaders are after their selfish interests, even though they enjoy tax free business transactions. Most of them, particularly the Pentecostal Churches, did not believe that COVID-19 Pandemic existed, hence they indulged in prophecies based on false hood for monetary gains. It is imperative to note that, religious leaders are not operating in abstraction but in an environment within Nigerian territorial integrity without paying taxes. They cannot eat their cakes, and still have their penny at all times. To this end, Federal government of Nigeria should sponsor an executive bill titled “Development Tithes” as part of social responsibilities in which any religious institution having 100 members and above shall pay 10% of the total income into the federation account. The funds should be dedicated specifically for the development and revitalization of four key sectors of the Nigerian economy such as; Health, Power, Agriculture and Education. If these key sectors of Nigerian economy are well developed and harnessed, poverty reduction, extreme hunger and unemployment rate will reduce to a manageable proportion, and by extension, the foundational Goals: 1, 2, 3, 4, 8 and 10 would have been achieved before the 2030 deadline. Religious leaders should be closely monitored as some of them are now involved in all sort of criminalities and exploitation of their members for selfish gains. As a matter of urgency, each state of the federation should sponsor an executive bill to various State House of Assembly for the enactment of an Edict to be titled “‘Ethical Conduct and Limitations of Religious 56 European Modern Studies Journal, 2023, 7(4) European Modern Studies Journal journal-ems.com Leaders 2024 to forestall their penchant for: Child abuse; Sexual molestation; Ritualism, Monetization of prayers; Self-kidnap, and Fake prophecies. Each State government of the federation should replicate Federal government’s policy implementation on the matter. These measures will act as a deterrence to other institutions in Nigeria. Leaders 2024 to forestall their penchant for: Child abuse; Sexual molestation; Ritualism, Monetization of prayers; Self-kidnap, and Fake prophecies. Each State government of the federation should replicate Federal government’s policy implementation on the matter. These measures will act as a deterrence to other institutions in Nigeria. 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https://openalex.org/W390868286	https://digitalcommons.uri.edu/cgi/viewcontent.cgi?article=1411&context=theses	English	null	RACE AND ENVIRONMENTAL EQUITYA GIS BASED SPATIAL ANALYSIS OF PROVIDENCE, RHODE ISLAND	null	2,020	cc-by	19,814	University of Rhode Island University of Rhode Island DigitalCommons@URI DigitalCommons@URI Open Access Master's Theses 1995 RACE AND ENVIRONMENTAL EQUITYA GIS BASED SPATIAL RACE AND ENVIRONMENTAL EQUITYA GIS BASED SPATIAL ANALYSIS OF PROVIDENCE, RHODE ISLAND ANALYSIS OF PROVIDENCE, RHODE ISLAND Sangeeta Jain University of Rhode Island Open Access Master's Theses RACE AND ENVIRONMENTAL EQUITYA GIS BASED SPATIAL RACE AND ENVIRONMENTAL EQUITYA GIS BASED SPATIAL ANALYSIS OF PROVIDENCE, RHODE ISLAND ANALYSIS OF PROVIDENCE, RHODE ISLAND Sangeeta Jain University of Rhode Island Follow this and additional works at: https://digitalcommons.uri.edu/theses Terms of Use All rights reserved under copyright Recommended Citation Recommended Citation Jain, Sangeeta, "RACE AND ENVIRONMENTAL EQUITYA GIS BASED SPATIAL ANALYSIS OF PROVIDENCE, RHODE ISLAND" (1995). Open Access Master's Theses. Paper 409. https://digitalcommons.uri.edu/theses/409 Recommended Citation Recommended Citation This Thesis is brought to you by the University of Rhode Island. It has been accepted for inclusion in Open Access Master's Theses by an authorized administrator of DigitalCommons@URI. For more information, please contact digitalcommons-group@uri.edu. For permission to reuse copyrighted content, contact the author directly. Approved: Major Professor Acknowledged: Director MASTER OF COMMUNITY PLANNING RESEARCH PROJECT OF SANGEETA JAIN Dr. Farhad Atash Dr. Marcia Marker Feld MASTER OF COMMUNITY PLANNING RESEARCH PROJECT OF SANGEETA JAIN RACE AND ENVIRONMENTAL EQUITY- A GIS BASED SPATIAL ANALYSIS OF PROVIDENCE, RHODE ISLAND BY SANGEETA JAIN BY SANGEETA JAIN A RESEARCH PROJECT SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF COMMUNITY PLANNING MASTER OF COMMUNITY PLANNING RESEARCH PROJECT OF SANGEETA JAIN Major Professor Acknowledgments This study owes its genesis to my exposure to the world of Geographic Information Systems (GIS) during the past one year. I would like to thank several people for their help in this research project. First of all I would like to thank Dr. Farhad Atash for his encouragement and timely comments on the "just-in time" drafts. I would also like to thank Maia Peck, for her sincere prodding and help wi~h conceptual and technical GIS issues from time to time. Dr. Marcia Feld, as my second outside reader, was also a source of encouragement and deserves my thanks. Clint Webb, at Vannasse Hangen and Brustlin, deserves my special thanks for his cooperation and understanding on the days I would vanish from work and pursue completion of my thesis instead. Finally, I would like to dedicate this study to my family: my children Selina and Kabir and my husband Kapil for the many nights when I was around but not really around and yet they accepted it. ii ii Table of Contents ACKNOWLEDGMENTS LIST OF TABLES LIST OF FIGURES ACKNOWLEDGMENTS LIST OF TABLES LIST OF FIGURES vi CHAPTER 1: INTRODUCTION CHAPTER 1: INTRODUCTION CHAPTER 4: ANALYSIS AND RESULTS CHAPTER 4: ANALYSIS AND RESULTS 34 UNIVARIATE ANALYSIS CORRELATION ANALYSIS COMPARISON OF BLOCK GROUPS WITH AND WITHOUT TRI SITES MULTIVARIATE ANALYSIS - LOGIT ANALYSIS USING DOMAINS OF INFLUENCE DESCRIPTIVE ANALYSIS CHAPTER 5: DISCUSSION AND CONCLUSIONS DISCUSSION LIMITATIONS POLICY IMPLICATIONS FURTHER RESEARCH APPENDIX LIST OF ALL THE TRI SITES IN PROVIDENCE BIBLIOGRAPHY 34 46 52 55 60 63 71 71 72 73 75 77 77 79 CHAPTER 3: HYPOTHESIS AND METHODOLOGY STUDY HYPOTHESIS METHODOLOGY DATA SOURCES VARIABLES OVERVIEW OF ANALYSIS iii CHAPTER 1: INTRODUCTION Environmental equity has recently emerged as an important issue both in the media and within the Federal government. The issue of environmental equity refers to whether people bear the burden of our technological advances - environmental pollution - evenly across society. In particular, the issue addresses whether or not racial minority and low-income communities bear a disproportionate share of exposure to pollution and environmental risk. CHAPTER 5: DISCUSSION AND CONCLUSIONS 79 iv iv List of Tables Table 1: Racial Composition by Block Groups------------- 36 Table 2: Bivariate Pearson Correlations Among Key Variables 47 Table 3: Summary Statistics of Key Variables by Block Groups 53 Table 4: Block Groups Based LOGIT Analysis - Model 1 58 Table 5: Block Groups Based LOGIT Analysis - Model 2 59 Table 6: DOis Contrasted With Non-TRI Blocks 64 Table 7: DOis Contrasted With Non-DOI Block Groups 66 Table 8: DOis and Non-TRI Block Groups- LOGIT Analysis 68 Table 9: DOis and Non-DOI Block Groups-LOGIT Analysis 70 Table 1: Racial Composition by Block Groups------------- 36 Table 2: Bivariate Pearson Correlations Among Key Variables 47 Table 3: Summary Statistics of Key Variables by Block Groups 53 Table 4: Block Groups Based LOGIT Analysis - Model 1 58 Table 5: Block Groups Based LOGIT Analysis - Model 2 59 Table 6: DOis Contrasted With Non-TRI Blocks 64 Table 7: DOis Contrasted With Non-DOI Block Groups 66 Table 8: DOis and Non-TRI Block Groups- LOGIT Analysis 68 Table 9: DOis and Non-DOI Block Groups-LOGIT Analysis 70 v List of Figures List of Figures Figure 1: Study Area - Providence, RI 5 Figure 2: Locations of TRI Sites 35 Figure 3: Population Density 37 Figure 4: Frequency Distribution of Minority Proportions 38 Figure 5: Spatial Distribution of Minorities 40 Figure 6: Spatial Distribution of Blacks 41 Figure 7: Spatial Distribution Of Hispanics 42 Figure 8: Median Per Capita Income 44 Figure 9: Median Housing Values 45 Figure 1 0: Scatter Plots - Density and Population groups 49 Figure 11: Scatter Plots - Median Per Capita Income and Population Groups 50 Figure 12: Scatter Plots - Median Housing Values and Population groups 51 Figure 13: Domains Of Influence (DOis) 62 vi vi PROBLEM STATEMENT The environmental movement and the civil rights/ anti-discrimination movements both began contemporaneously in the 1960s. Each, in its own way, has since made measurable strides with tangible results in both public awareness and legislation. However, the question of social equity and distribution in clean-environment efforts is still significant. Recent studies strongly suggest the presence of a disproportionate exposure of minorities to environmental hazards through their proximity to waste dumps, landfills, and commercial toxic releases. This project seeks to expand on this growing literature by using GIS, a relatively new and powerful methodology, to study the extent of environmental equity with respect to race and income in Providence. Rhode Island. The environmental movement and the civil rights/ anti-discrimination movements both began contemporaneously in the 1960s. Each, in its own way, has since made measurable strides with tangible results in both public awareness and legislation. However, the question of social equity and distribution in clean-environment efforts is still significant. Recent studies strongly suggest the presence of a disproportionate exposure of minorities to environmental hazards through their proximity to waste dumps, landfills, and commercial toxic releases. This project seeks to expand on this growing literature by using GIS, a relatively new and powerful methodology, to study the extent of environmental equity with respect to race and income in Providence. Rhode Island. Providence provides an interesting case study from many perspectives. An urban area in the New York .- Boston corridor, it has a history of hiyh industrial activity. In addition, it is presently experiencing a rise in its proportion of minority residents, largely through immigration. The geo-demographic composition of Providence offers a rich distribution of socio-economic strata that, coupled with a large number of toxic waste sites, offers a suitable site for studying the question of environmental equity. Previous studies of environmental equity have included both bivariate and multivariate analyses. However, the use of Geographic Information Systems (GIS), in this study, will provide the opportunity for a more accurate look at the impact of environmental hazardous sites on populations within their "domain of influence (DOI)" rather than on just census defined geographic bounds. STUDY OBJECTIVES This study has two main objectives: This study has two main objectives: 1 ) to determine the extent of environmental inequity in the city of Providence and to provide a measure of the hazardous exposure to various socio-economic strata. 1 ) to determine the extent of environmental inequity in the city of Providence and to provide a measure of the hazardous exposure to various socio-economic strata. to study whether inequity, if present, is related to race. 2) to compare the results of analysis, based on (a) the traditional approach using census block groups as the unit of analysis, with (b) the GIS based approach using the "domains of influence" of each toxic release site as the unit of analysis. 2) to compare the results of analysis, based on (a) the traditional approach using census block groups as the unit of analysis, with (b) the GIS based approach using the "domains of influence" of each toxic release site as the unit of analysis. 2 2 STUDY SIGNIFICANCE Ever since a report, produced by the US General Accounting Office (US General Accounting Office, 1983), found that three of the four landfills in the Southeast (that were studied) were located in predominantly poor and African-American communities, a variety of other reports have documented wide-spread environmental inequity. "Toxic Wastes and Race in the United States" (United Church of Christ Commission for racial Justice, 1987), another prominent report, found that minorities, mainly African and Hispanic Americans, are strikingly over represented in communities with commercial hazardous waste facilities. The study found that more than fifteen million African Americans and eight million Hispanic Americans lived in communities with one or more hazardous waste sites. The disproportionate impact on minority racial communities were also found for uncontrolled toxic waste sites. Providence has a large and growing minority population consisting of African Americans, Hispanics, and Asian immigrants. In this respect, if national trends hold true, one should expect to find evidence of environmental inequity in Providence as well. Further, previous studies support a linkage between environmental hazard exposure and the community's economic state. Poorer communities cannot afford the luxury of being concerned about the state of their environment when confronted by basic survival-related economic problems. They are more likely to accept the trade-off in return for employment and economic development with or without the knowledge of associated risks. Such a trade-off clearly carries longer-term risks whose costs, in fact, must often be borne by the larger community, both the minority and the majority. 3 Identifying and eradicating such discrimination is clearly in the interests of the public and should be a public policy imperative. This study will yield not only a measure but also a graphic representation of the environmental inequity, if any, in Providence. Such data should serve public policy makers well, in their efforts to make Providence equally hospitable to all residents. STUDY AREA DESCRIPTION Providence is a city made up of physically, economically, and socially diverse neighborhoods. It was developed near a natural harbor by its early settlers, in the 17th century, to establish a coastal trading route. The city kept gaining economic significance, and by the turn of the 20th century it had become the economic center of the most industrialized state in the nation. The rich history of about 200 years has contributed to a mix of architectural forms, economic functions, and social groups, characterizing the city today. Figure 1 shows the location of Providence within the State of Rhode Island. The city of Providence reached its peak population of 253,504 in 1940. Since then, the population has been declining and reached its lowest level of 156,804, in 1980. This major loss, occurred partly due to the suburbanisation taking place across the nation. The city of Providence reached its peak population of 253,504 in 1940. Since then, the population has been declining and reached its lowest level of 156,804, in 1980. This major loss, occurred partly due to the suburbanisation taking place across the nation. The 1990 census, however, recorded a minor increase in the population of Providence at 160, 199. The median age increased from 29.9 years in 1980 to J3.8 years in 1990. In 1980, about 19 percent of the population was classified as non white, whereas this 4 Study Area - Providence, RI Study Area - Providence, RI Study Area - Providence, RI RI y , - City of Providence D Town Boundaries 0 10 20 Kilometers N + Data Sources: RIGIS Map produced by Sangeeta Jain CPAD, URI (1995) Figure# 1 City of Providence Town Boundaries Map produced by Sangeeta Jain CPAD, URI (1995) 20 Kilometers Figure# 1 figure was 23 percent in 1990. The rapid growth of the non-white population was primarily caused by an increase in the numbers of migrant Southeast-Asians. REPORT OUTLINE The rest of this report is arranged in chapters that flow logically beginning with a discussion of previous efforts in this area, followed by the hypotheses and methodology for this study and, finally, the results and conclusions from the empirical analysis. Chapter 2: Environmental Equity - A Review This chapter will provide a review of past research on environmental equity, This chapter will provide a review of past research on environmental equity, summarizing the contexts studied, methods employed, and findings. Also discussed will be the role of government in both, initiating such studies and incorporating research findings into legislation, and in hazardous site location decisions. Chapter 3: Hypotheses and Methodology This chapter will provide a detailed description of the hypothesis and assumptions used in this study. It will also provide a discussion of data sources and accuracy issues, details on the operationalization of variables, and a description of the analytical methods applied. GEOGRAPHIC INFORMATION SYSTEMS (GIS) Geographic information system are being used for a variety of applications. It has made valuable contributions to the understanding and solution of key socio-economic and environmental problems. It allows us to access data based on geographical locations. GIS could be best described as a database system that allows the manipulation and analysis of geographic data. It is a collection of computer hardware and software that integrates computer graphics with a relational database, for the purposes of managing data about geographic locations (Garson, 1992). GIS comprises of three distinct but overlapping views - maps, database, and spatial analysis (Maguire: 1991). The map view focuses on the cartographic aspects of GIS, and could be seen as map processing . The second view emphasizes the importance of a well designed and implemented database that allows for complex analytical operations. The third view, emphasizes the importance of spatial analysis. 6 Chapter 4: Analysis and Results Chapter 4: Analysis and Results This chapter will include a detailed analysis of the results of the study. The first part includes a descriptive analysis of all the key variables at the block group level. The second part analyzes and compares the relationship between racial composition and 7 7 hazardous exposure with the census block groups and DOis as alternative units of analysis. Chapter 5: Discussion and Conclusions This chapter will continue discussion of the results in light of the findings made by previous research, and derive implications for public policy and land-use decisions in Providence. It will conclude with a discussion of this study's limitations and recommendations for future research. 8 8 CHAPTER 2: ENVIRONMENTAL EQUITY -- A REVIEW Evidence of environmental inequity comes from various studies, that show how hazardous waste sites have been located in communities with higher proportions of disadvantaged (in terms of race/income) population groups. This chapter includes a review of literature on environmental inequity and various federal and state initiatives to address this particular problem. The term 'environmental inequity' refers to the uneven distribution of any known or potential environmental risks, across different demographic groups. When environmental risks are higher in residential areas with higher proportions of minority populations, the inequity is referred to as 'environmental racism'. Another commonly used term- 'environmental justice' -refers to a political movement aimed at achieving environmental equity (Burke 1993). MAJOR PREVIOUS STUDIES The awareness and concern about inequity in the distribution of hazardous waste sites have been increasing steadily in the past decade. The first event to focus the nation's attention on the issue of environmental injustice occurred in Warren County, North Carolina incident, in 1982. Residents of Warren County, dominated by African- Americans, protested for four years against the siting of a polychlorinated biphenyl 9 9 9 (PCB) landfill (Lee 1990). The campaign was unsuccessful in preventing the siting of the unwanted landfill, but was successful in many other ways. The most important outcome was that it lead to a further investigation of racial demographics and hazardous site locations. GAO Study The first major study of the relationship between community demographics ant the siting of toxic waste was conducted by the US Government's General Accounting Office. The objective of this study was to determine the correlation between the location of hazardous waste landfills and the racial and economic status of the surrounding communities (GAO 1983). The GAO report found that three out of the four hazardous waste landfills in the Southeast part of the country (EPA's Region IV - Alabama, Florida, Georgia, Kentucky, Mississippi, North Carolina, South Carolina, and Tennessee) were located in black dominated communities. The percentage of black population in the region was about 20, but for the communities with the landfills, the corresponding percentages ranged from 38 to 90 percent. The GAO study was limited in scope, in as much it researched only four landfills in one region of the nation. It was not designed to study the relationship between the location of hazardous waste sites and the socio-economic status of the people residing around those sites, throughout the United States (Lee 1990). Since the GAO report was the first study of its kind, it could not be an indication of any national pattern. Nonetheless, it served a critical purpose in initiating more systematic inquiries into environmental inequity. 10 United Church of Christ Report The next major study "Toxic Wastes and Race in the United States: A National Report on the Racial and Socio-Economic Characteristics of Communities Surrounding Hazardous Waste Sites" was published by the United Church of Christ Commission (UCCC) for Racial Justice (UCCC 1987). This study, released in 1987, was the first comprehensive, national-level study, to document the location of hazardous waste sites in racial and ethnic communities. This report was very influential in raising public awareness about the disproportionate burden of pollution on minority population groups. The report, a culmination of over five years of work, found that the racial composition was the single most important variable in explaining the presence of commercial hazardous waste facilities in any community. The UCCC report comprised of two studies - the first analyzed the relationship between demographic patterns and "commercial hazardous waste" sites, and the second study studied the relationships between the demographic patterns and "uncontrolled toxic waste" sites. Commercial hazardous waste sites are facilities that accept hazardous wastes from a third party for a fee, while uncontrolled toxic waste sites are those that have been closed and abandoned by US Environmental Protection Agency (EPA). The first study, focusing on commercial hazardous waste sites, used five digit zip code level demographic data as its unit of analysis. The locational data, on the then operating hazardous waste sites, were collected from the EPA's 1986 Hazardous Waste Data Management System. The study tested five variables - minority percentage of the 11 population, mean household income, mean value of owner-occupied homes, number of uncontrolled toxic waste sites per 1,000 persons, and pounds of hazardous waste generated per person. The major findings of the first study were the uncovering of a consistent national pattern in the location of hazardous waste sites. The pattern consisted of: • Race proved to be the most significant variable in location of hazardous waste facilities, • Although socio-economic status appeared to play an important role, race proved to be more significant, and • Communities with a higher percentage of minorities had a greater number of commercial hazardous waste facilities (UCCC 1987). The second study in the report, focused on the communities surrounding the over 18,000 uncontrolled toxic waste sites from the EPA's Comprehensive Environmental Response, Compensation, and Liability Act Information System (CERCLIS). United Church of Christ Report This study was aimed at quantifying the number of persons from different racial and ethnic groups, living in communities with uncontrolled toxic waste sites. The findings of the second study in reported alarming statistics: • Three out of five African· Americans live in communities with abandoned toxic waste sites, • Three out of five African· Americans live in communities with abandoned toxic waste sites, • Minorities comprised 24 percent of the total population in communities with one hazardous facility, compared to only 12 percent in communities with no toxic facility, 12 • Three of the five largest commercial hazardous waste landfills are located in predominantly African-American or Latino communities, and account for 40 percent of the nation's total estimated landfill capacity in 1986, and • Three of the five largest commercial hazardous waste landfills are located in predominantly African-American or Latino communities, and account for 40 percent of the nation's total estimated landfill capacity in 1986, and • African Americans are heavily over-represented in the population of cities with the largest number of abandoned toxic waste sites. • African Americans are heavily over-represented in the population of cities with the largest number of abandoned toxic waste sites. Burke's Study of Los Angeles County Burke conducted a case study to examine environmental equity in Los Angeles County, California (Burke 1993). The objective of the study was to determine whether minorities suffer a greater degree of environmental pollution. Data was collected for more than 700 toxic release sites (TRI) in the county. The socio-economic data collected at the census tract level included median per capita income, population density, and the racial composition of the population. The results of the study indicated that on average the number of toxic waste sites increases with the increase in minority population or a decrease in per capita income. The study treated all toxic waste sites as uniform, and census tracts were used as the unit of analysis. Mohai and Bryant Study Another prominent empirical study was conducted by Mohai and Bryant in 1992 to provide additional evidence on the issue of environmental equity (Mohai & Bryant 1992). The main objective of the study was to assess the effect of race on the distribution of commercial hazardous waste sites. They used a sample survey to collect data on race and income from the residents of three counties surrounding the city of Detroit area. The locations of 16 hazardous waste facilities and the 289 respondents were mapped and distances between the two were measured to the nearest tenth of a mile. The study found that in the Detroit area, 48 percent of the minorities sampled lived within a mile of a hazardous waste site. As the distance increased to a mile and a half, the ratio dropped to 18 percent. The study also found that the relationship between race and location of hazardous waste sites is independent of income. However, when the data were analyzed for the entire population of three counties. The results showed that on average 4 percent of the population live within a mile of a hazardous site, and the number is 11 percent for blacks and 3 percent for all whites. 13 Other Case Studies Numerous other studies have been done that show evidence of environmental racism. In 1979, a major empirical study was conducted that linked municipal solid waste siting with the race of adjacent area residents in the city of Houston, Texas. The study reported that until the 1970s, all the publicly owned landfills and six out of eight garbage incinerators were located in African American neighborhoods (Bullard 1992). From 1970 to 1978, three of the four privately owned landfills were located in African American neighborhoods. Although the population of African American neighborhoods made up of 28 percent of total population, 82 percent of total solid waste sites were located in the neighborhoods dominated by them. 14 In 1992, the National Law Journal (NLJ) published results of an analysis of environmental lawsuits. The NLJ conducted a study of all suits held in the previous seven years in the United States. The major finding of the study was that the penalties imposed for environmental law violations in areas inhabited by people of color, whether they are rich or poor, are lower than those imposed for violations in areas largely inhabited by White. The average penalties were six times more in White dominated areas ($336,000 versus $55,000) than African American dominated areas, under the Resource Conservation & Recovery Act's hazardous waste law enforcement (Lavalle & Coyle 1991 ). ENVIRONMENTAL EQUITY - GOVERNMENT INITIATIVES EPA's Position The EPA set up the Environmental Equity Workgroup to assess the available evidence on whether the disadvantaged population groups bear a higher environmental burden than the general population. The group released its report, Environmental Equity - Reducing Risk for all Communities in 1992. The report reviewed existing data on the distribution of environmental exposures and risks across population groups (Reilly 1992). 15 • The workgroup's report provides a cautious, conservative balance to the more assured conclusions of discrimination provided by many other studies. Some of the major findings of the study included : • Although there are clear differences between racial groups in terms of disease and death rates, there is an absence of data to document the environmental contribution to these differences. • It is possible to document differences in observed and potential exposure to some environmental pollutants by socio-economic factors and race. • There is a lack of data on health risks posed by multiple industrial facilities, cumulative and synergistic effects, and multiple paths of exposure. EPA's stand on the issue can be summed up as follows" ... Racial minority and low-income populations experience higher than average exposures to selected air pollutants, hazardous waste facilities, contaminated fish, and agricultural pesticides in the work place. Exposure does not always result in immediate or acute health effects. High exposure and the possibility of chronic effects, are nevertheless a clear cause for health concerns." (Burke 1993) Federal Policies Until very recently, there were no government procedures established to address the environmental equity issue. At best, Resource Conservation and Recovery Act of 1976, (RCRA) creates comprehensive federal guidelines for the production, management, transport, treatment or any other kind of hazardous materials handling. This is a very 16 basic procedure, leaving states with broad guidelines from EPA and RCRA to handle hazardous waste issues (Godsil 1992). basic procedure, leaving states with broad guidelines from EPA and RCRA to handle hazardous waste issues (Godsil 1992). A major achievement at the federal level was the Presidential Order in 1994. President Clinton signed an Executive Order- Federal Actions To Address Environmental Justice in Minority Populations and Low-Income Populations, in 1994, emphasizing protection of disadvantaged population groups. The Order directly addresses the issue of environmental justice: " .... each Federal agency shall make achieving environmental justice part of its mission by identifying and addressing as appropriate, disproportionately high and adverse human health or environmental health of its programs, policies, and activities on minority populations in the United States ... " (Executive Order 12898 of February 11, 1994). State Initiatives The strength of public opposition is the major obstacle faced by the states attempting to evenly distribute the burden of hazardous waste facilities. The Not In My Back Yard (NIMBY) syndrome is one of the important reasons that minorities are disproportionately burdened by hazardous waste facilities. Many states have set up hazardous waste management programs to bypass any local opposition that may arise (Godsil 1991 ). There are four general approaches that states take to address the location of any type of hazardous sites - super review, site designation, local control, and incentives approach. As per the super review process, the developer chooses a site and applies 17 for a permit. The states will typically look for natural resources or constraints, like topography, geology, soils among others around the area for environmental compatibility. The states focus only on the site chosen by the developer. Since the developer chooses a site, it often tends to be in an area with lower land values and that is inhabited by the poor. Under the second approach, some states create an inventory of possible sites that could be used for locating such sites. Since the state is not motivated by the profit motive, the possibility of siting these sites in poor neighborhoods is less likely. However, there are two syndromes that limit the ability of this approach to address equity issues - "Not In My Term Of Office" (NIMTOF) and "Not In My Election Year" (NIMEY) (Godsil 1992). Therefore, a community least able to sustain the NIMBY syndrome, usually a poor and minority community, becomes a host to the site. The local control approach permits local communities to develop regulations that control the siting of hazardous waste sites and these regulations cannot be preempted by state regulations. California and Florida are the only two states currently practicing this approach. The state thus delegates responsibility for waste site location to the local communities. As a result, the NIMBY syndrome actually gets accentuated. To counter this, states have to offer incentives to communities. The incentives control approach, essentially rests on the belief that hazardous waste sites cause an undue burden on neighboring communities while the rest of the state enjoys the benefits. Hence, cooperating communities should be offered economic 18 incentives. Economic incentives are clearly more desirable to poorer communities which, in turn, are often minority communities. State Initiatives Minority communities, hence, become more susceptible to the location of hazardous waste sites. Therefore, current state hazardous waste management programs do not adequately address the equity issue. CONCLUSION The studies described above were performed at differing scales (ZIP code, county, census tract, etc.), used a wide range of analytical methods (correlational, regression, LOGIT models, etc.), and examined a variety of types of hazardous sites (commercial, uncontrolled, landfills, etc.). Together, these studies and reports provide significant evidence of environmental inequity. This project will focus on the extent of environmental inequity in Providence, Rhode Island. 19 19 CHAPTER 3: HYPOTHESIS AND METHODOLOGY This chapter provides a description of the study hypotheses and the methodologies employed to test them. It begins with a description of the study hypothesis and assumptions, discussion of data sources, variables used in the analysis, and description of the analytical methods applied. STUDY HYPOTHESIS The relationship between race and hazardous exposure can be modeled from at least two perspectives. The economic perspective can be used to understand locations of hazardous sites from a cost-benefit trade-off by the concerned parties. From a slightly different perspective, although not mutually exclusive, location of such sites can also be modeled on the basis of the political unity and action-orientation of the residents of affected areas. Classic economic theory would predict that poverty plays a role. The economically disadvantaged people disproportionately suffer a greater share of pollution than those better-off economically. Since poorer people have lesser economic resources, they have more limited choices regarding places to live in. Given limited monetary resources, one might choose to better one's self in a multitude of ways including better housing, better education, healthier environments, or even more consumer goods. How 20 20 a particular person chooses to use his/her buying power is not the issue. Rather, the fact remains that greater buying power affords one a greater set of choices for all purchases, including the choice of where to live. The poorer one is, the less choice one has regarding where to live. Hence, a fundamental assumption of this perspective is that money offers choice. A corollary of this is that wealthier people tend to live in cleaner neighborhoods. a particular person chooses to use his/her buying power is not the issue. Rather, the fact remains that greater buying power affords one a greater set of choices for all purchases, including the choice of where to live. The poorer one is, the less choice one has regarding where to live. Hence, a fundamental assumption of this perspective is that money offers choice. A corollary of this is that wealthier people tend to live in cleaner neighborhoods. Over and above the set of feasible choices is the issue of trade-offs one makes in spending one's buying power. It is certainly possible to maximize on the cleanliness of one's living environment while sacrificing other benefits, say consumer goods, within one's budget constraints. However, poorer people generally can not afford the luxury of being concerned about the state of their environment when confronted by basic survival- related economic problems and are more likely to accept a trade-off for an uncleaner living environment in return for employment and/ or cheaper housing. STUDY HYPOTHESIS NIMBY (Not In My Back Yard) implies an acceptance of the need for the given facility, as long as it is sited outside of the relevant area or "backyard" of those resisting the facility. (This differs from the more ideological "Not In Anybody's Back Yard" which encourages fundamental changes in industry and waste management, thereby eliminating the need for some facilities.) If a facility is perceived as undesirable, there is the chance that there will be resistance to its siting. Facility siting often follows the path of least resistance, coupled with considerations of minimizing costs and maximizing profits. The path of least resistance could include targeting areas where people are not politically active or well connected, have not indicated tendencies toward environmental activism, or perhaps do not have English as their first language (Dear, 1992). All other things being equal, the siting of facilities which are perceived as undesirable often follows the path of least resistance. Minorities, in general, tend to be less politically active and are therefore much more vulnerable to new hazardous facilities being located in their backyards. phenomenon. NIMBY (Not In My Back Yard) implies an acceptance of the need for the given facility, as long as it is sited outside of the relevant area or "backyard" of those resisting the facility. (This differs from the more ideological "Not In Anybody's Back Yard" which encourages fundamental changes in industry and waste management, thereby eliminating the need for some facilities.) If a facility is perceived as undesirable, there is the chance that there will be resistance to its siting. Facility siting often follows the path of least resistance, coupled with considerations of minimizing costs and maximizing profits. The path of least resistance could include targeting areas where people are not politically active or well connected, have not indicated tendencies toward environmental activism, or perhaps do not have English as their first language (Dear, 1992). All other things being equal, the siting of facilities which are perceived as undesirable often follows the path of least resistance. Minorities, in general, tend to be less politically active and are therefore much more vulnerable to new hazardous facilities being located in their backyards. The two perspectives discussed above suggest different causal orders. STUDY HYPOTHESIS In fact, the criteria used in judging the relative merits of choice options may also differ substantially among poorer and wealthier communities. Poorer communities may be less knowledgeable of the implications of the various choice options. Additionally, when survival is a salient issue, long-term implications of choices may appear totally irrelevant. Minorities are typically more economically disadvantaged than Whites for a myriad of reasons. These often include being new arrivals to an area, language differences, educational disadvantages, and the restructuring and suburbanization of industry (Eggers & Massy, 1992). Being more economically disadvantaged, minorities face a narrower set of choices of places to live in and are more limited to lower-cost housing. 21 Land values are also cheaper in polluted neighborhoods and, hence, minorities tend to live in neighborhoods that are polluted by hazardous waste sites. The locations of industrial facilities in a landscape are the result of a complex set of physical, economic, and political factors working together, in addition to an element of chance. The location of transportation corridors and waterways; zoning regulations; local taxes; property values; land availability; and proximity to a market, labor force and input resources are among the plethora of factors that affect where a facility might be located. Due in part to industrial zoning, industries are often located in the less densely populated parts of an urban landscape, and often along transportation arteries. Residential property values are typically lower near industrial areas than in areas without industry (Asabere & Huffman, 1991 ). For this reason, low income communities could develop in and around industrial areas, as the industry offers the dubious appeal of making the area a less desirable place to live and lowering property values and rents. This premise, that areas with polluting industries tend to be less expensive places to live accounts for the potential circumstance of people with low incomes choosing to live in areas with industrial facilities. The political-clout perspective is much more appropriate in cases involving the siting of new facilities. As mentioned previously, many factors affect locational choice. But, as environmental awareness has increased over the last twenty years, so has community resistance to the siting of new potentially polluting facilities such as the NIMBY 22 22 phenomenon. STUDY HYPOTHESIS The economic approach, by itself, does not distinguish between the situation wherein a poorer resident chooses to locate himself/herself in the proximity of an existing hazardous facility and the situation wherein a poorer resident chooses to accept a new facility in his/her backyard. The political perspective, on the other hand, focuses on the acceptance of new facilities only. Distinguishing between these two models would require longitudinal data that permits one to test the implied causality. 23 In this study, the objective is not to test the appropriateness of a process model that would explain the relationship between race and hazardous exposure. Rather, the objective is simply to determine whether race is a significant factor in the relationship with environmental pollution when the effects of other significant variables are removed. As stated earlier, minorities tend to be more economically disadvantaged, have less choices about where to live, and for reasons for affordability might choose to live in areas close to industry. For these socio-economic reasons alone, one would expect to see a disproportionately higher number of minority residents in areas exposed to hazardous emissions. But, the question of interest here is whether or not there is an effect related to race over and above that which can be expected from economic factors alone. To state the hypothesis of this study in statistical terms: If one were to control for the effect of variables such as income level and population density and then examine the numbe~ of polluting facilities in a geographic area as a function of race, the null hypothesis states that the coefficients associated with the racial variable(s) are equal to zero. The alternative hypothesis states that for at least one of the racial variables, the coefficient is non-zero, with a statistically significant degree of confidence. This statistical analysis examines the relationship between the explanatory variables and the dependent variable for a single point in time. As such, this analysis provides no information on changes over time, but rather, quantifies the current relationship. 24 METHODOLOGY In the last fifteen years, a number of studies have investigated the relationship between disadvantaged population groups and hazardous exposure (Anderson 1986, Asch and Seneca 1978, Bryant and Mohai 1992, United Church of Christ 1987, Zimmerman 1986). This literature has indicated that minorities and the poor are likely to be more exposed to environmental hazards than their white and/or richer counterparts. Further, these studies have also established that the costs of environmental degradation are also borne more heavily by minorities and the poor than by other groups. Most of the work in this area, including a review of the studies, is compiled in a recent work by Bryant and Mohai (1992). In their review, Bryant and Mohai (1992a) have tabulated over a dozen studies that addressed the issue of environmental inequity. The majority of these studies, looking at the relationship of income and race with environmental hazard exposure, found both income and race to be significantly related. More importantly, race usually had a greater effect than income after removing out the effect of income. Besides the uniform findings of all these studies, the other theme common to them all is their mode(s) of analysis. Typically, various measures of environmental hazard level are correlated with corresponding socio-economic data using either bivariate or multiple regression methods. The link between the hazard and the exposed population is some measure of geographical location such as zip codes, census tracts, or counties. The exposed population is thus defined as the population in the relevant zip code area, census tract, or county, as the case may be. 25 This mode of matching hazardous site location to affected population opens the door to inaccuracies. For example, assume that a minority dominated census tract abuts a more white dominated tract. A hazardous site located on the fringe of the minority census tract would be considered, in the previous studies, to affect only the minority population. In reality, though, the white population in the adjacent census tract are equally exposed. Such a phenomenon, when aggregated across many sites could have led to the results obtained in previous studies. Ideally then, one needs to better define the hazardous site's DOI and focus on the socio-economic characteristics of this area. This study, used a Geographic Information Systems (GIS) approach to link sources of environmental hazards with affected populations. 1 The term Domain of Influence (DOI) has been coined for this study to define buffers around each TRI site. METHODOLOGY A geographic information system can be defined as a constellation of hardware and software that integrates computer graphics with a relational database for the purpose of managing data about geographic locations (Garson: 1992). These geographic data are both spatial and descriptive in nature. GIS based methods link spatial location information with data bases, allowing population elements to be analyzed I combined with hazardous waste sites. The spatial information can be used to create a buffer, which is an area of measured distance from a selected map element such as a point, line, or a polygon. In the context of this study, the spatial information can be used to demarcate a particular site's Domain of Influence 1 (DOI), which can be linked to socio-economic characteristics of the resident population, GIS based methods link spatial location information with data bases, allowing population elements to be analyzed I combined with hazardous waste sites. The spatial 26 allowing study of the relationship between extent of hazardous exposure and minority characteristics. Burke (1993) used GIS to study race and environmental equity in Los Angeles. However, her analysis was limited to modeling at the census tract level and did not use GIS to identify buffers, and she recommends that future analysis" be implemented by buffering facilities, and then analyzing the characteristics of the "affected population" within the buffer." Burke (1993). The rest of this chapter outlines the methodology used to test the relationship between environmental exposure and race for the City of Providence. A description of the various data sources that are used is followed by a discussion of the variables and their operationalizations. Finally, an overview of the analysis phases is presented. DATA SOURCES Three major data sources were used in this analysis - the U.S. Census Bureau, U.S. Environmental Protection Agency (EPA), and Rhode Island Geographic Information System (RIGIS). The 1990 census data was combined with the census block group boundaries from the RIGIS coverages. The EPA's Toxic Release Inventory system (TRI) provided data on the sites that handle toxic chemicals in substantial amounts in the study area for the year 1992 First, block group boundaries were created in ARC/INFO using U.S. Census Bureau TIGER files. Next, demographic information at the block group level was merged from 27 the RIGIS coverages with these block group boundaries. Census data was used to evaluate the socio-economic characteristics of Providence at the census block group level. The data includes information on population size, race, ethnicity, median housing prices, and median per capita income at the level of block groups. 195 census block groups are included in the analysis. EPA's TRI data, which is the most comprehensive source of data on facilities releasing toxic substances to the environment was used to locate the distribution of toxic emissions sites within Providence. The TRI system was required by the Emergency Planning and Community Right to Know provisions (Title Ill) of the Superfund Amendments and Reauthorization Act (SARA). The Act provides for the collection and public release of information about the presence and release of toxic chemicals in the communities. The goal is to help citizens, officials, and community leaders to be better informed about toxic and hazardous materials in their communities. The TRI data base also provides information on the type of facility by industrial category and type and amount of chemical(s) released into the air, water, and land. There are 41 TRI sites distributed among the census block groups within the scope of this study. Within the GIS, two data sets were developed for analysis. First, a point in polygon overlay was performed in order to determine which census block group each TRI facility lies in. The resulting data set permits a comparison of the demographic characteristics of TRI site affected block groups with those that do not contain TRI sites. By 1992, 21 out of the 195 census block groups ( 11 % ) contained TRI sites. 28 Second, a buffer zone of one-fourth of a kilometer (250 meters) radius was created around each TRI site2• This buffer zone is defined as the particular TRI site's DOI. 2 There is no general consensus on the appropriate radius for the buffer. Bryant and Mohai (1992) in their study in the Detroit area used a 1 mile radius. However, their study focused on only a single hazardous materials facility. In this study, the analysis includes multiple sites, all located within the city of Providence, and the level of analysis is the census block group. Most block groups are far smaller than one mile in any orientation. Using a buffer as large as one mile would have nullified the advantages of a finer-grained analysis obtained by working at the block group level. 3 Using the weighted average approach assumes that all variables at the block group level are uniformly distributed across the block group. The smaller the unit of analysis, block compared to block group compared to census tract, the more reasonable such an assumption is. 4 Variables are described as independent and dependent on the basis of an implicit causal model, although the methodology does not strictly permit us to test any causal relationship. This is in the same spirit as classification of variables in regression procedures. VARIABLES Three kinds of variables are of interest in this study - dependent, independent, and control. The dependent variables4 represent the sources of hazardous emissions. The independent variables are descriptors of the minority population in the block group or DOI, as the case may be. Control variables represent other socio-economic characteristics that can be expected to correlate significantly with the presence/absence of TRI sites but whose effects should be separated out from the pure relationship of race with hazardous exposure. As described earlier, the relationship between hazardous exposure and race will be analyzed with two distinct units of analysis - the block group level and the DOI. Hence, each of the following variables was separately operationalized at both the block group level and the DOI level. DATA SOURCES Note that each DOI can overlap several census block groups. The socio-economic characteristics for the DOI were computed as a weighted average of the socio-economic features of the overlapped block groups with weights being equal to the percentage of the block group area included in the DOl3. When two or more TRI sites are in very close proximity (less than 250 meters apart), their DOis are merged to create a single DOI with multiple TRI sites contained within. Hence, for both data sets, number of TRI sites is a relevant variable for the unit of analysis - the block group or the DOI. This data set permits a comparison of the socio- economic characteristics of geographic areas that are affected by TRI site emissions through uniform proximity to the site with unaffected areas. 29 Hazardous Sites Hazardous Sites Exposure to toxic emissions was captured by noting the presence or absence of TRI sites within the block group. This created a dichotomous variable. In addition, the degree of exposure was operationalized in terms of the number of TRI sites within the block group or DOI. Control Variables Three control variables were used to distinguish the relationship between hazardous exposure and race from the effects of economic factors. Median per capita income and median housing value were used as indicators of the economic well-being of the block groups' residents. Also, the population density (people per sq. km.) was used as a surrogate for both income and land-use factors. Minority Presence Minority presence was operationalized in multiple ways. The census data provides counts of whites, blacks, American Indians, Asians, and Pacific Islanders. For each block group or DOI, the percentage of non-whites was calculated as an aggregate measure of the proportion of minorities. In addition, black and hispanic populations, the 30 significant minorities in Providence, were separately measured both in terms of their count and their proportion. OVERVIEW OF ANALYSIS Several statistical techniques are applied in order to examine the influence of race on proximity to polluting facilities. Data sets created using ARC/INFO and ARCVIEW are analyzed using the Statistical Analysis System (SAS) procedures. The analysis can be described as consisting of three phases. 31 In the descriptive phase, summary statistics and simple bivariate relationships are computed for all the explanatory and dependent variables. This information allows us to get a feel for the data, identify anomalies if any, and also check whether the use of statistical procedures assuming linearity will be valid. In the second phase, the relationship between race and exposure to toxic emissions is examined using the block group as the unit of analysis. Two separate tests are conducted. First, the presence or absence of TRI sites is used as the classification variable. This provides a comparison of the minority proportion of affected versus unaffected block groups using t-tests. Next, the presence I absence of TRI sites is used as the dependent variable in a regression-like model. The intent of this analysis is to determine the relationship between the independent variables {population density, income, housing values, and one or more variables representing racial composition) and the dependent variable (presence or absence of TRI facilities in a block group). However, the dependent variable , presence or absence of TRI sites, is a binary variable and its distribution does not in any way resemble a statistical normal distribution. Of the 195 block groups under study, only 21 of them contain at least one TRI site while the balance 174 have no TRI site. Hence, a simple regression model using Ordinary Least Squares is not acceptable. Following the approach adopted by Burke (1993), the presence I absence of TRI sites is therefore modeled as a Logistic regression problem instead and the relationship with explanatory and control variables tested within a general linear model framework. 32 Several combinations of explanatory variables are evaluated within this model structure. The influence of population density, median per capita income and median housing value is always included in the models. The explanatory variables used as an indication of race /ethnicity vary. A variable reflecting the total Minority Proportion is used in the basic model, while two separate variables reflecting the black and hispanic proportions of each block group are evaluated in a second model. CHAPTER 4: ANALYSIS AND RESULTS The relationship between racial composition and hazardous exposure was analyzed using multiple approaches. This chapter, first presents a descriptive analysis of all the key variables at the block group level. Next, analyzes the relationship between racial composition and exposure to hazardous materials with the census block group as the unit of analysis. This approach parallels previous studies in that the unit of analysis is a geographic zone determined by criteria independent of the location of TRI sites. The final section, presents the results obtained by using the DOI as the unit of analysis. DOis were determined by creating a buffer zone of 250 meters around TRI sites using GIS procedures. Unlike census block groups, DOis are anchored to the existing TRI sites and their utilization as the unit of analysis reflect the unique contribution of this study. OVERVIEW OF ANALYSIS In the third phase, the analysis approach duplicates that of the second phase. However, the unit of analysis is the DOI rather than the census block group. A comparison of the results from this phase with those obtained by using the block group as the unit of analysis (second phase) will permit us to detect whether the results of past studies were simply methodological artifacts. A hypothesis testing framework will be used in this analysis, and individual coefficients will be evaluated at the .05 level of significance for a two-tailed test. This level of significance affords a high degree of confidence that the coefficient of interest is non- zero. Although this framework is technically appropriate for inferring characteristics about the whole population from a sample, it is also commonly used in modeling an entire population, such as in analysis including all U.S. counties. 33 UNIVARIATE ANALYSIS There are 41 TRI sites located in Providence. The distribution of these sites across the block groups is shown in Figure 2. Two observations can be made about the locations of these sites. First, it appears that the sites are contained in band-like clusters that are adjacent to the major roads. Second, most of the sites are concentrated in the South Providence area. 34 Location of. TRI Sites Location of. TRI Sites • TRI Sites N Major Roads ·o Census Block Groups 0 1 2 3 Kilometers Oata Sources: Census, EPA, and RIGIS Map produced by: Sangeeta Jain CPAD, URI (1995) Figure #2 TRI Sites Major Roads Census Block Groups Oata Sources: Census, EPA, and RIGIS Map produced by: Sangeeta Jain CPAD, URI (1995) 3 Kilometers Providence has a total population of 160, 199 (1990 census), with a majority (about 70 %) being white. The total number of census block groups in the City is 195, out of which five have no population. Summary statistics are shown in Table 1. Providence has a total population of 160, 199 (1990 census), with a majority (about 70 %) being white. The total number of census block groups in the City is 195, out of which five have no population. Summary statistics are shown in Table 1. Table 1: Racial Composition by Block Groups City of Providence (1990) Variables White population Non white population Black population Hispanic population Total Population Total 111,921 48,278 23,820 24,973 160, 199 Percent 69.86 30.14 14.87 15.59 100.00 Table 1: Racial Composition by Block Groups The total population is distributed over 190 block groups with a mean population of 843 per block group. The most populated block group has 2461 residents, while the least populated block group has only 9 residents, and the median population per block group is 756. In this study, population density of block groups (population/ area in square kilometers) is of greater interest than the absolute population as it removes the effect of the block group area. The average population density for block groups is 4,445 persons per sq. km. Figure 3 displays the variation in density over block groups. Minority population 36 Population Density • TRI Sites Persons per Sq. Km. Q Less than 2,000 . ·.• 2,000 - 4,000 .. UNIVARIATE ANALYSIS 4,001 - 6,000 - Above 6,001 0 1 2 3 Kilometers Data Sources: Census, EPA, and RIGIS Map produoed by: Sangeeta Jain CPAD, URI (1995) F/gum t 3 Data Sources: Census, EPA, and RIGIS Map produoed by: Sangeeta Jain CPAD, URI (1995) 3 Kilometers 70 I/) Cl) 60 :;::; ~ 50 0 c 40 ~ - 30 c Cl) 20 u ... 10 Cl) ll. 0 - I/) 100 ~ u CV '° - 60 c Cl) ~ 40 Cl) ll. 20 0 100 I/) 90 u 80 c 70 CV c.. 60 I/) :f 50 - 40 c Cl) 30 u ... Cl) ll. 10 0 Frequency Distribution Of Minority Proportions Minority Population 10 20 30 40 50 60 70 80 90 100 Frequency intervals Black Population 10 20 30 40 50 60 70 80 90 100 Frequency intervals Hispanic Population 10 20 30 40 50 60 70 80 90 100 Frequency intervals Figure# 4 Frequency Distribution Of Minority Proportions Minority Population Frequency Distribution Of Minority Proportions 100 I/) 90 u 80 c 70 CV c.. 60 I/) :f 50 - 40 c Cl) 30 u ... Cl) ll. 10 0 10 20 30 40 50 60 70 80 90 100 Frequency intervals Hispanic Population 10 20 30 40 50 60 70 80 90 100 Frequency intervals Figure# 4 Hispanic Population Figure# 4 (non-whites)5 in block groups varies widely with the inter-quartile range extending from 6.9% to 49.1 %. The distribution of minorities across block groups has a bimodal distribution as shown in Figure 4. There are many block groups with under 10% minorities (i.e. largely white dominated block groups), and there is a cluster of block groups with 70% to 80% minority populations. Forty seven of the 190 populated block groups in Providence have a minority population in excess of 50%. Figure 5 shows the geographic distribution of minorities among the City's block groups. High-minority block groups tend to be clustered together in the South Providence area. A visual examination of the figure indicates that more TRI sites are located in the high- minority areas than in the low-minority areas. Blacks and hispanics comprise the bulk of the minority population in Providence. 5 In this report, the term minorities is used to refer to non-white residents. 5 In this report, the term minorities is used to refer to non-white residents. 39 UNIVARIATE ANALYSIS The mean proportion of blacks per block group is 15.2% but the median is only 6.5%, indicating that there are many more block groups with low black proportions than block groups with high black proportions (see Figure 4). The high-black proportion block groups are clustered in the South Providence area and generally tend to mirror the distribution of high-minority block groups (see Figure 6). 39 Spatial Distribution of Minorities p • TRI Sites Percent minorities by Block Groups [:=J 1st Quartile (0 - 7%) D 2nd Quartile (7 - 18%) ~ 3rd Quartile (18 - 49%) - 4th Quartile (49 - 100%) 0 1 2 3 Kilometers Data Sources: Census, EPA, and RIGIS Map produced by: Sanaeeta Jain CPAD, URI (1995) F/gul8 #5 • TRI Sites Percent minorities by Block Groups [:=J 1st Quartile (0 - 7%) D 2nd Quartile (7 - 18%) ~ 3rd Quartile (18 - 49%) - 4th Quartile (49 - 100%) Data Sources: Census, EPA, and RIGIS Map produced by: Sanaeeta Jain CPAD, URI (1995) 3 Kilometers Spatial Distribution of Blacks Spatial Distribution of Blacks • TRI Sites Percent Blacks by Block Groups f 7> ... H 1st Quartile (O - 2%) D 2nd Quartile (2- 7%) 3rd Quartile (7 - 23%) - 4th Quartile (23 - 79%) 0 1 2 3 Kilometers Data Sources: Census, EPA, and RIGIS Map produced by: Sangeeta Jain CPAD, URI (1995) Figure# ts • TRI Sites Percent Blacks by Block Groups f 7> ... H 1st Quartile (O - 2%) D 2nd Quartile (2- 7%) 3rd Quartile (7 - 23%) - 4th Quartile (23 - 79%) Data Sources: Census, EPA, and RIGIS Data Sources: Census, EPA, and RIGIS Map produced by: Sangeeta Jain CPAD, URI (1995) 3 Kilometers Spatial Distribution of Hispanics Spatial Distribution of Hispanics • TRI Sites Percent Hispanics by Block Groups a1l 1st Quartile (0 - 3%) 2nd Quartile (3 - 10%) .. 3rd Quartile (10 - 25%) .. 4th Quartile (25 - 63%) 0 1 2 3 Kilometers Oata Sources: Census, EPA, and R/GIS Map produoed by: Sangeeta Jain CPAD, URI (1995) Figure #1 • TRI Sites Percent Hispanics by Block Groups a1l 1st Quartile (0 - 3%) 2nd Quartile (3 - 10%) .. 3rd Quartile (10 - 25%) .. UNIVARIATE ANALYSIS 4th Quartile (25 - 63%) Oata Sources: Census, EPA, and R/GIS Map produoed by: Sangeeta Jain CPAD, URI (1995) 3 Kilometers Figure #1 There are approximately equal numbers of blacks and hispanics in Providence as shown in Table 1. Distribution of hispanics across block groups parallels the distribution of blacks with many low-hispanic block groups (median = 9.6%) but a few with higher hispanic proportions (mean = 16%). Figure 4 shows the frequency distribution of hispanic proportions in block groups, and Figure 7 displays the concentration of hispanics among Providence's block groups. Once again, the hispanic population appears to be generally located in the vicinity of the South Providence area. By comparing Figures 5, 6, and 7, one can see that there is substantial overlap in the minority, black, and hispanic concentrations. However, a closer examination of Figures 6 and 7 suggests that there are some minority dominated block groups that are either black dominated or hispanic dominated. More importantly, this observation suggests that the relationship of TRI site location with minority presence needs to be studied separately for blacks and hispanics. As discussed in previous chapters, racial composition of geographic areas tends to be highly correlated with both income and housing values. In order to determine the direct relationship of race with hazardous exposure, it is imperative that effects of income and housing value be isolated. Median per capita income and median home value for block groups were used as the income and housing value indicators for each block group. There is great variation in income within different block groups. The average per capita income is about $10,023 with minimum and maximum being $175 and $52,335 respectively. The average median housing value of owner occupied housing units is $103,400, ranging 43 Median per Capita Income • TRI Sites Per Capita Income by Block Groups ft« := I 1st Quartile (Below $6,933) · · 2nd Quartile ($6,933 - 9,926) 3rd Quartile ($9,926 - 13,588) .. UNIVARIATE ANALYSIS 4th Quartile ($13,588 - 52,335) 0 1 2 3 Kilometers Data Sources: Census, EPA, and RIGIS Map produced by: Sangeeta Jain CPAD, URI (1995) F/gul8 ti 8 Data Sources: Census, EPA, and RIGIS Map produced by: Sangeeta Jain CPAD, URI (1995) Data Sources: Census, EPA, and RIGIS Map produced by: Sangeeta Jain CPAD, URI (1995) Median Housing Values • TRI Sites Housing Values (in $'000) CJ 1st Quartile (Below 85) CJ 2nd Quartile (85 - 102) k;:,-. 1 3rd Quartile (102 - 120) if~ 4th Quartile (Above 120) 0 1 2 3 Kilometers Oata Sources: Census, EPA, and RIGIS Map produced by: Sangeeta Jain CPAO, URI (1995) FigUl8 # 9 Median Housing Values • TRI Sites Housing Values (in $'000) CJ 1st Quartile (Below 85) CJ 2nd Quartile (85 - 102) k;:,-. 1 3rd Quartile (102 - 120) if~ 4th Quartile (Above 120) • TRI Sites Housing Values (in $'000) CJ 1st Quartile (Below 85) CJ 2nd Quartile (85 - 102) k;:,-. 1 3rd Quartile (102 - 120) if~ 4th Quartile (Above 120) Oata Sources: Census, EPA, and RIGIS Map produced by: Sangeeta Jain CPAO, URI (1995) Oata Sources: Census, EPA, and RIGIS Map produced by: Sangeeta Jain CPAO, URI (1995) 3 Kilometers from $52,500 to $423,400. Figures 8 and 9 show the geographic distribution of income and housing value across all the block groups in the city. As expected, there is a substantial correlation between income and housing value for block groups. More importantly, a comparison of these Figures with Figure 5 shows a clear disparity between the high-minority areas and the high-income/high-value areas. Minority concentrations tend to be in low-income and low-value block groups. CORRELATION ANALYSIS Table 2 provides the significant (p < .05) bivariate Pearson correlations among all the key variables. The presence I absence of TRI sites (TYES), the main dependent variable, is a bivariate variable and, hence, all correlations with it are biserial correlations. Three conclusions can be drawn regarding its correlations with other variables. First, population and density are negatively correlated with the presence of TRI sites. Second, the greater the income, housing value, and number of white residents of a block group, lower the chances of a TRI site being located in that block group. The significant positive correlations among income, housing value, and number of white residents suggests that these three variables will tend to have a combined effect in most of the analysis. Third, TRI site locations are positively correlated with the proportion of minorities and hispanics, but not blacks. 46 Table 2: Bivariate Pearson Correlations Among Key Variables (Only correlations significant at p < 0.05) TYES TN um DNS VAL INC POP WH NWH BLK HSP PNW PBLK TN um 0.75 DNS -0.20 -0.27 VAL -0.12* INC -0.17 -0.26 0.60 POP -0.13* -0.20 0.55 WH -0.18 -0.18 0.27 0.24 0.18 0.78 NWH 0.50 -0.23 0.51 BLK 0.35 -0.23 0.42 0.93 HSP 0.52 -0.31 0.45 0.89 0.73 PNW 0.14 0.26 -0.37 0.77 0.76 0.67 PBLK 0.14* -0.33 0.65 0.77 0.47 0.92 PHSP 0.17 0.28 -0.43 0.61 0.51 0.74 0.83 0.63 *p < 0.1 Terminology. TYES Presence of TRI in block group NWH Total non whites TNum Number of TRI sites BLK Total blacks DNS Population density HSP Total hispanics VAL Value of owner occupied units PNW Proportion non whites INC Per capita income PBLK Proportion blacks POP Total population PHSP Proportion hispanics WH Total whites Focusing on the variables capturing racial composition, two observations are noteworthy. First, high-minority block groups tend to have high-black (r = 0.92) and 47 Table 2: Bivariate Pearson Correlations Among Key Variables (Only correlations significant at p < 0.05) Terminology. TYES Presence of TRI in block group NWH Total non whites TNum Number of TRI sites BLK Total blacks DNS Population density HSP Total hispanics VAL Value of owner occupied units PNW Proportion non whites INC Per capita income PBLK Proportion blacks POP Total population PHSP Proportion hispanics WH Total whites Focusing on the variables capturing racial composition, two observations are noteworthy. CORRELATION ANALYSIS First, high-minority block groups tend to have high-black (r = 0.92) and Focusing on the variables capturing racial composition, two observations are noteworthy. First, high-minority block groups tend to have high-black (r = 0.92) and 47 high-hispanic proportions (r = 0.83). However, the correlation between black and hispanic proportion is lower (r = 0.63), although significant. This, in conjunction with the fact that presence of TRI sites is significantly correlated with high-minority and high- hispanic, but not with high-black population proportions, reaffirms the need for separate analysis for blacks and hispanics, as noted previously. Second, the proportions of minorities, blacks, and hispanics have similar correlations with block group density, income, and housing value. The bivariate scatter plots shown in Figures 10, 11, and 12 provides a graphic depiction of these relationships. Minorities tend to be in block groups with higher density, lower median per capita income, and lower median housing values. Each of these three variables is, in turn, significantly related to the presence I absence of TRI sites. Hence, it is difficult to isolate the relationship between minority proportion of a block group and the presence I absence of a TRI site on the basis of correlation analysis alone. A multivariate procedure such as multiple regression is needed to isolate the effect of minority proportion. Finally, this study intended to also investigate the relationship between racial composition of block groups and the number of TRI sites in the block group. This analysis would obviously have been appropriate only after establishing a link between racial composition and the presence I absence of TRI sites. However, as shown in Table 2, the number of TRI sites (TNum) is not significantly correlated with any minority population related variables. This may be partly due to the very skewed distribution of number of TRI sites: the 41 TRI sites are spread over 21 block groups with 15 block groups having only one TR I 48 Scatter Plots - Density and Population Groups 0 ? 14000 -; 12000 ~ 10000 Q. Ill 8000 c 0 6000 Ill ... GI e:. 4000 ?:' iii 2000 c GI 0 Q 0 ? 14000 ~ 12000 - Ill ~ 10000 Q. Ill 8000 - c 0 6000 - Ill ... ! CORRELATION ANALYSIS 4000 ?:' iii 2000 c GI 0 Q 0 Scatter Plots - Density and Population Groups • Density and Minority Population • • • •• • • • ••• ••• ••• . .,.,. , .. ·• • • ••••• ·' .. • :• • 10 20 30 40 50 60 70 80 90 Proportion Minorities Density and Black Population • • , . •• •• , ..... • •• • • • .. . . ,. ,. • •.... ~ ... • • • . -.· . •• •• .... 10 • • 10 ••• .. . • • • • • • •• • • ... , • 20 30 40 50 60 Proportion Blacks Density and Hispanic Population • .. - • I • • • .• • • r. • • .. ... • =- ....... ~ .. - ,,, • .._ I . -.. .. . ...... . . .-. ... 20 30 40 50 Proportion Hispanics Figure# 10 • 70 • • 60 100 • 80 70 0 Scatter Plots Density and Population Groups • Density and Minority Population • • • •• • • • ••• ••• ••• . .,.,. , .. ·• • • ••••• ·' .. • :• • 10 20 30 40 50 60 70 80 90 Proportion Minorities 100 ? 14000 -; 12000 ~ 10000 Q. Ill 8000 c 0 6000 Ill ... GI e:. 4000 ?:' iii 2000 c GI 0 Q 0 Density and Black Population • • , . •• •• , ..... • •• • • • .. . . ,. ,. • •.... ~ ... • • • . -.· . •• •• .... 10 ••• .. . • • • • • • •• • • ... , • 20 30 40 50 60 Proportion Blacks • 70 • 80 Density and Black Population ? 14000 ~ 12000 - Ill ~ 10000 Q. Ill 8000 - c 0 6000 - Ill ... ! 4000 ?:' iii 2000 c GI 0 Q 0 • • 10 Density and Hispanic Population • .. - • I • • • .• • • r. • • .. ... • =- ....... ~ .. - ,,, • .._ I . -.. .. . ...... . . .-. ... CORRELATION ANALYSIS 20 30 40 50 Proportion Hispanics Figure# 10 • • 60 70 Density and Hispanic Population Figure# 10 Scatter Plots - Median Per Capita Income and Population Groups Scatter Plots - Median Per Capita Income and Population Groups Scatter Plots - Median Per Capita Income and Population Groups Income and Minority Population ~ 60000 ~-------------------------.... 50000 • .!!! g. 30000 - 0 ~ 20000 0. ~ 10000 • " t.wl1' I ~ O+----+--t.--+----+-------w11-----1----t----+--~--- 0 10 20 30 40 50 60 70 80 90 100 Proportion Minorities Income and Black Population E 60000 -.......----------------------------. G> E 50000 0 u .: 40000 .!!! g. 30000 - 0 .. 20000 G> 0. c 10000 CQ =g ~ 0 0 E 60000 G> 50000 E 0 u .: 40000 .!!! g. 30000 0 .. 20000 G> 0. c 10000 CQ '6 G> 0 ~ 0 . -. ...._• . ...-.... ·.. . . .. ....... ~ .. --- ._ ... -.· .. -,.. 10 20 30 40 50 60 Proportion Blacks Income and Hispanic Population ...... 10 20 30 40 50 Proportion Hispanics Figure# 11 • 70 80 60 70 Income and Minority Population ~ 60000 ~-------------------------.... 50000 • .!!! g. 30000 - 0 ~ 20000 0. ~ 10000 • " t.wl1' I ~ O+----+--t.--+----+-------w11-----1----t----+--~--- 0 10 20 30 40 50 60 70 80 90 100 Proportion Minorities Income and Black Population E 60000 -.......----------------------------. G> E 50000 0 u .: 40000 .!!! g. 30000 - 0 .. 20000 G> 0. c 10000 CQ =g ~ 0 0 E 60000 G> 50000 E 0 u .: 40000 !!! . -. ...._• . ...-.... ·.. . . .. ....... ~ .. --- ._ ... -.· .. -,.. 10 20 30 40 50 60 Proportion Blacks Income and Hispanic Population • 70 80 50000 • .!!! g. 30000 - 0 ~ 20000 0. ~ 10000 • " t.wl1' I ~ O+----+--t.--+----+-------w11-----1----t----+--~--- 0 10 20 30 40 50 60 70 80 90 100 Proportion Minorities Income and Black Population E 60000 -.......----------------------------. G> E 50000 0 u .: 40000 .!!! g. 30000 - 0 .. 20000 G> 0. c 10000 CQ =g ~ 0 0 E 60000 G> 50000 E 0 u .: 40000 .!!! g. 30000 0 .. 20000 G> 0. c 10000 CQ '6 G> 0 ~ 0 . -. ...._• . ...-.... ·.. . . .. ....... ~ .. --- ._ ... -.· .. CORRELATION ANALYSIS -,.. 10 20 30 40 50 60 Proportion Blacks Income and Hispanic Population ...... 10 20 30 40 50 Proportion Hispanics Figure# 11 • 70 80 60 70 Income and Black Population E 60000 G> 50000 E 0 u .: 40000 .!!! g. 30000 0 .. 20000 G> 0. c 10000 CQ '6 G> 0 ~ 0 Income and Hispanic Population ...... 10 20 30 40 50 Proportion Hispanics Figure# 11 60 70 Income and Hispanic Population Figure# 11 Scatter Plots - Median Housing Values and Population Groups Housing Values and Minority Population 450000----------------------------. 400000 ' • ' ~ 350000 : gi 300000 -.... ~ 250000 •• • I C) 200000 -· c: u; 150000 :J ~ 100000 50000 0 10 20 30 40 50 60 70 80 90 100 Proportion Minorities Housing Values and Black Population 450000 --------------------------- 400000 E 350000 gi 300000 :J ~ 250000 C) 200000 - c: u; 150000 :J ~ 100000 50000 • ~.....-~.~ -~ .· ..:.__. o--~---+----11.------------i---+--------+----'f 0 10 20 30 40 50 60 70 80 Proportion Blacks Housing Values and Hispanic Population 450000 400000 ••• E 350000 • gi 300000 ~· :J ~ 250000 • C) 200000 c: u; 150000 :J 0 100000 J: 50000 • 0 0 10 20 30 40 50 60 70 Proportion Hispanics Scatter Plots - Median Housing Values and Population Groups Housing Values and Minority Population 450000----------------------------. 400000 ' • ' ~ 350000 : gi 300000 -.... ~ 250000 •• • I C) 200000 -· c: u; 150000 :J ~ 100000 50000 0 10 20 30 40 50 60 70 80 90 100 Proportion Minorities Scatter Plots - Median Housing Values and Population Groups Scatter Plots - Median Housing Values and Population Groups Housing Values and Black Population 450000 --------------------------- 400000 E 350000 gi 300000 :J ~ 250000 C) 200000 - c: u; 150000 :J ~ 100000 50000 • ~.....-~.~ -~ .· ..:.__. o--~---+----11.------------i---+--------+----'f 0 10 20 30 40 50 60 70 80 Proportion Blacks Housing Values and Black Population Housing Values and Hispanic Population 450000 400000 ••• E 350000 • gi 300000 ~· :J ~ 250000 • C) 200000 c: u; 150000 :J 0 100000 J: 50000 • 0 0 10 20 30 40 50 60 70 Proportion Hispanics Figure# 12 Housing Values and Hispanic Population Figure# 12 site, one block group having 3 TRI sites, four having 4 TRI sites, and one having 7 TRI sites. COMPARISON OF BLOCK GROUPS WITH AND WITHOUT TRI SITES In this phase, block groups are identified as either containing at least one TRI site or containing no TRI sites. Comparisons of the two sets of block groups are then undertaken using univariate as well as multivariate procedures. CORRELATION ANALYSIS In conclusion, the correlation analysis suggests that locations of TRI sites are related to the proportions of minority and hispanic population in the affected areas. Further analysis is needed to separate the effect of minority populations from the effects of density, income, and housing value. Also, analysis of the relationship between minority population and number of TRI sites will not be pursued since there were no significant correlations in this category. Univariate Analysis: t-tests T-tests can provide indications of significant differences between the two sets of block groups. Summary statistics are presented in Table 3 separately for block groups with TRI sites, for those without TRI sites, and for all the block groups with non-zero populations. Results oft-tests for the hypothesis of no difference between the two sets are also provided. 52 Table 3: Summary Statistics of Key Variables by Block Groups Presence of TRI t-test Variables Statistic No Yes Overall t df p > ltl Population Count 169 21 190 2.8 188 0.0056 density Mean 4754 3019 4445 Std Dev 2690 2542 2783 Total Count 169 21 190 1.8639 188 0.0639 population Mean 866 659 843 Std Dev 471 553 484 Median Count 164 19 183 3.3778 57.8 0.0013 housing Mean 131175 99984 127937 value (in$) Std Dev 80532 29473 77365 Median per Count 167 21 188 4.6315 68.2 0.0001 capita Mean 12833 8087 12303 income (in $) Std Dev 9226 3369 8890 White Count 169 21 190 2.4881 188 0.0137 population Mean 615 377 589 Std Dev 413 409 418 Black Count 169 21 190 -0.2489 188 0.8037 population Mean 124 134 125 Std Dev 178 139 174 Hispanic Count 169 21 190 -0.9652 188 0.3357 population Mean 127 163 131 Std Dev 154 195 159 Proportion Count 169 21 190 -1.9901 188 0.048 non-white Mean 0.28 0.41 0.30 population Std Dev 0.28 0.28 0.28 Proportion Count 169 21 190 -1.511 188 0.1325 black Mean 0.14 0.21 0.15 population Std Dev 0.18 0.17 0.18 Proportion Count 169 21 190 -2.4067 188 0.0171 hispanic Mean 0.15 0.23 0.16 population Std Dev 0.15 0.17 0.15 Table 3: Summary Statistics of Key Variables by Block Groups 53 Block groups with TRI sites have a lower population density (mean = 3019 people/sq. km) than those with TRI sites (mean= 4754 people/sq. km), and this difference is significant (t = 2.8, df = 188, p < 0.01 ). Similarly, the average population tends to be lower in block groups containing TRI sites (t = 1.86, df = 188, p < 0.1 ). In terms of income and housing values, block groups with TRI sites have higher median per capita incomes (t = 4.63, df = 68.2, p < 0.0001) and lower median housing values (t = 3.3778, df = 57.8, p < 0.05) than block groups without any TRI sites. Univariate Analysis: t-tests Considering the racial composition of block groups, TRI containing block groups do not differ from block groups with no TRI sites in the absolute number of either black residents or hispanic residents. However, there are significantly fewer white residents in TRI affected block groups (t = 2.4881, df = 188, p < 0.05). More relevant than the absolute number of residents of any racial category are the proportions of each category in a block group. Overall, TRI affected block groups have a significantly greater proportion of non-white residents (t = -1.99, df = 188, p < 0.05). However, this difference is true only for the proportion of hispanics (t = -2.41, df = 188, p <0.05) and not for the proportion of black residents. In conclusion, t-test based analysis suggests that th~ higher the proportion of hispanics and minorities in a block group per se, the greater is the chance of the block group containing at least one TRI site. However, the presence or absence of TRI sites is also significantly related to the block groups' population density, median per capita income, 54 and median housing value, which in turn were correlated with minority proportions. Hence, while it cannot be concluded from this analysis that racial inequity exists, there is clear evidence of the possibility and the need to pursue analysis that will yield an estimate of the pure effect of racial composition, isolated from income, density, and housing value. MULTIVARIATE ANALYSIS - LOGIT This study initially intended to analyze both the presence/absence of TRI sites and the number of TRI sites as independent variables. However, correlation analysis showed that the number of TRI sites in a block group was not significantly correlated with any independent variables of interest. Hence, this analysis will focus, instead, on the presence/absence of TRI sites in the block group. Presence/absence of TRI sites is a binary dependent variable and, therefore, not suitable for traditional multiple regression. LOGIT analysis is more suited for the analysis of binary dependent variables and has been used previously in investigations of environmental equity (Burke, 1993). If pis the probability of a TRI site being present, then (1-p) is the probability of a TRI site being absent and (p/(1-p)) becomes the odds ratio of a TRI site being present. In LOGIT modeling, the dependent variable is defined as the natural log of the odds ratio or LOGIT (p) = log ( p I (1-p) ). 55 The advantage of recasting the binary dependent variable into this form is that while the probability p can only vary between O and 1, the LOGIT(p) can vary between minus and plus infinity and is hence more suitable for an assumption of a normal distribution. The independent variables are still framed in terms of a linear model similar to that in multiple regression. Hence, an example of the model estimated below is: LOGIT (p) = bO + b1 (BGINCOME) + b2 (BGDNSITY) + b3 (BGTOTPOP) where bO, b1, b2, and b3 are coefficients to be estimated and BGINCOME, BGDNSITY, and BGTOTPOP are block group income, block group density, and block group total population respectively. LOGIT (p) = bO + b1 (BGINCOME) + b2 (BGDNSITY) + b3 (BGTOTPOP) where bO, b1, b2, and b3 are coefficients to be estimated and BGINCOME, BGDNSITY, and BGTOTPOP are block group income, block group density, and block group total population respectively. The LOGIT model was estimated using the Logistic Procedure in SAS (Version 6) using different combinations of the independent variables for all 195 block groups in the study area6. The dependent variable BGTRIYES is a binary variable for which the value 1 indicates at least one TRI site in the block group. The independent variables fall into two classes: (i) indicators of minority residents, and (ii) relevant covariates. 6 As noted previously, 5 of the block groups had no population. Since each of the LOGIT models estimated used at least one population related independent variable, these 5 block groups consistently had missing data. MULTIVARIATE ANALYSIS - LOGIT Minority population was determined either by considering the proportion of non-whites in the block group (BGPNWH) or the combination of black and hispanic proportions in the block group (BGPBLK and BGPHSP respectively). Covariates were variables that, as per the correlation analysis, were significantly related to both the presence/absence of TRI sites on the one hand and minority population indicators on the other hand. The 56 covariates included income, housing value, population density, and total population. By incorporating these covariates into the model, this study will be able to identify the uncontaminated link between racial characteristics of the block group and presence/absence of TRI sites. The first model estimated used BGNWH as the independent variable of interest and all the covariates. The intention here was to allow for any possible effects. Table 4 shows the results of estimation. 182 block groups had non-missing data on these independent variables with 19 of the 21 block groups containing TRI sites included in the estimation. The Log Likelihood ratio (-2LOG L) tests the incremental predictive power of the independent variables over using the intercept alone, and the Score statistic gives a test for the joint significance of the explanatory variables. The model fit very well, as is evident from the highly significant chi-squares for both -2LOG L (p < 0.001) and Score (p < 0.005). However, among the independent variables in the model, only block group density - BGDNSITY (p < 0.005) had a significant effect. The second model estimated used block group density (BGDNSITY) as the covariate and proportions of blacks (BGPBLK) and hispanics (BGPHSP) as the explanatory variables of interest. As shown in Table 5, once again the model fit extremely well in terms of both Score and -2LOG L (p < 0.001 ). In terms of the independent variables, BGDNSITY was again significantly (p < 0.001) related to the probability of a TRI site being present in the block group. The negative sign in its estimated coefficient suggests that as density increases, the probability of a TRI site being present decreases. This is consistent with previous results using correlation analysis and t-tests. MULTIVARIATE ANALYSIS - LOGIT 57 Table 4: Block Groups Based LOGIT Analysis - Model 1 Analysis unit is block groups Dependent variable: BGTRIYES: Presence of TRI site(s) in block group Independent variables: BGINCOME, BGVALUE, BGDNSITY, BGPNWH, BGTOTPOP Response Profile BGTRIYES COUNT 0 163 19 Total 182 (13 observation(s) were deleted due to missing values) Criteria for Assessing Model Fit Criterion Intercept Intercept and Chi-Square for Independents -2 LOG L Score Variable INTERCPT BG INCOME BGVALUE BGDNSITY BGTOTPOP BGPNWH Variable INTERCPT BG INCOME BGVALUE BGDNSITY BGTOTPOP BGPNWH Only Independents 121.807 100.841 20.966 with 5 df {p=0.0008) 19.17 with 5 df (p=0.0018) Analysis of Maximum Likelihood Estimates df Parameter Standard Wald Chi- Pr> Chi- Standardized Estimate -0.0335 -0.0001 1 . -0.00000221 -413.4 0.000239 1.7053 Odds Ratio Odds Ratio 0.967 1 1 0 5.503 Error Square Square 1.2085 0.0008 0.9779 0.000078 1.6104 0.2044 0.0000066 0.1098 0.7404 144 8.2407 0.0041 0.000628 0.1454 0.703 1.1979 2.0266 0.1546 Variable Label Intercept Median Block group income Median home value in block group Population density of block group Total population of block group Proportion non-whites in block group Estimate -0.46~432 -0.094655 -0.608736 0.06195 0.261626 Table 4: Block Groups Based LOGIT Analysis - Model 1 alysis of Maximum Likelihood Estimates 58 Table 5: Block Groups Based LOGIT Analysis - Model 2 Analysis unit is block groups Dependent variable: BGTRIYES: Presence of TRI site(s) in block group Independent variables: BGDNSITY, BGPBLK, BGPHSP Response Profile BGTRIYES COUNT 0 169 1 21 Total 190 (5 observation(s) were deleted due to missing values) Criterion -2 LOG L Score Variable INTERCP BGDNSIT BGPBLK BGPHSP Variable INTERCP BGDNSIT BGPBLK BG PH SP Criteria for Assessing Model Fit Intercept Only 132.093 Intercept and Chi-Square for Independents Independents 113.127 18.967 with 3 df (p=0.0003) 18.51 with 3 df (p=0.0003) Analysis of Maximum Likelihood Estimates df Parameter Standard Wald Chi- Pr> Chi- Estimate Error Square Square -1.5365 0.4816 10.1801 0.0014 -388.4 118.4 10.7648 0.001 0.3545 1.6076 0.0486 0.8255 4.6937 1.9407 5.8491 0.0156 Odds Ratio Odds Ratio Variable Label 0.215 Intercept 0 Population density of block group 1.426 Proportion blacks in block group 109.252 Proportion hispanics in block group Standardized Estimate -0.583045 0.035166 0.390586 Table 5: Block Groups Based LOGIT Analysis - Model 2 Table 5: Block Groups Based LOGIT Analysis - Model 2 59 BGPBLK and BGPHSP were the two variables representing proportion of minority residents in the block group. MULTIVARIATE ANALYSIS - LOGIT The estimated coefficients suggest that the proportion of blacks in a block group is not related to the absence I presence of a TRI site. However, the proportion of hispanics is significantly related to the odds of a TRI site in the block group (p < 0.05). The coefficient for hispanic proportion (4.69) is far greater than that for the black proportion (0.35) although both variables are measured on the same scale. In conclusion, there is evidence for environmental inequity in Providence in that block groups with a higher proportion of Hispanics, a large minority group in Providence, are more likely to contain at least one TRI site compared with block groups containing smaller proportions of hispanics. This relationship exists over and above the impact of income, housing values, and population density on TRI site locations. ANALYSIS USING DOMAINS OF INFLUENCE The approach followed in the previous section parallels past research in that the unit of analysis, the census block group in this case, is defined using criteria external to the location of a TRI site. Hence, an underlying assumption is that all residents within the TRI affected block group, no matter how distant from the actual TRI site, are exposed to the hazardous emissions. Similarly, all residents in an adjacent non-TRI block group are not exposed to the hazardous exposure, no matter how close they actually are to the TRI site. In an effort to minimize the impact of this assumption, smaller geographic units such as block groups were chosen rather than the larger census tracts. 60 The use of GIS is extremely valuable to identify the area around a TRI site within which residents face exposure to hazardous materials independent of the block group. Contrasting the socio-economic characteristics of residents of a DOI with residents outside the DOI will provide a more meaningful analysis of environmental equity. Using the polygon over point overlay procedure available in ARC/INFO, a buffer zone of 250 meters radius was created around each of the 41 TRI sites in the City of Providence. As noted earlier, TRI sites in Providence tend to cluster together, resulting in a fairly substantial overlap of the obtained buffer zones. Overlapping buffers were combined into a single DOI, yielding a total of 17 DOis for the study area (see Figure 13). A series of steps were performed using ARC/INFO to obtain the socio-economic profile of a DOI. First, all the DOis were intersected with the census block groups to obtain a set of fragmented blocks. Then for each such fragmented block, the population mix was calculated assuming a uniform distribution of the population in the parent block group. Hence, if a DOI included one-fourth of a block group containing 100 hispanic residents, it was assumed that twenty-five of those hispanic residents were in the DOI. The cumulation of such intersection populations was used to derive the racial composition, in absolute numbers and proportions, of each DOI. ANALYSIS USING DOMAINS OF INFLUENCE For computing the DOI income, a weighted average of the block group median incomes reflecting the proportion of DOI a 61 Domains Of Influence (DOis) Domains Of Influence (DOis) • TRI Sites l illt1~rnw1 001s I >A Census block groups 0 1 2 3 Kilometers Data Sources: Census, EPA, and RIGIS Map produced by: Sangeeta Jain CPAD, URI (1995) Figure# 13 TRI Sites 001s Census block groups Data Sources: Census, EPA, and RIGIS 3 Kilometers area common to the block group was used. Income was the only covariate used in the DOI based analysis. Two approaches were used to identify the non-DOI areas. First, all block groups that did not contain a TRI site (non-TRI block groups) were classified as the contrast area. In this approach, the contrast area remains the same as that used in the previous analysis of TRI affected versus non-TRI block groups. However, it is important to note that the DOI can actually include sections of block groups that did not contain a TRI site but were adjacent to other block groups that did. Hence, there can be common elements in the DOI and the non-TRI block groups, thus resulting in a diminishing of the contrast potential. In the second approach, the contrast set was defined as all block groups that did not overlap with the DOI (non-DOI block groups) and, hence, had no common elements with the DOI. This results in a cleaner contrast. DESCRIPTIVE ANALYSIS Table 6 provides descriptive statistics and t-test based contrasts for comparing DOis with non-TRI block groups. DOis have significantly lower median per capita income (p < 0.005) and numbers of white residents (p < 0.05). DOis also have more total minorities and more blacks, but the difference is only significant at the 0.1 O level. With respect to hispanics, although DOis on average have more hispanics per DOI (mean = 63 Table 6: DOis Contrasted With Non-TRI Blocks Table 6: DOis Contrasted With Non-TRI Blocks Variables Statistic Non-TRI Domain of t-tests block group Influence t df Prob> ltl Total Count 169 17 0.6967 184 0.4869 population Mean 866 781 Std Dev 471 546 Median per Count 169 17 3.0446 36.2 0.0043 income Mean 12833 9128 Std Dev 9227 4063 White Count 169 17 2.2156 184 0.0279 population Mean 615 387 Std Dev 414 291 Non-white Count 169 17 -1.7782 184 0.077 population Mean 251 394 Std Dev 307 401 Black Count 169 17 -1.6933 184 0.0921 population Mean 124 202 Std Dev 178 203 Hispanic Count 169 17 -1.4707 17.5 0.1591 population Mean 128 210 Std Dev 154 227 Proportion Count 169 17 1.889 184 0.0605 white Mean 0.72 0.58 population Std Dev 0.28 0.27 Proportion Count 169 17 -1.8889 184 0.0605 non-white Mean 0.28 0.42 population Std Dev 0.28 0.27 Proportion Count 169 17 -1.7316 184 0.085 black Mean 0.14 0.22 population Std Dev 0.18 0.18 Proportion Count 169 17 -1.6962 184 0.0915 hispanic Mean 0.15 0.21 population Std Dev 0.15 0.14 64 210) than non-TRI block groups (mean= 128), the difference is not statistically significant due to the high variance in hispanic population among the DOis. Focusing on the racial composition in terms of proportion of each race, DOis have greater proportions of minorities, blacks, and hispanics than non-TRI block groups although, once again, the differences are statistically significant at only the 0.1 O level. The lower significance levels could be attributed to the fact that DOis and non-TRI block groups contain some common elements thus dampening the contrast. Table 7 shows the results of comparing DOis with non-DOI block groups. In this case there are no common elements. All the comparisons are now much sharper. DOis have significantly lower incomes and proportions of white residents. They also have more minority, black, and hispanic residents. 7 21 of the 195 block groups in Providence have TRI sites. DESCRIPTIVE ANALYSIS In terms of the racial mix of population, DOis have a significantly greater proportion of minorities, both blacks and hispanics. These differences are extremely significant (p < 0.005). In conclusion, results based on DOis echo the results reported in the previous section using TRI affected block groups as the unit of analysis. In general, proportions of minorities are greater in the vicinity of TRI sites than in areas distant from the TRI sites. The t-tests focused on a single variable at a time and do not, therefore, take into account correlations among the socio-economic variables. Once again, LOGIT analysis was used to identify the pure relationships between minority residents and exposure to environmental hazards. 65 Table 7: DOis Contrasted With Non-DOI Block Groups Variables Statistic Non-DOI Domain of t-tests block group Influence t df Prob> ltl Total Count 116 17 0.8226 131 0.4122 population Mean 882 781 Std Dev 461 546 Median per Count 116 17 4.0737 48.5 0.0002 capita Mean 14545 9128 income Std Dev 9617 4063 White Count 116 17 2.9594 131 0.0037 population Mean 696 387 Std Dev 415 291 Non-white Count 116 17 -2.079 17.9 0.0523 population Mean 186 394 Std Dev 251 401 Black Count 116 17 -2.233 18.3 0.0383 population Mean 88 202 Std Dev 140 203 Hispanic Count 116 17 -2.0764 17.6 0.0528 population Mean 93 210 Std Dev 131 227 Proportion Count 116 17 3.3734 131 0.001 white Mean 0.79 0.58 population Std Dev 0.24 0.27 Proportion Count 116 17 -3.3733 131 0.001 non-white Mean 0.20 0.42 population Std Dev 0.24 0.27 Proportion Count 116 17 -3.1935 131 0.0018 black Mean 0.10 0.22 population Std Dev 0.15 0.18 Proportion Count 116 17 -3.054 131 0.0027 hispanic Mean 0.11 0.21 population Std Dev 0.13 0.14 Table 7: DOis Contrasted With Non-DOI Block Groups 66 Multivariate Analysis - LOGIT LOGIT modeling was used to estimate the unique relationship between race and hazardous exposure. Once again, all DOis were contrasted separately with all non-TRI block groups and with all non-DOI block groups. In the first case, this amounted to comparing the 17 DOis with 171 non-TRI block groups7. In the second case the 17 DOis were compared to the 116 block groups that did not overlap with a DOI, independent of whether they contained a TRI site or not. The dependent variable was a binary variable with a value of 1 if the area under consideration was a DOI and 0 otherwise. In contrasting DOis with non-TRI block groups, various LOGIT models with different combinations of the independent variables were estimated. None of these models fit the data with an acceptable level of significance. This finding prompts the conclusion that racial composition does not adequately distinguish between TRI affected areas (DOis) and unaffected areas (non-TRI blocks). However, as mentioned earlier, there are some non-TRI block groups that are included in both comparison sets by virtue of their adjacency to block groups containing TRI sites, and hence, in the resulting DOI. In the next step, DOis were contrasted with all block groups that did not overlap with a DOI (non-DOI block groups). In a spatial sense, this represents a cleaner comparison of residents living in the vicinity of TRI sites with those living further away. Multivariate Analysis - LOGIT Table 8 contains the LOGIT results of the first model using income, total population, 67 Table 8: DOis and Non-TRI Block Groups- LOGIT Analysis Analysis unit is DOis and block groups Dependent variable: BUFFYES: Is analysis area a DOI or a non-TRI lock group Independent variables: BGINCOME, BGPNWH, BGTOTPOP Response Profile BGTRIYES 0 Total COUNT 116 17 182 (1 observation(s) were deleted due to missing values) Criterion -2 LOG L Score Variable INTERCPT BG INCOME BGPNWH BGTOTPOP Variable INTERCPT BG INCOME BGPNWH BGTOTPOP Criteria for Assessing Model Fit Intercept Intercept and Chi-Square for Independents Only Independents 101.671 89.403 12.268 with 3 df (p=0.0065) 12.836 with 3 df (p=0.0050) Analysis of Maximum Likelihood Estimates df Parameter Standard Wald Chi- Estimate Error Square -1.0427 1.0961 0.9051 -0.00008 0.000065 1.3636 1.9055 1.1536 2.7283 -0.0007 0.000623 1.2554 Odds Ratio Odds Ratio Variable Label 0.352 1 6.723 0.999 Intercept Median block group income Proportion non-whites in block group Total population of block group Pr> Chi- Square 0.3414 0.2429 0.0986 0.2625 Standardized Estimate -0.389171 0.263395 -0.181605 Table 8: DOis and Non-TRI Block Groups- LOGIT Analysis Table 8: DOis and Non-TRI Block Groups- LOGIT Analysis Analysis unit is DOis and block groups Dependent variable: BUFFYES: Is analysis area a DOI or a non-TRI lock group Independent variables: BGINCOME, BGPNWH, BGTOTPOP Response Profile BGTRIYES 0 Total COUNT 116 17 182 (1 observation(s) were deleted due to missing values) (1 observation(s) were deleted due to missing values) 68 and proportion of minorities as the independent variables. This time the LOGIT model fit the data significantly well as indicated by the log likelihood ratio (Chi-Square = 12.268, df = 3, p < 0.01) and the Score statistic (Chi-Square = 12.836, df = 3, p < 0.005). On examining the relative influence of the independent variables, this study found that proportion of minority population in an analysis area had a marginally significant influence (p < 0.1) on the odds of the area being in the close vicinity of a TRI site, or in a TRI site's DOI. Next, the relative influence of black and hispanic proportions along with income and total population as covariates was estimated. Table 9 provides the results. Once again the model fit significantly well as seen in the log likelihood ratio and score statistics. However, none of the independent variables had a significant effect. Multivariate Analysis - LOGIT In conclusion, based on the contrast of DOis with non-DOI areas, one can suggest that minority proportion in an area is positively related to the likelihood of hazardous exposure, but one can not draw the same conclusion for greater levels of specificity - proportions of blacks and proportions of hispanics in an area. The next chapter, discusse$ the findings of this study, the limitations within which the results must be viewed, public policy implications of these results, and avenues for further research in the area. 69 Table 9: DOis and Non-DOI Block Groups-LOGIT Analysis Analysis unit is DOis and block groups Dependent variable: BUFFYES: Is analysis area a DOI or a non-DOI block group Independent variables: BGINCOME, BGTOTPOP, BGPBLK, BGPHSP Response Profile BGTRIYES 0 COUNT 116 17 Total 182 (1 observation(s) were deleted due to missing values) Criteria for Assessing Model Fit Criterion Intercept Intercept and Chi-Square for Independents Only Independents -2 LOG L 101.671 89.598 12.073 with 4 df {p=0.0168) Score 12.786 with 4 df (p=0.0124) Analysis of Maximum Likelihood Estimates Variable df Parameter Standard Wald Chi- Pr> Chi- Estimate Error Square Square INTERCPT -1.0449 1.1625 0.808 0.3687 BGINCOME -0.00008 0.000069 1.2654 0.2606 BGTOTPOP -0.00062 0.000618 0.9972 0.318 BGPBLK 1.9619 1.779 1.2162 0.2701 BG PH SP 1.4033 2.3362 0.3608 0.5481 Odds Ratio Variable Odds Ratio Variable Label INTERCPT 0.352 Intercept BG INCOME 1 Median block group income BGTOTPOP 0.999 Total population of block group BGPBLK 7.113 Proportion blacks in block group BGPHSP 4.069 Proportion hispanics in block group Standardized Estimate -0.395647 -0.160593 0.168247 0.106128 Table 9: DOis and Non-DOI Block Groups-LOGIT Analysis Analysis unit is DOis and block groups Dependent variable: BUFFYES: Is analysis area a DOI or a non-DOI block group Independent variables: BGINCOME, BGTOTPOP, BGPBLK, BGPHSP Analysis of Maximum Likelihood Estimates 70 DISCUSSION The guiding factor then has to be the researcher's theoretical underpinnings for choosing one unit over the other. This study argues in favor of using the domain of influence. In fact, it is recommend that the domain of influence unit be improved for further studies by permitting non-uniform shapes that more closely reflect actual hazardous impact of the enclosed sites. GIS approaches give us the power to minimize approximations and one should exploit this potential more extensively. Even though the results from block group analysis are not directly comparable with the DOI based findings, consistency across the approaches lends further credence to the presence of environmental inequity in Providence. DISCUSSION Every method of analysis used yielded a strong relationship between the proportion of minorities and presence of hazardous sites. This pattern is present in simple correlations and in more sophisticated multivariate procedures. Clearly, there is reason to be concerned about environmental inequity in Providence. Further, as was anticipated, racial categories are closely interlinked with economic indicators. In fact, relationships that were present in simple bivariate analysis often vanished when analyzed within a multivariate framework. The multivariate logistics regression analysis showed that racial composition of the area is related to hazardous exposure independent of the residents' economic characteristics. Of equal, if not greater, interest is the uneven impact on hispanics compared to blacks. Relative to hispanics, blacks appear to be more integrated with the majority white residents in terms of residential location. This results in hispanics being at greater risk of hazardous exposure than blacks. The uneven impact strongly suggests that, in future, research focus should be defined not at macro groupings such as minorities but rather at more specific racial categories such as hispanic and south-east Asians. Given recent immigration trends in the Providence area, it is not inconceivable that a study ten years from today will find south-east Asians at even greater risk than hispanics. 71 71 Finally, the difference in findings that result from changing the unit of analysis - census block group versus DOI - is an important reminder of the analysis' sensitivity to spatial · units. With no objective standard to use as a reference, it is difficult to compare the validity of block group based versus domain of influence based findings. The guiding factor then has to be the researcher's theoretical underpinnings for choosing one unit over the other. This study argues in favor of using the domain of influence. In fact, it is recommend that the domain of influence unit be improved for further studies by permitting non-uniform shapes that more closely reflect actual hazardous impact of the enclosed sites. GIS approaches give us the power to minimize approximations and one Finally, the difference in findings that result from changing the unit of analysis - census block group versus DOI - is an important reminder of the analysis' sensitivity to spatial · units. With no objective standard to use as a reference, it is difficult to compare the validity of block group based versus domain of influence based findings. LIMITATIONS Certain limitations must be borne in mind as one considers the results of this study. Most importantly, the data used in this study represent a snap-shot in time and, hence, while they permit investigation of association they can not be used to draw causal inferences. Longitudinal study of key areas, such as South Providence neighborhoods, is needed to distinguish between the case of deciding to locate a TRI site in high- minority areas in anticipation of low resistance versus the case of minority residents 72 migrating toward TRI site vicinities in search of jobs and residential facilities that the majority white population considers undesirable. The analysis presented in this report applies only to toxic release sites reported under TRI data base, and only to Providence, and is not generalizable beyond this case study. Many other environmental hazards exist within Providence, which were not included in this analysis. Environmental hazards such as landfills and conventional air pollutants from automobiles and industrial emissions have not been considered. POLICY IMPLICATIONS The bivariate analysis employed in this analysis, including the correlation analysis and LOGIT models, confirmed the strong correlation between income and minority population. There is a clear pattern showing that greater the proportion of minorities in a block group, the poorer the block group tends to be. To understand whether race or income has a more important effect on the distribution of environmental hazards may be less relevant than understanding the underlying causes that lead to it, and addressing and remedying the situation. The findings of this report have relevance for potential policies aimed at addressing the disproportionately high minority residents in close proximity to TRI sites. Until very recently, there were no public policies in place that required monitoring equity in the distribution of environmental quality. While there are many, and increasingly more, 73 73 policies to help control pollution, there is at the same time very little knowledge of the equity consequences of such control measures. Risk-based approaches, which focus on areas of high pollution and identify the greatest risk to the population generally treat all residents equally. Even if such policies are eventually successful in limiting the extent of pollution, there success will be constrained by their lack of focus on socio-economic characteristics of affected populations. In most cases, such as with South Providence in this study, a closer examination would show that residents of such affected areas are generally racial minorities with lower incomes. To be truly effective, policies on environmental protection must address both priority setting and socio-economic descriptors of the residents and do so independently of political interests. These patterns also have important implications because of the distribution of the costs and benefits of these facilities. The benefits in terms of product-value generated at these sites may be benefiting the individuals around the world but the burden of pollution related health is borne by the adjacent residents of these sites. This study confirms that in the Providence area, residents adjacent to hazardous sites are more likely to be hispanics. Racism remains a major social problem in this country, as so the degradation of the environment Minority groups are under represented on the local, state, and federal regulatory boards that determine and I or approve the location of hazardous waste sites. Also very few minority citizens occupy leadership positions among mainstream green organizations. POLICY IMPLICATIONS Clearly, this imbalance between the impacted and the ones with the voice needs to be urgently addressed. 74 And, finally, the quality of environmental education is poor in general, and even worse in inner-city schools. Environmental education should be improved in both primary and secondary schools as a means of improving environmental awareness and of improving people's ability to address environmental concerns within their communities. These education projects should be targeted to minority population groups. 3) Finally, another extension could be an in depth neighborhood analysis, especially South Providence area. A longitudinal research of the history of the neighborhood may yield more insightful findings. FURTHER RESEARCH This research examined the relationship between the population and TRI facility occurrence within census block groups and their DOI, defined as a buffer of a quarter mile around the site. This analysis could be expanded in three ways: 1) Within this analysis, all TRI facilities were treated as equivalent. The analysis could be enhanced by evaluating the relative risk posed by individual facilities in terms of type and volume (or overall toxicity) of emissions. 2) Additionally, other sources of pollution could be included in order to get a broader picture of environmental equity in Providence. Sources such as incinerators, municipal and hazardous waste landfills, and Superfund sites could be included, in addition to information on concentrations of conventional pollutants. 75 3) Finally, another extension could be an in depth neighborhood analysis, especially South Providence area. A longitudinal research of the history of the neighborhood may yield more insightful findings. 3) 76 LIST OF ALL THE TRI SITES IN PROVIDENCE LIST OF ALL THE TRI SITES IN PROVIDENCE No. Name of the Facility Street Address City State Zip Alan Jewelry Co. 1280 Eddy St. Providence RI 02905 2 Anson Inc. 100 Dupont Dr. Providence RI 02907 3 Antonelli Plating Co. 50 Valley St. Providence RI 02909 4 Arconium Specialty Alloys 400 Harris Ave. Providence RI 02909 5 Armbrust Chain Co. 735 Allens Ave. Providence RI 02905 6 B. B. Greenberg Co. 333 W. River St. Providence RI 02904 7 Darmet Corp. 960 Broad St. Providence RI 02905 8 Eastern Color & Chemical Co. 35 Livingston St. Providence RI 02904 9 Eastern Wire Prods. Co. 498 Kinsley Ave. Providence RI 02909 10 Electrolizing Inc. 1 O Houghton St. Providence RI 02904 11 Esposito Jewelry Inc. 225 Dupont Dr. Providence RI 02907 12 Excell Mfg. Co. 200 Chestnut St. Providence RI 02903 13 Federal Prods. Corp. 1139 Eddy St. Providence RI 02940 14 Ferguson Perforating & Wire Co. Inc. 130-140 Ernest St. Providence RI 02905 15 Ge Co. Providence Base Plant 586 Atwells Ave. Providence RI 02909 16 George Mann & Co. Inc. Harborside Blvd. Providence RI 02905 17 H & H Prods. Co. Inc. 148 W. River St. Providence RI 02904 18 International Etching Inc. 7 Ninigret Ave. Providence RI 02907 19 lnterplex Metals 45 Salem St. Providence RI 02907 20 Klitzner Ind. Inc. 44 Warren St. Providence RI 02901 21 Mainelli Tool & Die Inc. 30 Houghton St. Providence RI 02904 22 Microfin Corp. 555 Valley St. Providence RI 02908 8 Eastern Color & Chemical Co. 9 Eastern Wire Prods. Co. 10 Electrolizing Inc. 11 Esposito Jewelry Inc. 12 Excell Mfg. Co. 16 George Mann & Co. Inc. 17 H & H Prods. Co. Inc. 18 International Etching Inc. 21 Mainelli Tool & Die Inc. 22 Microfin Corp. n 23 Monarch Metal Finishing Co. Inc. 189 Georgia Ave. Providence RI 02905 24 National Plating Corp. 946 Eddy St. Providence RI 02905 25 Oster Alloys 50 Sims Ave. Providence RI 02909 26 Patton-Macguyer Inc. 17 Virginia Ave. Providence RI 02905 27 Pilgrim Screw Corp. 120 Sprague St. Providence RI 02907 28 Providence Chain Co. 225 Carolina Ave. Providence RI 02905 29 Quality Spraying & Stenciling Co. 150 Park Ln. Providence RI 02907 30 Quebecor Printing Providence Inc. 99 W. River St. Providence RI 02904 31 Rau Fastener Inc. 102 Westfield St. Providence RI 02907 32 Regal Plating Co. Inc. 85 S. Source: EPA's Toxic Release Inventory, 1992 LIST OF ALL THE TRI SITES IN PROVIDENCE St. Providence RI 02903 33 Ribco Mfg. Inc. 192 Georgia Ave. Providence RI 02905 34 Rolo Mfg. Co. Inc. 274 Pine St. Providence RI 02903 35 Spectrum Coatings Labs. Inc. Inc. 217 Chapman St. Providence RI 02905 36 Technic Inc. 1 Spectacle St. Cranston RI 02910 37 Textron Inc. Speidel Div. 70 Ship St. Providence RI 02903 38 Uncas Mfg. Co. 623 Atwells Ave. Providence RI 02909 39 Universal Engravers 695 Eddy St. Providence RI 02903 40 Victory Finishing Tech. Inc. Inc. 145 Globe St. Providence RI 02903 41 Victory Pearl Inc. 50 Agnes St. Providence RI 02909 189 Georgia Ave. Providence RI 02905 946 Eddy St. Providence RI 02905 50 Sims Ave. Providence RI 02909 17 Virginia Ave. Providence RI 02905 120 Sprague St. Providence RI 02907 225 Carolina Ave. Providence RI 02905 150 Park Ln. Providence RI 02907 99 W. River St. Providence RI 02904 102 Westfield St. Providence RI 02907 85 S. St. Providence RI 02903 192 Georgia Ave. Providence RI 02905 274 Pine St. Providence RI 02903 217 Chapman St. Providence RI 02905 1 Spectacle St. Cranston RI 02910 70 Ship St. Providence RI 02903 623 Atwells Ave. Providence RI 02909 695 Eddy St. Providence RI 02903 145 Globe St. Providence RI 02903 50 Agnes St. Providence RI 02909 23 Monarch Metal Finishing Co. 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https://openalex.org/W4392909647	https://worldcustomsjournal.scholasticahq.com/article/91371.pdf	English	null	TRS as a Measurement of Trade Facilitation: Customs’ Experience in the Asia-Pacific Region	World customs journal	2,009	cc-by	5,422	World Customs Journal World Customs Journal TRS AS A MEASUREMENT OF TRADE FACILITATION: THE EXPERIENCE OF CUSTOMS IN THE ASIA PACIFIC REGION Shujie Zhang1 Abstract A Time Release Study (TRS), a systematic and standard way to measure the time taken to release cargo, can substantially help Customs, other governmental agencies and the private sector to measure effectiveness, identify bottlenecks and find possible solutions for improvement in clearance and logistics. The World Customs Organization (WCO) has promoted the TRS and concentrated efforts to develop guidelines and software to address the emerging trade facilitation agenda. In the Asia Pacific Region, a number of TRS programs have been, and will be, conducted both at national and regional level with capacity building support. Critical findings and significant lessons can be drawn from regional experience to show how to roll out and use the TRS effectively. 1. Introduction The development of the Time Release Study (TRS) dates back to the early 1990s. In 1994, the World Customs Organization’s (WCO) Permanent Technical Committee (PTC) adopted a study to measure the time required for the release of goods, based on similar initiatives undertaken by Japan and the United States. In 2001, the PTC reviewed and updated the study based on pilot projects in an effort to simplify its application. Subsequently, the ‘Guide to Measure the Time Required for the Release of Goods’ (TRS Guide) was developed and recommended to member administrations. In 2005, TRS software was jointly developed by the WCO and the World Bank.2 Intended for measurement of trade and transportation facilitation (TTF), TRS serves as a multipurpose tool for Customs, other government agencies (OGAs) and the private sector involved in the trade supply chain. As the TRS Guide defines it, TRS is a systematic and standardised way to measure the average time taken between the arrival and release of goods and can also be used at each step. It is a diagnostic tool, providing concrete baseline data for identifying any bottlenecks in the clearance process and logistics. It helps to evaluate the impact of reform or modernisation initiatives taken by the public and private sectors. It provides a mechanism to further improve national trade competitiveness by enhancing the national TTF. It is also a persuasive indicator to demonstrate progress made and further requirements to the budgetary authority and the donor community. As the sole intergovernmental organisation competent in customs matters, the WCO endeavours, through its instruments and tools, to help Members enhance their effectiveness and efficiency in facilitating legitimate trade and safeguarding the security and safety of society. The WCO has been promoting TRS as a key tool in its trade facilitation package, using all available opportunities including international occasions both formal and informal. The audiences have included Customs and the trade community with the TRS message pitched at both policy and technical levels. Technical assistance is provided to Volume 3, Number 2 Volume 3, Number 2 125 International Network of Customs Universities Members through the WCO Secretariat and regional structures such as the Regional Offices for Capacity Building (ROCB). In recent years, awareness of TRS has increased and the number of TRS projects has expanded significantly with the TTF agenda progressing both nationally and internationally, especially in World Trade Organization (WTO) trade facilitation negotiations. 1. Introduction The rationale behind TRS is a continuous improvement cycle; TRS is never a stand-alone activity. The imperatives to enhance TTF call for honest and accurate analysis of the clearance process. Conducting a TRS will identify the bottlenecks in clearance and assist in finding solutions for delays., and necessary measures can then be taken. In this cycle, the key is how to conduct a TRS. Drawing on the relevant experiences of customs administrations in the Asia Pacific Region, the following discussion focuses on how to apply and tailor the WCO TRS tools in a local context. 2. Regional rollout and capacity building Under the WCO umbrella, the Asia Pacific Region comprises 32 members. In terms of development level and customs capacity, these members are diverse. In developed countries/regions like Australia, Japan, Korea, New Zealand, Singapore and Hong Kong, customs modernisation is well advanced and these countries could be regarded as amongst the leading economies. There are also large developing countries like China and India, and some less developed countries like Bhutan and Lao PDR, where customs capacity needs a deal of enhancement. Such regional disparity provides potential for improved trade facilitation through capacity building. In relation to the TRS, an appropriate approach would be targeted capacity building by more developed members or regional organisations such as the ROCB which would assist less developed regional members. 2.1 Phased approach A TRS project usually spans several months, and a phased approach should be adopted to ensure smooth implementation and systematic application of the research findings. In relation to the conduct of a TRS, the TRS Guide covers only three stages: preparation (data collection and recording), data analysis and report. However, it is preferable to see TRS in a broader context. The ‘Initiation’ stage, that is, the proposal and decision to implement a TRS project, should be regarded as the first phase. Only if the decision to proceed with a TRS project has been taken do the other steps follow. Some experts propose that a fifth phase, ‘Action’, be added to the steps. In the TRS Guide, the preparation phase is critically important. Naturally, when conducting a TRS for the first time, the preparation stage is critical since much time and energy is needed to ensure that knowledge transfer and capacity building occur. From the perspective of project management, each phase is equally important as any omission of a minor step may undermine the whole project. The parties involved in the TRS extend well beyond Customs. Close communication and cooperation among all the stakeholders such as the national government, Customs, OGAs, donors, and the private sector, are integral to smooth implementation. Clear project boundaries, objectives, and the responsibilities of each party need to be stipulated. As the focal point in border management and control with unique expertise in clearance procedures, Customs is usually recommended to lead and monitor the project. A TRS Working Group should be established, with responsibility for the overall project. In some countries, a Steering Committee at policy level or a TRS Reference Group involving the private sector may be set up to ‘serve as a channel of engagement with the industry sectors and OGAs involved in the supply chain’.3 The following table shows the main activities and possible parties involved. 2.1 Phased approach Volume 3, Number 2 126 World Customs Journal Table 1: Five Phases in TRS Phase Main activities Stakeholders 1 Initiation Prepare proposal to conduct a TRS Receive acceptance by the government Decide to conduct a TRS Government Customs Donors WCO & ROCB Private sector OGAs 2 Preparation of the study Establish a TRS Working Group4 Conduct specific workshop (if necessary) Plan (scope, methodology, questionnaire) Hold pre-meetings Conduct test run Customs Donors ROCB Private sector OGAs 3 Data collection and compilation Collect data Input data (using WCO TRS software) Verify data Working Groups Private sector ROCB 4 Data analysis and report Analyse data (using WCO TRS software) Follow up interview/research (if necessary) Prepare final report with recommendations Working Groups Private Sector Donors ROCB 5 Action for improvement Distribute report and publicise findings Consider recommendations and initiate action for improvement in clearance Government Customs & OGAs Donors Private sector Public Source: Adapted from the WCO TRS Guide and Tadatsugu Matsudaira 2006.5 Source: Adapted from the WCO TRS Guide and Tadatsugu Matsudaira 2006.5 urce: Adapted from the WCO TRS Guide and Tadatsugu Matsudaira 2006.5 2.2 Regional situation of conducting TRS Based on information available to ROCB, the rollout of TRS in the Asia Pacific Region is summarised in Table 2 below. Against the geographical scope of data collection, the projects can be categorised into two groups: national level and regional level. Except in Korea, all relevant customs administrations collect data by using a data collection form or questionnaire. This does not mean that all the data are captured manually. In practice, a combination of extracting data from existing computer systems and obtaining other necessary data through the questionnaire is used, with the extent of the use of electronic data depending on the technological context of the countries concerned and cooperation from stakeholders. Generally, the more electronic data used, the better. In this paper, the author treats such methodology as ‘manual’. Use of the WCO TRS software is still in a pilot stage, although some countries like Japan and China have developed specific computer systems to record and analyse the data. It should be noted that, at this stage, in most countries, the target of the TRS is generally limited to import cargo. Volume 3, Number 2 Volume 3, Number 2 127 International Network of Customs Universities The process of initiating a project varies from country to country. Regional blocs like the Asia-Pacific Economic Cooperation (APEC) and donors like the World Bank and the Asian Development Bank (ADB) may play a ‘catalyst’ role, but external requirements should complement internal commitment. Besides those conducted in Japan and Korea, regional TRS projects that have been conducted in recent years have been initial projects or pilot projects, which is consistent with the global picture of TRS application. 2.2 Regional situation of conducting TRS Table 2: Regional Rollout of TRS Level Countries/ Projects Time Method Use of WCO TRS Software First Time Initiation Support Country Level Australia 2007 Manual No Yes APEC Japan, Korea China 2006 Manual No Yes APEC ROCB Indonesia 2006 Manual No Yes NA JICA Japan Since 1991 Manual No Periodical Self - Korea Since 1997 Auto- matic No Periodical Self - Lao PDR 2008 Manual Yes Yes World Bank ROCB Philippines 2002 Manual No Yes Self JICA Malaysia 1995, 2007 Manual Yes No Self WCO Thailand 2006 Manual No Yes Self ROCB Regional Level GMS CBTA EWC TRS6 2009 Manual Yes No7 ADB ROCB BIMP-EAGA TRS8 2009 Manual Yes Yes ADB ROCB Table 2: Regional Rollout of TRS At a national level, Japan and Korea provide two different models for the collection of data and their experience is significant for other regional members. As mentioned earlier, Japan conducted the first pilot TRS in 1991, and since 1993, the study has been conducted every two or three years with implementation being consistent with the TRS Guide. Up to now, nine studies have been conducted with the latest conducted in 2008. With strong support from all the relevant government ministries and the trade and transportation communities, following careful analysis of the survey results, Japan Customs introduced a series of modernisation initiatives, procedures and techniques such as computer-based risk management, an automatic clearance system such as Single Window and pre-arrival declaration, and more recently the Authorised Economic Operator (AEO) program. Japan Customs also works closely with OGAs and the private sector to facilitate procedures and update facilities. As a result, substantial improvement has been made: from 1991 to 2006, the average release time for sea cargo has been reduced from 7 days to 2.7, and for air cargo, from 2.2 days to 0.6 day. In total, there has been a reduction in nearly two-thirds of the clearance time, and statistics from the Japan Ministry of Finance show that 40 billion Japanese yen could be saved in one year. Japan’s case demonstrates that a TRS can yield substantial benefits both for the public and the private sectors if it is used effectively. Korea has also followed the WCO’s principles of undertaking a TRS and, taking advantage of their strong expertise in ICT application, key elements of the TRS are automated. ce: Takashi Matsumoto and Sang-Hyup Lee, ‘Regional Best Practice TRS’, ROCB, 2007 The ROCB is also involved in two regional projects. In 2008, Mr Kunio Mikuriya, the current Secretary- General of the WCO, stressed that ‘If the Time Release Study is implemented at the regional level using equivalent methods, it could offer a useful insight on the way to improve border procedures in a comparable way’.9 This is because the data will be collected and analysed from the perspective of an extended supply chain and control chain. In this regard it should also be noted that Regional TRS, especially along economic corridors, can be extended to all checkpoints along the corridor. For the Greater Mekong Subregion (GMS), Cross-border Transport Agreement (CBTA) East-West Corridor (EWC) TRS Survey project, ROCB, while consulting ADB and concerned countries such as Lao PDR, Thailand, and Vietnam, recommended the use of a regional approach under which the release time will combine time consumed both by export and import countries. This approach may be problematic, however, where there is a lack of consistency between the procedures on both sides of the frontier and a lack of consistency in the definitions of various components. 2.2 Regional situation of conducting TRS The current Client-orientated Volume 3, Number 2 128 World Customs Journal Logistics Information System (CLIS) now provides reliable, fast and comprehensive information about clearance time and logistical status of goods (see Example 1 below). Embedding the TRS in current computer systems and running TRS automatically is clearly a model for other administrations. Example 1: Client-orientated Logistics Information System in Korea Customs Service Korea Customs Service (KCS) completed the EDI-based import cargo management system and import clearance system in 1997 and, utilising the processing time recorded in the systems, developed a method of calculating, at major logistics stages, the average processing time of all imported cargoes brought into Korea for a specific time. In 2006, KCS updated the existing system into an independent, web-based TRS system, which enables automatic TRS measurement of all processes and scope on a real-time basis of average processing time, standard deviation and performance of individual logistics participants, and information sharing among stakeholders. KCS named the system ‘Client-orientated Logistics Information System’ (CLIS), highlighting its client-orientated function and automated, independent information system. CLIS is differentiated from existing EDI-based TRS measurement systems in that first, its statistics are based on complete enumerations rather than sampling; second, all measurement scope and processes are done by an automated, independent system; and third, clients can get all related logistics information through the internet free of charge. In 2006, the government of Korea awarded CLIS the Presidential Prize as a best innovation practice. 2.3 Role of capacity building The existence of the WCO tools and instruments in itself does not necessarily ensure automatic adoption and wide application by Member administrations; in the case of the TRS, the WCO has been promoting the initiative for nearly 15 years. The number of Member administrations that have actually conducted a TRS is comparatively small considering its 174 Members, the reasons for which no doubt include a lack of capacity in the individual Member states. Generally, however, the situation in the Asia Pacific Region is reasonable, and can be partly attributed to the drivers discussed at the beginning of this paper, and partly to the promotion of the TRS and capacity building efforts within the region. Capacity building can be delivered as a comprehensive package or as a specific element of technical assistance, with support being offered by bilateral partners like Australia as well as by international institutions. In the case of Lao PDR, the World Bank has been instrumental in providing such assistance, and for two of the regional TRS projects, the ADB has initiated the project and provided the funding. Volume 3, Number 2 129 International Network of Customs Universities As the regional arm of the WCO, the ROCB Asia Pacific was established in 2004 to enhance the effectiveness and efficiency of regional customs administrations. In the past five years, the ROCB has conducted various capacity building activities based on the needs and requests of members. As a rule of thumb, the ROCB focuses on knowledge transfer and skill building and promotes ownership by the recipient administrations. In relation to TRS, the ROCB aims to help members to build their expertise which is essential for conducting the necessary follow-up studies. It should be noted that ROCB support runs through all five phases and by promoting TRS in various forums, the ROCB is seeking to increase awareness and solicit requests for capacity building. In the preparation stage, for example, the ROCB is able to provide basic suggestions and conduct a dedicated TRS workshop in the recipient administration. During the data collection phase, the ROCB may dispatch experts to assist on site, and assistance with data analysis and report drafting can also be provided as the ROCB keeps ‘open-line’ communication. Then, the ROCB would provide recommendations on how to improve trade facilitation. In 2007, the ROCB developed a Regional Best Practice Compendium on TRS for members’ reference. 2.3 Role of capacity building In addition to the various types of support that can be provided, the ROCB considers the centrepiece to be the TRS workshop which runs for two or three days. To maximise the benefits of the workshops, several considerations are necessary. First, the right persons need to be invited. Generally, all the representatives of the stakeholders (for example, Customs, OGAs, trade and transport sectors) are invited to attend and it is essential that all members of the TRS Working Group participate. Second, a logical and balanced program should be designed and implemented, which should include the opportunity for interactive sessions (see Table 3 below). Table 3: Model design of a TRS Workshop Contents Objectives Training Methods Brief on the WCO TRS Guide and Regional Rollout Learn background, principles, phases, tasks on TRS Presentation Application of the WCO TRS software Learn how to use all the functions, such as creating a questionnaire, input data, data analysis, report generation, etc. Presentation Simulation exercises Regional best practices Expand horizon Draw on lessons from other countries Presentation Mapping the flowchart of clearance process Gain clear picture of each step in clearance process Prepare to draft questionnaire Presentation Discussion Decide scope and planning Discuss thoroughly and decide the key aspects of TRS Group discussion and panel session Develop draft questionnaire Discuss and agree on defining data elements Develop the questionnaire by using the WCO TRS software Group discussion Panel session Practice Field simulation Simulate the actual data collection process Discuss and agree the logistical flow of the questionnaire Identify potential problems in actual conduct of the survey Field study Discussion Source: Shujie Zhang, drawing on ROCB past experience (2009). Table 3: Model design of a TRS Workshop Volume 3, Number 2 130 World Customs Journal World Customs Journal 1.1 Common findings Although the results of a TRS vary from country to country, significant findings can be distilled. First, Customs may not necessarily represent a major impediment to cargo flow. On the contrary, Customs is very often found to be one of the more efficient agencies, although there are always opportunities for Customs to further modernise and collaborate more closely with OGAs and the private sector. Second, the entities and variables which are positive and negative on TTF are numerous and are usually intertwined and interdependent. Therefore, a holistic approach for correction and improvement should be adopted, and partnership and cooperation among all the key players along the trade supply chain is a pre-condition. Third, the private sector may be responsible for some delays. This may occur at various stages from arrival to declaration, and from release permission to physical removal. Problems of capacity and port management also require resolution. Fourth, regular conduct of a TRS or ongoing measurement of trade facilitation is recommended. 1.2 Concerned dilemmas In actually conducting a TRS, some dilemmas emerge and it is important to clarify these issues. Customs only vs whole process. TRS can be approached in two ways. The first is to cover only the customs procedures, and the second is to cover the whole process of clearance. The first appears to be simple, as complicated coordination with other stakeholders can be bypassed. However, it is recognised by experts and demonstrated from past experiences that it is better that a comprehensive TRS be conducted. The reasons are twofold: first, a comprehensive TRS will yield more useful findings, especially in helping to identify the bottlenecks in the procedures that are out of Customs’ control. This is in line with the concept and practice of supply chain management. Second, coordination and communication among stakeholders during TRS will provide a sound basis for possible efforts to seek solutions and to take action to reform the process. Looks easy vs actually difficult. In learning to conduct a TRS, a ‘U-shape’ learning curve is common in many countries. This is natural because many international instruments and tools are, in essence, not so complex and difficult. However, considerable efforts are needed to absorb and apply those instruments and tools in a local context. The TRS Guide provides comprehensive guidelines. In one administration, for example, a workshop was conducted, an overall plan formulated and a questionnaire drafted. Unfortunately, and for a variety of reasons, the study was postponed for one year, reflecting the fact that it was not so ‘easy’ to progress the project. Standalone vs integral. A TRS project stands independently in terms of data collection and analyses. However, TRS should not be conducted for its own purposes. It is wasteful for such a comprehensive report to be produced without further actions being planned. TRS therefore represents one critical stage of a continuous improvement cycle. Quantitative vs qualitative. In conducting a TRS, most of the data are quantitative, especially time data. Naturally, analysis will be conducted mainly on a quantitative basis, for example, calculating elapsed processing time. However, numbers are just numbers, and on their own they cannot tell the whole or true story. A qualitative probe is necessary, especially when seeking to identify the causes for delays. This can be done by including open questions in the TRS questionnaire and conducting follow-up contacts or interviews on ‘outstanding’ cases as is the practice in Japan. 1.2 Concerned dilemmas Volume 3, Number 2 131 International Network of Customs Universities Performance measurement or not? In collecting data, it is stressed that all the officers involved should be informed that the TRS is not intended as a measurement of performance. However, when the results are interpreted and/or used, they do relate to the overall performance of organisations. This is a dilemma and this author’s view is that, for individual officers, we should not stress that the TRS is not intended to measure their personal performance to avoid intentionally or unintentionally distorting the data. Organisationally, a TRS should be treated as an index of performance. Benchmarking tool or not? The TRS Guide emphasises that the ‘TRS should not be considered as a competition between Members or as an effort to place a value judgment on the operations of an administration’. However, a customs administration is inclined to compare its performance with the TRS results of other countries. It is therefore advisable to leave it to Members to decide whether they would like to conduct a TRS as a benchmark tool. If such a decision is prevalent amongst Members, the WCO could consider providing a platform to share TRS findings. The quicker, the better? It seems that for clearance times, the shorter, the better. However, it may not be reasonable to interpret survey findings in this way. First, we should never forget the tenet for border control: the balance of facilitation and control. Second, too much focus on ‘speed’ would incur undesired consequences. As observed in one country, competition to reduce clearance time among different customs houses has invited complaints from the private sector. 3.3 What makes a successful TRS? The ‘successful’ story always follows a similar plot. Drawing on the ‘happy’ and ‘painful’ experiences of regional members, the following elements can be extracted to ensure a sound and helpful TRS project. Political will and concrete commitment should be in place. It may be a cliché to highlight the importance of commitment for any initiative. This is true with a TRS. Since many stakeholders are outside Customs, high-level support is critical. In countries where Trade and Transportation Facilitation Committees (TTFC) have been established, it is recommended that the TTFC steer the TRS project. Necessary resources including personnel and finances should be readily available. Buy-in of stakeholders should be obtained. The buy-in of stakeholders is needed in each phase, especially in collecting data and implementing measures for improvements. Such buy-in is critical to the success of any TRS project in the Asia Pacific Region. Project management approach should be adopted. The ROCB observes that many TRS Working Group members may not have effective skills in project management and monitoring. In one administration, data was collected under difficult circumstances, the reason being that insufficient people were assigned to the task. Defining types and components of clearance process is critical. Thorough discussions should be held to gain a clear picture of the different types of clearance and their components. This is critical to the design of the questionnaire and its subsequent analysis. Principle of 3W & H should be followed. The biggest challenge for many developing countries in conducting a TRS may be how to collect honest, reliable data. Therefore, in designing the questionnaire and collecting data, the following questions should be clear for each data element: What does this data element mean? When should the data be collected? Who is responsible? By what method or means should the data be collected? The results should be communicated properly and utilised wisely. It is a tragedy that, after long and painstaking efforts, a well-drafted report based on valid data and scientific analysis is only made available to limited audiences. The TRS Guide stresses that ‘In the spirit of transparency and cooperation, the Volume 3, Number 2 132 World Customs Journal results of the study should be made available to all participating and relevant parties to stimulate any necessary further action on their part’. Like any diagnostic study, the value lies in how to reform and improve administration by using the findings wisely and effectively. 3.3 What makes a successful TRS? results of the study should be made available to all participating and relevant parties to stimulate any necessary further action on their part’. Like any diagnostic study, the value lies in how to reform and improve administration by using the findings wisely and effectively. In the latest WCO Train-the-Trainer Workshop on TRS,10 participants discussed the challenges in conducting a TRS. Their findings mirror the above points. 4. Conclusions In times of financial turmoil and economic slowdown, the global international customs community has reiterated a firm stance on free, fast and secure trade.11 A TRS can contribute to the continuous improvement of trade and transportation facilitation. The methodology devised and recommended by the WCO can provide sound guidance. The TRS project should be incorporated into the national trade and transport facilitation strategy and customs modernisation program. The findings should be used wisely and various reform measures should be implemented to reduce clearance time and cost. This will eventually lead to enhanced trade facilitation and rationalised border management. For many developing countries which have finished the diagnostic phase of the WCO Columbus Programme and are implementing action plans, a TRS will help review the impact of reform measures. Since a TRS has been included as a proposal12 to the WCO trade facilitation negotiations, more countries may consider TRS in their national TTF strategy. Capacity building is critical to apply WCO instruments and tools including TRS in a local context. ROCB, working closely with members, the WCO Secretariat and other partners, can provide targeted assistance. Endnotes 1 The author values the comments made by Mr Rob Preece and Ms Sandra Caligari on an earlier draft of this article. 1 The author values the comments made by Mr Rob Preece and Ms Sandra Caligari on an earlier draft of this article. 2 The WCO TRS software is an internet-based application for creating a database for WCO TRS projects. As a web-based service, Members can use the software free of charge to develop the questionnaire and analyse data in a more convenient manner. Members can access the software at http://members.wcoomd.org/trs/index.asp. 2 The WCO TRS software is an internet-based application for creating a database for WCO TRS projects. As a web-based service, Members can use the software free of charge to develop the questionnaire and analyse data in a more convenien manner. Members can access the software at http://members.wcoomd.org/trs/index.asp. 3 See Time Release Study 2007, Australian Customs and Border Protection Service, www.customs.gov.au/webdata/resources/ files/time-release-study.pdf. 3 See Time Release Study 2007, Australian Customs and Border Protection Service, www.customs.gov.au/webdata/resources/ files/time-release-study.pdf. 3 See Time Release Study 2007, Australian Customs and Border Protection Service, www.customs.gov.au/webdata/resources/ files/time-release-study.pdf. 4 For the functions and responsibilities of the TRS Working Group, see the WCO TRS Guide. 4 For the functions and responsibilities of the TRS Working Group, see the WCO TRS Guide. 5 See Tadatsugu Toni Matsudaira 2006, ‘WCO Time Release Study Guide: an analytical tool for TF’, Presenta UNECE Workshop, November, Geneva. 5 See Tadatsugu Toni Matsudaira 2006, ‘WCO Time Release Study Guide: an analytical tool for TF’, Presentation at UNCTAD/ UNECE Workshop, November, Geneva. 6 GMS: the Greater Mekong Subregion Program, mainly supported by the Asian Development Bank (ADB); CBTA: Agreement Between and Among the Governments of the Lao People’s Democratic Republic, the Kingdom of Thailand, and the Socialist Republic of Viet Nam for Facilitation of Cross-border Transport of Goods and People; EWC: East-West Corridor Project under CBTA. For details, see www.adb.org/GMS/about.asp. 6 GMS: the Greater Mekong Subregion Program, mainly supported by the Asian Development Bank (ADB); CBTA: Agreement Between and Among the Governments of the Lao People’s Democratic Republic, the Kingdom of Thailand, and the Socialist Republic of Viet Nam for Facilitation of Cross-border Transport of Goods and People; EWC: East-West Corridor Project under CBTA. For details, see www.adb.org/GMS/about.asp. Endnotes 6 GMS: the Greater Mekong Subregion Program, mainly supported by the Asian Development Bank (ADB); CBTA: Agreement Between and Among the Governments of the Lao People’s Democratic Republic, the Kingdom of Thailand, and the Socialist Republic of Viet Nam for Facilitation of Cross-border Transport of Goods and People; EWC: East-West Corridor Project under CBTA. For details, see www.adb.org/GMS/about.asp. 7 In 2005, the ADB conducted a small-scale TRS in Mukdahan-Savannakhet Check Points. 7 In 2005, the ADB conducted a small-scale TRS in Mukdahan-Savannakhet Check Points. 8 BIMP-EAGA: Brunei Darussalam-Indonesia-Malaysia-Philippines East ASEAN Growth Area, mainly supported by the ADB. For details, see www.bimp-eaga.org/. 8 BIMP-EAGA: Brunei Darussalam-Indonesia-Malaysia-Philippines East ASEAN Growth Area, mainly supported by the ADB. For details, see www.bimp-eaga.org/. 9 See Kunio Mikuriya, Keynote Speech, International Forum on the Role of Customs Administrations on Promoting and Facilitating Trade among Silk Road Countries, October 2008, Turkey. 10 In July 2009, to help developing members to enhance capacity in conducting a TRS in their administrations, two sessions of Train-the-Trainer on TRS were conducted in the WCO Secretariat. The participants learned how to apply the WCO TRS tools including the TRS Guide and the TRS Software. 11 See WCO 2008, Communiqué from the International Customs Community: the global financial crisis of 2008, 60th Session of the WCO Policy Commission. 12 See WTO 2007, Proposal on Release Time of Goods, TN/TF/W/139/Rev.1, Negotiation Group on Trade Facilitation. Volume 3, Number 2 133 International Network of Customs Universities Shujie Zhang Shujie Zhang, Technical Attaché of the WCO, has been working in the WCO Asia Pacific Regional Office for Capacity Building (ROCB) in Bangkok since March 2007. He was actively involved in international cooperation, training and research while serving in the Shanghai Customs College, China Customs, prior to his appointment to the ROCB. He has been engaged in projects sponsored by the World Bank and the Asian Development Bank in relation to TRS. Shujie Zhang, Technical Attaché of the WCO, has been working in the WCO Asia Pacific Regional Office for Capacity Building (ROCB) in Bangkok since March 2007. He was actively involved in international cooperation, training and research while serving in the Shanghai Customs College, China Customs, prior to his appointment to the ROCB. He has been engaged in projects sponsored by the World Bank and the Asian Development Bank in relation to TRS. Endnotes Shujie Zhang, Technical Attaché of the WCO, has been working in the WCO Asia Pacific Regional Office for Capacity Building (ROCB) in Bangkok since March 2007. He was actively involved in international cooperation, training and research while serving in the Shanghai Customs College, China Customs, prior to his appointment to the ROCB. He has been engaged in projects sponsored by the World Bank and the Asian Development Bank in relation to TRS. 134 Volume 3, Number 2
https://openalex.org/W2834121948	https://europepmc.org/articles/pmc7800508?pdf=render	English	null	The complete chloroplast genome sequence of Histiopteris incisa (Dennstaedtiaceae)	Mitochondrial DNA. Part B. Resources	2,018	cc-by	2,021	CONTACT Ting Wang tingwang@scau.edu.cn College of Life Sciences, South China Agricultural University, Wushan 483, Guangzhou 510642, China; Yingjuan Su suyj@mail.sysu.edu.cn School of Life Sciences, Sun Yat-sen University, Xingang Xi Lu 135, Guangzhou 510275, China Xinyao Sun and Ziqi Kang contributed equally to this paper. 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ARTICLE HISTORY Received 29 May 2018 Accepted 18 June 2018 The fern is up to 2 m tall with long-creeping rhizome covered with chestnut-brown scales (Yan et al. 2013). Known as bat's wing fern or water fern, its fronds are soft and green and have deeply lobed segments. The plant is usually found in the moist areas, where it may form extensive colonies. It is wide- spread across Bhutan, NE India, S Japan, pantropical areas, islands near Antarctica, and Madagascar (Yan et al. 2013). Histiopteris incisa is a core member to explore the phylogen- etic relationships between Histiopteris and Pteris due to their morphological similarity (Yan et al. 2013). Hence, acquirement of its whole chloroplast (cp) genome sequence will lay solid foundation to address this issue. (Schattner et al. 2005) were used to conduct the annotation. The complete chloroplast genome of H. incisa (GenBank accession number: MH319942) is a quadripartite structure of 16,0567 bp in length, with two inverted repeat regions (IRs) of 27,119 bp each, a large single-copy (LSC) region of 84,897 bp and a small single-copy (SSC) region of 21,432 bp. It harbors 132 genes, including 88 protein-coding genes, eight rRNA genes, 35 tRNA genes, and one pseudogene. A total of 18 genes have introns. Among them, ndhB, rps16, atpF, rpoC1, petB, petD, ndhA, rpl16, and rpl2 have a single intron, whereas ycf3, clpP, and rps12 contain two introns. Fourteen genes occur as two copies, including four protein- coding genes (rps12, rps7, psbA, and ycf2), six tRNA genes (trnN-GUU, trnH-GUG, trnI-GAU, trnA-UGC, trnT-UGU, and trnR- ACG), and four rRNA genes (rrn4.5, rrn5, rrn16, and rrn23). The total GC content was 43.0% and the corresponding val- ues in LSC, IRs, and SSC are 41.2%, 46.5%, and 39.1%, respectively. Sample of H. incisa was provided by South China Botanical Garden, Chinese Academy of Sciences (2319’28.21’’N, 11337’47.46’’E). The specimen is stored in Herbarium of Sun Yat-sen University (SYS; voucher: SS Liu 20161012). Total genomic DNA was isolated by Tiangen Plant Genomic DNA Kit (Tiangen Biotech Co., Beijing, China). Average 300 bp Illumina paired-end genomic library was pre- pared and sequenced on an Illumina Hiseq 2500 platform (Illumina Inc., San Diego, CA). After trimming the sequences using Trimmomatic v0.32 (Bolger et al. 2014), we obtained high quality clean reads and assessed their quality by FastQC v0.10.0 (Andrews 2010). MITOCHONDRIAL DNA PART B 2018, VOL. 3, NO. 2, 772–773 https://doi.org/10.1080/23802359.2018.1491344 MITOCHONDRIAL DNA PART B 2018, VOL. 3, NO. 2, 772–773 https://doi.org/10.1080/23802359.2018.1491344 The complete chloroplast genome sequence of Histiopteris incisa (Dennstaedtiaceae) Xinyao Suna, Ziqi Kanga, Shanshan Liua, Ruixiang Xua, Zhen Wangb, Ting Wangc and Y Xinyao Suna, Ziqi Kanga, Shanshan Liua, Ruixiang Xua, Zhen Wangb, Ting Wangc and Yingjuan Sua,d aSchool of Life Sciences, Sun Yat-sen University, Guangzhou, China; bCollege of Life Sciences, Nanjing Agricultural University, Nanjing, China; cCollege of Life Sciences, South China Agricultural University, Guangzhou, China; dResearch Institute of Sun Yat-sen University in Shenzhen, Shenzhen, China aSchool of Life Sciences, Sun Yat-sen University, Guangzhou, China; bCollege of Life Sciences, Nanjing Agricultural University, Nanjing, China; cCollege of Life Sciences, South China Agricultural University, Guangzhou, China; dResearch Institute of Sun Yat-sen University in Shenzhen, Shenzhen, China ARTICLE HISTORY Received 29 May 2018 Accepted 18 June 2018 KEYWORDS Histiopteris incisa; chloroplast genome; phylogenetic analysis ARTICLE HISTORY Received 29 May 2018 Accepted 18 June 2018 incisa (GenBank accession number: MH319942) is a quadripartite structure of 16,0567 bp in length, with two inverted repeat regions (IRs) of 27,119 bp each, a large single-copy (LSC) region of 84,897 bp and a small single-copy (SSC) region of 21,432 bp. It harbors 132 genes, including 88 protein-coding genes, eight rRNA genes, 35 tRNA genes, and one pseudogene. A total of 18 genes have introns. Among them, ndhB, rps16, atpF, rpoC1, petB, petD, ndhA, rpl16, and rpl2 have a single intron, whereas ycf3, clpP, and rps12 contain two introns. Fourteen genes occur as two copies, including four protein- coding genes (rps12, rps7, psbA, and ycf2), six tRNA genes (trnN-GUU, trnH-GUG, trnI-GAU, trnA-UGC, trnT-UGU, and trnR- ACG), and four rRNA genes (rrn4.5, rrn5, rrn16, and rrn23). The total GC content was 43.0% and the corresponding val- ues in LSC, IRs, and SSC are 41.2%, 46.5%, and 39.1%, respectively. Phylogenetic analysis was carried out using the com- plete genome sequence of 14 ferns and Osmundastrum cin- namomeum as outgroup by Maximum likelihood (ML) method. After sequence alignment by MAFFT (Katoh et al. 2002), phylogenetic tree was constructed using RAxML (Stamatakis 2014) with 1000 bootstrap replicates. The results revealed that H. incisa was closely related to Pteridium aquilinum subsp. aquilinum (Figure 1). The CONTACT Ting Wang tingwang@scau.edu.cn College of Life Sciences, South China Agricultural University, Wushan 483, Guangzhou 510642, China; Birney 2008). DOGMA (Wyman et al. 2004) and tRNAscan-SE (Schattner et al. 2005) were used to conduct the annotation. Histiopteris incisa is only one accepted species of Histiopteris in the family Dennstaedtiaceae (Yan et al. 2013). The fern is up to 2 m tall with long-creeping rhizome covered with chestnut-brown scales (Yan et al. 2013). Known as bat's wing fern or water fern, its fronds are soft and green and have deeply lobed segments. The plant is usually found in the moist areas, where it may form extensive colonies. It is wide- spread across Bhutan, NE India, S Japan, pantropical areas, islands near Antarctica, and Madagascar (Yan et al. 2013). Histiopteris incisa is a core member to explore the phylogen- etic relationships between Histiopteris and Pteris due to their morphological similarity (Yan et al. 2013). Hence, acquirement of its whole chloroplast (cp) genome sequence will lay solid foundation to address this issue. Histiopteris incisa is only one accepted species of Histiopteris in the family Dennstaedtiaceae (Yan et al. 2013). ARTICLE HISTORY Received 29 May 2018 Accepted 18 June 2018 ARTICLE HISTORY Received 29 May 2018 Accepted 18 June 2018 ABSTRACT Histiopteris incisa is a core member to address the issue of relationship between Histiopteris and Pteris. Its complete chloroplast genome was generated by de novo assembly using Illumina sequencing. The complete chloroplast genome is a quadripartite structure of 16,0567 bp in length, with two inverted repeat regions (IRs) of 27,119bp each, a large single-copy (LSC) region of 84,897bp and a small single- copy (SSC) region of 21,432bp. A total of 132 genes were predicted, involving in 88 protein-coding genes, eight rRNA genes, 35 tRNA genes, and one pseudogene. ML phylogenetic analysis showed that H. incisa was closely related to Pteridium aquilinum subsp. aquilinum. KEYWORDS Histiopteris incisa; chloroplast genome; phylogenetic analysis Histiopteris incisa is only one accepted species of Histiopteris in the family Dennstaedtiaceae (Yan et al. 2013). The fern is up to 2 m tall with long-creeping rhizome covered with chestnut-brown scales (Yan et al. 2013). Known as bat's wing fern or water fern, its fronds are soft and green and have deeply lobed segments. The plant is usually found in the moist areas, where it may form extensive colonies. It is wide- spread across Bhutan, NE India, S Japan, pantropical areas, islands near Antarctica, and Madagascar (Yan et al. 2013). Histiopteris incisa is a core member to explore the phylogen- etic relationships between Histiopteris and Pteris due to their morphological similarity (Yan et al. 2013). Hence, acquirement of its whole chloroplast (cp) genome sequence will lay solid foundation to address this issue. Sample of H. incisa was provided by South China Botanical Garden, Chinese Academy of Sciences (2319’28.21’’N, 11337’47.46’’E). The specimen is stored in Herbarium of Sun Yat-sen University (SYS; voucher: SS Liu 20161012). Total genomic DNA was isolated by Tiangen Plant Genomic DNA Kit (Tiangen Biotech Co., Beijing, China). Average 300 bp Illumina paired-end genomic library was pre- pared and sequenced on an Illumina Hiseq 2500 platform (Illumina Inc., San Diego, CA). After trimming the sequences using Trimmomatic v0.32 (Bolger et al. 2014), we obtained high quality clean reads and assessed their quality by FastQC v0.10.0 (Andrews 2010). These clean reads were further de novo assembled into contigs by Velvet v1.2.07 (Zerbino and Birney 2008). DOGMA (Wyman et al. 2004) and tRNAscan-SE (Schattner et al. 2005) were used to conduct the annotation. The complete chloroplast genome of H. ma UK Limited, trading as Taylor & Francis Group. under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, um, provided the original work is properly cited. References Andrews S. 2010. FastQC: a quality control tool for high throughput sequence data; [accessed 2017 Sep 2]. http://www.bioinformatics.bab- raham.ac.uk/projects/fastqc/. Bolger AM, Lohse M, Usadel B. 2014. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 30:2114–2120. ARTICLE HISTORY Received 29 May 2018 Accepted 18 June 2018 These clean reads were further de novo assembled into contigs by Velvet v1.2.07 (Zerbino and Phylogenetic analysis was carried out using the com- plete genome sequence of 14 ferns and Osmundastrum cin- namomeum as outgroup by Maximum likelihood (ML) method. After sequence alignment by MAFFT (Katoh et al. 2002), phylogenetic tree was constructed using RAxML (Stamatakis 2014) with 1000 bootstrap replicates. The results revealed that H. incisa was closely related to Pteridium aquilinum subsp. aquilinum (Figure 1). The MITOCHONDRIAL DNA PART B 773 773 0.08 Osmundastrum cinnamomeum (outgroup) KF225592.1 Cystopteris chinensis KY427337.1 Asplenium prolongatum KY427332.1 Lepisorus clathratus KY419704.1 Ceratopteris richardii KM052729.1 Pteridium aquilinum subsp. aquilinum HM535629.1 Rhachidosorus consimilis KY427356.1 Dryopteris decipiens KY427348.1 Deparia viridifrons KY427340.1 Adiantum capillus-veneris AY178864.1 Athyrium sheareri KY427330.1 Histiopteris incisa Diplazium unilobum KY427347.1 Cheilanthes lindheimeri HM778032.1 100 100 70 80 100 100 100 100 99 100 100 Athyriaceae Aspleniaceae Rhachidosoraceae Cystopteridaceae Dryopteridaceae Polypodiaceae Dennstaedtiaceae Pteridaceae Osmundaceae Figure 1. ML phylogenetic tree of Histiopteris incisa with 14 ferns and Osmundastrum cinnamomeum as outgroup based on complete chloroplast genome sequen- es. Numbers in the nodes are support values with 1000 bootstrap replicates. Figure 1. ML phylogenetic tree of Histiopteris incisa with 14 ferns and Osmundastrum cinnamomeum as outgroup based on complete chloroplast genome sequen- ces. Numbers in the nodes are support values with 1000 bootstrap replicates. Figure 1. ML phylogenetic tree of Histiopteris incisa with 14 ferns and Osmundastrum cinnamomeum as outgroup based on c ces. Numbers in the nodes are support values with 1000 bootstrap replicates. complete chloroplast genome of H. incisa provides power- ful data to further elucidate phylogenetic relationships of dennstaedtioid ferns. Disclosure statement Katoh K, Misawa K, Kuma K, Miyata T. 2002. MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier trans- form. Nucleic Acids Res. 30:3059–3066. The authors declare no conflict of interests. The authors alone are responsible for the content and writing of the paper. Schattner P, Brooks AN, Lowe TM. 2005. The tRNAscan-SE, snoscan and snoGPS web servers for the detection of tRNAs and snoRNAs. Nucleic Acids Res. 33:W686–W689. Funding Stamatakis A. 2014. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics. 30:1312–1313. This work was supported by the National Natural Science Foundation of China [31370364, 31570652, 31670200, and 31770587], the Natural Science Foundation of Guangdong Province, China [2016A030313320 and 2017A030313122], Science and Technology Planning Project of Guangdong Province, China [2017A030303007], Project of Department of Science and Technology of Shenzhen City, Guangdong, China [JCYJ20160425165447211 and JCYJ20170413155402977], and Science and Technology Planning Project of Guangzhou City, China [201804010389]. Wyman SK, Jansen RK, Boore JL. 2004. Automatic annotation of organ- ellar genomes with DOGMA. Bioinformatics. 20:3252–3255. Yan YH, Qi XP, Liao WB, Xing FW, Ding MY, Wang FG, Zhang XC, Wu ZH, Serizawa S, Prado J, et al. 2013. Dennstaedtiaceae. In: Wu ZY, Raven PH, Hong DY, eds., Flora of China. Vol. 2–3 (Pteridophytes). Beijing: Science Press; p. 147–168. Zerbino DR, Birney E. 2008. Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res. 18:821–829.
https://openalex.org/W4205494402	https://digital.csic.es/bitstream/10261/258537/1/Zamanillo_et_al_2021.pdf	English	null	Uncoupled seasonal variability of transparent exopolymer and Coomassie stainable particles in coastal Mediterranean waters	Elementa	2,021	cc-by	17,782	Uncoupled seasonal variability of transparent exopolymer and Coomassie stainable particles in coastal Mediterranean waters: Insights into sources and driving mechanisms Marina Zamanillo1,2, Eva Ortega-Retuerta3, Carolina Cisternas-Novoa2, Ce`lia Marrase´ 1, Carles Pelejero1,4, Josep Pascual5, Josep M. Gasol1,6, Anja Engel2, and Rafel Simo´ 1,* Marina Zamanillo1,2, Eva Ortega-Retuerta3, Carolina Cisternas-Novoa2, Ce`lia Marrase´ 1, Carles Pelejero1,4, Josep Pascual5, Josep M. Gasol1,6, Anja Engel2, and Rafel Simo´ 1,* Transparent exopolymer particles (TEP) and Coomassie stainable particles (CSP) are gel-like particles, ubiquitous in the ocean, that affect important biogeochemical processes including organic carbon cycling by planktonic food webs. Despite much research on both groups of particles (especially TEP) over many years, whether they exist as distinctly stainable fractions of the same particles or as independent particles, each with different driving factors, remains unclear. To address this question, we examined the temporal dynamics of TEP and CSP over 2 complete seasonal cycles at 2 coastal sites in the Northwestern Mediterranean Sea, the Blanes Bay Microbial Observatory (BBMO) and the L’Estartit Oceanographic Station (EOS), as well as their spatial distribution along a coast-to-offshore transect. Biological, chemical, and physical variables were measured in parallel. Surface concentrations (mean + standard deviation [SD]) of TEP were 36.7 + 21.5 mg Xanthan Gum (XG) eq L–1 at BBMO and 36.6 + 28.3 mg XG eq L–1 at EOS; for CSP, they were 11.9 + 6.1 mg BSA eq L–1 at BBMO and 13.0 + 5.9 mg BSA eq L–1 at EOS. Seasonal variability was more evident at EOS, where surfaceTEP and CSP concentrations peaked in summer and spring, respectively, and less predictable at the shore-most station, BBMO. Vertical distributions between surface and 80 m, monitored at EOS, showed highest TEP concentrations within the surface mixed layer during the stratification period, whereas CSP concentrations were highest before the onset of summer stratification. Phytoplankton were the main drivers of TEP and CSP distributions, although nutrient limitation and saturating irradiance also appeared to play important roles. The dynamics and distribution of TEP and CSP were uncoupled both in the coastal sites and along the transect, suggesting that they are different types of particles produced and consumed differently in response to environmental variability. ransparent exopolymer particles, Coomassie stainable particles, Mediterranean Sea, Time series, Keywords: Transparent exopolymer particles, Coomassie stainable particles, Mediterranean Sea, Time series, Phytoplankton Keywords: Transparent exopolymer particles, Coomassie stainable particles, Mediterranean Sea, Time series, Phytoplankton of dissolved organic matter (approximately 660 Gt; Hedges, 2002). 1Department of Marine Biology and Oceanography, Institut de Cie`ncies del Mar, CSIC, Barcelona, Catalonia, Spain 2GEOMAR Helmholtz Centre for Ocean Research Kiel, Kiel, Germany 3CNRS, Sorbonne Universite´ , UMR 7621, Laboratoire d’Oce´anographie Microbienne, Banyuls–sur–Mer, France 4ICREA, Institucio´ Catalana de Recerca i Estudis Avançats, Barcelona, Catalonia, Spain 5Estacio´ Meteorolo` gica de L’Estartit, Girona, Catalonia, Spain 6Center for Marine Ecosystems Research, School of Science, Edith Cowan University, Joondalup, WA, Australia * Corresponding author: Email: rsimo@icm.csic.es Zamanillo, M, et al. 2021. Uncoupled seasonal variability of transparent exopolymer and Coomassie stainable particles in coastal Mediterranean waters: Insights into sources and driving mechanisms. Elem Sci Anth, 9: 1. DOI: https://doi.org/10.1525/elementa.2020.00165 Zamanillo, M, et al. 2021. Uncoupled seasonal variability of transparent exopolymer and Coomassie stainable particles in coastal Mediterranean waters: Insights into sources and driving mechanisms. Elem Sci Anth, 9: 1. DOI: https://doi.org/10.1525/elementa.2020.00165 Uncoupled seasonal variability of transparent exopolymer and Coomassie stainable particles in coastal Mediterranean waters: Insights into sources and driving mechanisms Understanding the mechanisms that pro- duce and recycle organic matter in the ocean is essential to predicting further changes in CO2 capture. Marine gel- like organic particles, such as transparent exopolymer par- ticles (TEP) and Coomassie stainable particles (CSP), have gained considerable attention in oceanography over the past years due to their involvement in biological, chemi- cal, and physical processes such as carbon cycling, air–sea interactions, and microbial growth (Engel et al., 2020). TEP are relevant players in the carbon cycle as their production rate is estimated to represent 5%–10% of primary pro- duction in the ocean (Mari et al., 2017), and they favor the aggregation and sinking of suspended particles (Engel et al., 2004; Burd and Jackson, 2009), thus impacting the efficiency of the biological carbon pump. In addition, due to their low density (Azetsu-Scott and Passow, 2004), TEP 1Department of Marine Biology and Oceanography, Institut de Cie`ncies del Mar, CSIC, Barcelona, Catalonia, Spain 1Department of Marine Biology and Oceanography, Institut de Cie`ncies del Mar, CSIC, Barcelona, Catalonia, Spain 2GEOMAR Helmholtz Centre for Ocean Research Kiel, Kiel, Germany 1. Introduction Owing to the lack of similar studies with CSP, only nutrient avail- ability is known to affect CSP production (Radic et al., 2006), while physiological stress did not enhance CSP production by diatoms and cyanobacteria in culture (Thornton and Chen, 2017). The northwestern (NW) Mediterranean Sea is a temper- ate oligotrophic sea, characterized by the enhancement of water stratification in late spring and summer due to the increase in solar radiation, which leads to low nutrient concentrations in surface waters (Sala et al., 2002; Duarte et al., 2004; Lucea et al., 2005) and minimum chlorophyll a (Chla) concentrations, and by vertical mixing in fall through winter. Chla concentration in the NW Mediterra- nean Sea is usually highest in late winter or early spring, triggered by the onset of thermal stratification after win- ter mixing, coinciding with nutrient availability and high solar radiation (Marty et al., 2002; Gasol et al., 2016). However, the specific conditions that prompt the onset of the bloom in the Mediterranean Sea and other regions are still under debate (Smetacek and Cloern, 2008; Beh- renfeld, 2010; Taylor and Ferrari, 2011). Previous studies of the temporal dynamics of TEP in the Mediterranean Sea, other than those conducted in eutro- phic coastal areas or zones heavily influenced by the pres- ence of seagrass meadows (Radic et al., 2005; Scoullos et al., 2006; Iuculano et al., 2017a), showed maximum TEP concentrations in summer, both at coastal and offshore sites (Beauvais et al., 2003; Ortega-Retuerta et al., 2018), with a temporal mismatch between TEP and Chla (Ortega- Retuerta et al., 2018). In contrast, there are no published studies of CSP distributions in the Mediterranean Sea. This study was motivated by the paucity of empirical studies on the distribution and seasonality of CSP and their relationship with those of TEP (Cisternas-Novoa et al., 2015; Thornton et al., 2016; Thornton and Chen, 2017; Thornton, 2018). Our main objective was to elucidate whether or not these 2 pools of organic particles follow similar trends over the year and across horizontal and vertical scales. Based on a previous study (Cisternas- Novoa et al., 2015), we hypothesized that CSP distribu- tions and their temporal dynamics would be closely related to those of phytoplankton biomass, while TEP dis- tribution and dynamics would be related to the combina- tion of phytoplankton biomass and taxonomic composition, solar radiation, and nutrient limitation. 1. Introduction One of these studies was conducted in a low-salinity temperate coastal site in the Baltic Sea, where CSP concentration, measured in both the SML and the subsurface water (SSW), increased in summer and was generally similar to that of TEP (Dresh- chinskii and Engel, 2017). In studies in the Sargasso Sea (Cisternas-Novoa et al., 2015) and the Fram Strait (von Jackowski et al., 2020), TEP and CSP presented different temporal and vertical distributions. The 2 particle types were also temporally uncoupled during the course of a phytoplankton bloom in a mesocosm study (Cisternas- Novoa et al., 2015). Phytoplankton are the main source of TEP and presum- ably also of CSP in the ocean (Passow, 2002b; Thornton and Chen, 2017), although prokaryotic heterotrophs can also produce them (Stoderegger and Herndl, 1999; Radic et al., 2006). Diatoms and cyanobacteria have been shown to produce both CSP and TEP (Passow and Alldredge, 1994; Grossart et al., 1998; Grossart et al., 2006; Endres et al., 2013). Although CSP production by other phyto- plankton groups has not yet been examined (reviewed in Thornton, 2018), dissolved organic nitrogen, including protein (stained by the dye Coomassie brilliant blue [CBB]), is known to be released by phytoplankton into the surrounding medium (Hu and Smith, 1998; Suratman et al., 2008; Sarmento et al., 2013). Similarly, TEP precursors, which generally are acidic polysaccharides, are released mostly by phytoplankton (Decho, 1990) and then assem- bled abiotically into TEP through ionic bonding (Alldredge et al., 1993; Thornton, 2004). Such assembly is presum- ably also the case for CSP (Cisternas-Novoa et al., 2015), but this process has not yet been tested directly. Zooplank- ton (Ling and Alldredge, 2003) and prokaryotic hetero- trophs (Passow, 2002a; Grossart et al., 2006; Azam and Malfatti, 2007) can use TEP, which is probably also the case for CSP (Radic et al., 2006; Endres et al., 2013; Cisternas-Novoa et al., 2015; Engel et al., 2015). Environ- mental and biological variables, other than the taxonomic composition of phytoplankton and prokaryotic hetero- trophs, impact the production of TEP in the ocean. Phyto- plankton physiological state (Passow, 2002a), temperature (Claquin et al., 2008), light intensity (Trabelsi et al., 2008), and nutrient availability (Radic et al., 2006) may alter the production of TEP, while losses can occur through UV- induced photolysis (Ortega-Retuerta et al., 2009). 1. Introduction The ocean, which currently absorbs ca. 30% of the anthro- pogenic CO2, contains a vast amount of carbon in the form 2GEOMAR Helmholtz Centre for Ocean Research Kiel, Kiel, Germany * Corresponding author: Email: rsimo@icm.csic.es Zamanillo et al: Seasonality of TEP and CSP in the Mediterranean Sea Art. 9(1) page 2 of 23 Mari et al., 2017). Conversely, CSP were rarely described until the recent development of a spectrophotometric method (reviewed by Thornton, 2018), which is less time-consuming and labor-intensive than the classical microscopic quantification (Cisternas-Novoa et al., 2014). Our knowledge of the temporal dynamics of CSP re- mains limited to a handful of studies at sites where TEP and CSP were measured in parallel. One of these studies was conducted in a low-salinity temperate coastal site in the Baltic Sea, where CSP concentration, measured in both the SML and the subsurface water (SSW), increased in summer and was generally similar to that of TEP (Dresh- chinskii and Engel, 2017). In studies in the Sargasso Sea (Cisternas-Novoa et al., 2015) and the Fram Strait (von Jackowski et al., 2020), TEP and CSP presented different temporal and vertical distributions. The 2 particle types were also temporally uncoupled during the course of a phytoplankton bloom in a mesocosm study (Cisternas- Novoa et al., 2015). Mari et al., 2017). Conversely, CSP were rarely described until the recent development of a spectrophotometric method (reviewed by Thornton, 2018), which is less time-consuming and labor-intensive than the classical microscopic quantification (Cisternas-Novoa et al., 2014). can ascend through the upper water column, accumulate in the sea surface microlayer (SML), and influence air–sea gas exchanges (Calleja et al., 2009; Wurl et al., 2016; Jen- kinson et al., 2018). They can also be released to the atmo- sphere, contributing to organic aerosols (Aller et al., 2005) and impacting the Earth’s radiative budget (Brooks and Thornton, 2018). Regarding CSP, there is little evidence showing a significant impact on aggregation processes (Prieto et al., 2002; Cisternas-Novoa et al., 2015), but CSP also accumulate in the SML (Wurl et al., 2011; Engel and Galgani, 2016; Zancker et al., 2017; Sun et al., 2018) and have been observed in sea spray aerosols (Kuznetsova et al., 2005; Aller et al., 2017). Our knowledge of the temporal dynamics of CSP re- mains limited to a handful of studies at sites where TEP and CSP were measured in parallel. 2.1. Study sites and sampling Samples were collected within the Catalan Sea of the NW Mediterranean at (1) the BBMO site at 41.40N, 2.48E (http://bbmo.icm.csic.es); (2) the EOS site, 70 km further north at 42.05N, 3.25E, and (3) stations along a transect between the Catalan Coast near BBMO and Mallorca Is- land, on board the RV Garcı´a del Cid for a cruise desig- nated MIFASOL II (Figure 1). The BBMO is located around 800 m offshore, in an open bay with shallow waters (sandy bottom at 20 m depth) that only weakly stratifies in sum- mer and where most terrestrial inputs arrive as runoff from the surrounding coastal area (details in Guadayol et al., 2009a, and Gasol et al., 2012). The EOS is located 3.2 km off the main coast and 2 km off the Medes Islands, over a water column approximately 92 m deep (Figure 1) that strongly stratifies in summer and is heavily influ- enced by wind dynamics (Aparicio et al., 2017). oaded from http://online.ucpress.edu/elementa/article-pdf/9/1/00165/485853/elementa.2020.00165.pdf by guest on 24 January 2022 The BBMO and EOS samplings were performed at least once per month, from June solstice 2015 to October 2017, except during adverse weather conditions. At BBMO, water samples were taken with an acid-cleaned bucket from the surface, prefiltered through an acid-cleaned 200-mm mesh net to remove large plankton and kept in 20-L acid- cleaned polycarbonate carboys. Carboys were maintained under dim light, covered with black plastic bags to avoid light interference. Further processing of samples was done within 1.5 h at the laboratory. At EOS, 4 depths (surface, 20, 50, and 80 m) were sampled using 5-L Niskin bottles and then filtered through an acid-cleaned 200-mm nylon mesh. Water for TEP and CSP analyses was collected in 1-L acid-cleaned polycarbonate bottles, while those for the rest of variables were kept in 8-L acid-cleaned polyethyl- ene carboys, all of them covered with black plastic bags. Polycarbonate bottles were transported in a cooler box with pack ice. Sample processing was done at the home laboratory within 4 h. Figure 1. Map of the study area. The Blanes Bay Microbial Observatory (green circle), L’Estartit Oceanographic Station (pink circle), and the transect between the Catalan Coast and Mallorca Island (blue circles). DOI: https://doi.org/10.1525/elementa.2020. 00165.f1 abundance (flow cytometry). At the DCM, only TEP, CSP, and Chla were measured. 1. Introduction To test these hypotheses, we assessed the temporal dynamics TEP and CSP are defined by the specific dyes that stain them: TEP by Alcian Blue, which stains acidic polysacchar- ides (Alldredge et al., 1993; Passow and Alldredge, 1995), and CSP by CBB, which stains proteins (Long and Azam, 1996; Engel, 2009). CSP are thought to contain 5–7 times more nitrogen than TEP (Engel and Passow, 2001; Mari et al., 2001), although their content has not been measured directly. TEP have been thoroughly studied in the ocean (reviewed by Passow, 2002b, Bar-Zeev et al., 2015, and Zamanillo et al: Seasonality of TEP and CSP in the Mediterranean Sea Art. 9(1) page 3 of 23 Figure 1. Map of the study area. The Blanes Bay Microbial Observatory (green circle), L’Estartit Oceanographic Station (pink circle), and the transect between the Catalan Coast and Mallorca Island (blue circles). DOI: https://doi.org/10.1525/elementa.2020. 00165.f1 of TEP and CSP simultaneously over 2 complete seasonal cycles at 2 coastal sites of the NW Mediterranean Sea characterized by strong seasonality. At the shore-most site, the Blanes Bay Microbial Observatory (BBMO), we followed TEP and CSP concentrations near the surface; at the off- shore site, the L’Estartit Oceanographic Station (EOS), which presents a conspicuous deep chlorophyll maximum (DCM) between 20 and 80 m, we examined the seasonal variations in the vertical distribution of TEP and CSP. Hor- izontal variations of TEP and CSP were also analyzed at 2 depths in a nearby coast-to-offshore transect. Downloaded from http://online.ucpress.edu/elementa/article-pdf/9/1/00165/485853/elementa.2020.00165.pdf by guest on 24 January 2022 2.3. Chemical and biological variables 2.3.1. Particulate organic matter (TEP, CSP, partic- ulate organic carbon [POC], and particulate organic nitrogen [PON]) g TEP were analyzed following the spectrophotometric method proposed by Passow and Alldredge (1995). Sam- ples (150–500 mL) were filtered in duplicate under con- stant low pressure (approximately 150 mm Hg) onto 25-mm diameter 0.4-mm pore size polycarbonate filters (DHI Lab, Denmark). Immediately, the filters were stained with an Alcian Blue solution (500 mL, 0.02%, pH 2.5) for 5 s and rinsed with Milli-Q water. All sampling material was prewashed with HCl (10%) and Milli-Q water, and the first milliliters of sample were discarded. All filters remained frozen until further processing. Filters were soaked in 5 mL of 80% sulphuric acid for 3 h, shaking them inter- mittently, and absorbance of the solution was measured with the spectrophotometer at 787 nm (Varian Cary 100 Bio). The absorption of every batch of Alcian Blue was calibrated using an XG solution that was homogenized with a tissue grinder and measured by weight difference. A total of 7 calibrations of different Alcian Blue solutions were carried out during the temporal series. Calibration factors ranged from 123 to 231 mg XG eq A–1 (average + SD of 171 + 38 mg XG eq A–1, where eq refers to equiva- lents and A is absorbance) and detection limits ranged from 0.021 to 0.048 absorbance units. Duplicate blanks (empty filters stained with Alcian Blue) were also prepared with every batch of filtered samples. TEP values are re- ported as the average of analytical duplicates in units of mg XG eq L–1. We conducted TEP analyses in formalin-fixed (1% final concentration) samples, which were preserved at 4C until filtration (within 4 months at most). We decided to conduct TEP analyses on fixed samples, since formalin does not interfere with the measurement (Passow and Alldredge, 1995; Ortega-Retuerta et al., 2018), and in order to optimize the number of samples processed every time g In 4 of the BBMO samplings (November 2016 and April, August, and October 2017), TEP and CSP were also measured following the microscopy method (Alldredge et al., 1993; Passow and Alldredge, 1995). Duplicate samples of 50 mL were filtered onto 25-mm diameter 0.4-mm pore size polycarbonate filters (DHI), stained, and the excess dye removed. Blanks were prepared as stained empty fil- ters. 2.2. Physical variables A calibrated SAIVA/S SD204 sensor was used to measure vertical profiles of temperature and salinity at BBMO and EOS. In August 2016, at BBMO and from May to June 2016 at EOS, the sensor was inoperative. We calculated the mixed layer depth (MLD) as the depth where temperature changed more than 0.15C with respect to a reference depth of 1 m. At BBMO, we calculated a stratification index that we defined as the temperature difference between the surface and near the sea bottom (20 m). At EOS, the stratification index was defined as the tempera- ture difference between the surface and just below the thermocline.Water transparency (in meters) was measured The coast-to-offshore transect was conducted during the MIFASOL II cruise, from October 22, 2015, to October 24, 2015, reaching 145 km from shore, and including stations on the shelf (station 1, closest to the shore), con- tinental slope (stations 2–4), and deep basin (stations 5– 7). Seawater was collected at 2 depths: the surface (5 m) and, when present, the DCM, using Niskin bottles (12 L) on a 12-bottle rosette with conductivity–temperature– depth (CTD) casts. Surface variables measured during the transect were TEP, CSP, Chla, inorganic nutrients, prokary- otic heterotroph abundance, and picophytoplankton Zamanillo et al: Seasonality of TEP and CSP in the Mediterranean Sea Art. 9(1) page 4 of 23 a new calibration curve was obtained (1 every 4 months). To estimate TEP carbon content (TEP-C), we used the con- version factor of 0.51 mg TEP-C mg XG eq–1 (Engel and Passow, 2001). with a Secchi disk at the BBMO and with white-faced Ni- skin bottles (used as analogous to a Secchi disk) at the EOS. The light extinction coefficient (Kd, m–1) was calcu- lated as 1.7/water transparency (Kirk, 1994). Total irradi- ance at 2 m was recorded hourly by a pyranometer at the Malgrat de Mar station, situated at 41 380 57”N, 2 450 8”E, 520 m from the coast (approximately 5 km southwest of BBMO) and the Sant Pere Pescador station (approxi- mately 25 km north of EOS; Catalan Meteorological Ser- vice, www.meteo.cat). We measured the average solar radiation of the 24 h previous to the sampling and calcu- lated the daily-averaged solar radiation dose as: CSP concentration was determined by spectrophotom- etry following Cisternas-Novoa et al. (2014). 2.2. Physical variables Duplicate samples (200–350 mL) were filtered onto 25-mm diame- ter 0.4-mm pore size polycarbonate filters (Whatman and DHI) using a constant low filtration pressure (approxi- mately 150 mmHg). The samples were stained immedi- ately with 1 mL of CBB-G 250 solution (0.04%, pH 7.4) for 30 s, always prepared with the same filtered (0.2 mm) seawater from Medes, 80 m depth, and rinsed with Milli-Q water 3 times. The filters were stored frozen in 15-mL polypropylene tubes until further processing in the labo- ratory (within 4 months). Duplicate blanks (stained empty filters) were prepared with every batch of filtered samples. Both the sample and blank filters were soaked in 4 mL of extraction solution (3% sodium dodecyl sulfate in 50% isopropyl alcohol), and the tubes were incubated in a water bath for 2 h at 37C. The filters were shaken every 30 min during this period. Optimal water bath temperature and shaking conditions were determined prior to analyzing the samples. We avoided sonication as DHI filters do not withstand sonication. We used Whatman Nuclepore filters (apt for sonication) to test that the water bath and shaking were enough to remove the stain from the filters. We measured the absorbance after extraction in the water bath for 2 h and then sonicated the same filters for 2 h and measured the absorbance again. More than 90% of absorbance was recovered without sonication in our sam- ples. The samples were then measured spectrophotomet- rically at 615 nm (Varian Cary 100 Bio and Shimadzu UV– Vis spectrophotometer UV120). Concentrations of CSP, reported as averages of duplicate measurements, were determined relative to a bovine serum albumin standard and expressed in units of mg BSA eq L–1 after Cisternas- Novoa et al. (2014). A total of 3 calibrations of different CBB solutions were carried out during the temporal series, with calibration factors ranging from 51 to 111 mg BSA eq A–1 (average of 73 + 33 mg BSA eq A–1), and detection limits ranged from 0.0440 to 0.0609 absorbance units. Solar radiation dose ¼ I Kd MLD ð1 eðKdMLDÞÞ; where I is the average surface irradiance (W m–2) in the 24 h previous to sampling, Kd is the light extinction coef- ficient (m–1), and MLD is the mixed layer depth (m). 2.2. Physical variables During the MIFASOL II cruise, a SBE21 Sea Cat Thermo- salinograph was used to obtain surface temperature and salinity and a CTD SBE911 plus to record vertical profiles down to 400 m. 3. Results Picophytoplankton (Prochlorococcus, Synechococcus, and picoeukaryotes), nanophytoplankton (photosynthetic na- noeukaryotes), and heterotrophic prokaryotes were enu- merated by flow cytometry after fixation with 1% paraformaldehyde plus 0.05% glutaraldehyde (final con- centrations), following standard methods (Gasol and Mor- a´n, 2015). Abundances were converted to biomass (mg L–1) using average C-to-cell conversion factors gathered in Simo´ et al. (2009) of 51 + 18 fg C cell–1 for 2.3.2. Chla Chla measurements started by filtering 150 mL (BBMO) or 100 mL (EOS) of seawater on GF/F filters (Whatman, 25-mm diameter) and storing filters at –20C until further processing. The pigment was extracted in acetone (90% v: v) at 4C in the dark for 24 h. Fluorescence was measured with a calibrated Turner Designs fluorometer, following Yentsch and Menzel (1963). Analytical duplicates were taken, with average coefficient of variation between du- plicates of 3%. 2.3. Chemical and biological variables The filters were placed on the white side of a semi- transparent glass slide (CytoClear, Poretics Corp., Livermore, US) and stored frozen (–20C) until micro- scopic analysis. Following Engel (2009), abundance, area, and size–frequency distribution of TEP and CSP in the size range of 1–760 mm were determined using a light micro- scope (Olympus Bx61) connected to a camera (Olympus DP72). Filters were screened at 200 magnification. Thirty pictures were taken randomly from each filter along 2 perpendicular cross sections (15 pictures each; resolu- tion 1,360 1,024 pixel, 8-bit color depth). ImageJ image Art. 9(1) page 5 of 23 Art. 9(1) page 5 of 23 Zamanillo et al: Seasonality of TEP and CSP in the Mediterranean Sea Prochlorococcus (22 source studies), 175 + 73 fg C cell–1 for Synechococcus (28 source studies), 1,319 + 813 fg C cell–1 for picoeukaryotes (24 source studies), and fg C cell– 1 ¼ 220 V (mm3) for photosynthetic nanoeukaryotes (1 source study), where cell volume V was set to 34 mm3 (assuming a spherical model with a radius of 2 mm). The carbon content of prokaryotic heterotrophs was estimated empirically from the bead-standardized side scatter of the relevant populations following Calvo-Dı´az and Mora´n (2006). Size was converted to C content following Norland (1993), yielding the estimated average biomass per cell in our study of 19.0 + 0.5 fg C (n ¼ 30) at BBMO and 19.3 + 2.7 fg C (n ¼ 19) at EOS. analysis software (version 1.44, public domain, developed at the U.S. National Institutes of Health) was used to semi- automatically analyze particle numbers and area. For POC and PON analyses, seawater (1,000 mL) was filtered through combusted (4 h, 450C) GF/F glass fiber filters (Whatman) and filters frozen at –20C until pro- cessed. Prior to analysis, the filters were thawed in an HCl-saturated atmosphere for 24 h to remove inorganic compounds. The filters were then dried and analyzed with an elemental analyzer (Perkin-Elmer 2400 CHN). No repli- cates were collected due to water volume limitations. 2.4. Statistical analyses y The nonparametric Wilcoxon–Mann–Whitney test was used to check for statistical differences in the different environmental variables among seasons. The seasons were separated by the winter/summer solstices and the spring/ fall equinoxes. We also used the nonparametric Wilcoxon– Mann–Whitney test to assess statistical differences of the variables among regions (BBMO and EOS). We performed pairwise Spearman correlation analyses to test for covaria- tions between environmental and biological variables in the BBMO, EOS, and MIFASOL II data sets. Bivariate anal- yses (ordinary least squares) between TEP and CSP concen- trations and several biological, chemical, and physical variables were performed in EOS profiles. We log- transformed data to fulfill the requirements of parametric tests. A harmonic analysis of the annual component (period, 365 days) of the TEP and CSP time series was performed using the HarmonicRegression package in the R software (Lueck et al., 2015). The samplings of December 2016 were not used in the harmonic analysis because these anomalously high TEP and CSP values were thought to be a response to freshwater discharges. Statistical tests, calculations, and illustrations were performed with Micro- soft Office Excel 2010, Ocean Data View software version 4 (Schlitzer, 2017), and R (version 3.5.1). 2.3.4. Phytoplankton identification and biomass 2.3.4. Phytoplankton identification and biomass Phytoplankton > 5 mm were identified and counted with an inverted microscope. Seawater was fixed with hexamine-buffered formaldehyde solution (4% final con- centration) and 100 mL were allowed to settle in Uter- mo¨hl chambers at 4C until analysis (Utermo¨hl, 1958). The samples were stored for a maximum of 6 months, and we did not observe any signs of cell damage. Phytoplankton was identified to the species level when possible and finally classified into 4 groups: diatoms, dinoflagellates, coccolithophores, and “other microalgae.”We used conver- sion equations to calculate cell C content from volume measurements (Menden-Deuer and Lessard, 2000). For diatoms, we used log pg C cell–1 ¼ log –0.541 (0.099) þ 0.811 (0.028) log V; for the other algae groups, log pg C cell–1 ¼ log –0.665 (0.132) þ 0.939 (0.041) log V, where V is cell volume in mm3 and the values inside parentheses are the 95% confidence intervals. ( ) ( ) SD (s) was calculated using the following formula: s ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ X ðx xÞ2 ðn 1Þ s ; where x is the mean of the sample and n is the sample number. Standard error (SE) was calculated as: SE ¼ sﬃﬃﬃn p : 2.3.3. Inorganic nutrients Dissolved inorganic nutrients (nitrate, phosphate, and sil- icate) were measured with standard segmented flow anal- ysis with colorimetric detection (Hansen and Grasshoff, 1983), using a SEAL Auto Analyzer AA3 HR (BBMO) or Bran þ Luebe autoanalyser (EOS). 3.4. Variation of biological variables over the time series The higher concentration of TEP at EOS was observed between the surface and 20 m in summer, coinciding with shallow mixed layers, while TEP concentration was usually homogeneously distributed during the rest of the year, despite the presence of a mixed layer in certain months (April, May, September, and October 2017). Chla concentrations were similar at both stations (Table 1), varying between 0.13 and 1.52 mg L–1 (average + SD of 0.49 + 0.39 mg L–1) in the surface of BBMO (Figure 2), between 0.13 and 0.75 mg L–1(average of0.31 +0.19 mg L–1) in the surface of EOS (Figure 3), and between 0.05 and 0.84 mg L–1 (average of 0.34 + 0.19 mg L–1) in the whole water column of EOS (Figure S3). Maximum concentrations occurred in winter at both stations (Table 1). Total phyto- plankton biomass (Figure S3) was not correlated with Chla at either of the 2 stations (P > 0.05) and was maximum in late winter in both cases. Prokaryotic heterotroph abun- dance at the surface ranged from 5 105 to 17.26 105 cells mL–1 at BBMO (Figure 2) and from 6.69 104 to 6.90 105 cells mL–1 at EOS (Figure 3). CSP concentrations in the surface waters were similar at both stations (Table 1). In BBMO, surface CSP concen- tration varied between 4.7 and 24.8 mg BSA eq L–1 (aver- age + SD of 11.9 + 6.1 mg BSA eq L–1; Figure 2), while at EOS, it varied between 4.5 and 22.4 mg BSA eq L–1 (average of 13.0 + 5.9 mg BSA eq L–1; Figure 3). CSP concentra- tions only followed a significant seasonal pattern at EOS (harmonic analysis, P ¼ 0.02). Differences in vertical CSP distributions also occurred among seasons at EOS (Figure S3). In summer, CSP concentrations were higher at 20 m, that is, below the mixed layer depth. In spring, concentra- tions were higher within the mixed layer depth, although in April and May 2017, a peak was also observed at 60 m. In fall and winter, there was no clear pattern of CSP dis- tribution with depth (Figure S3). Phytoplankton communities were similar at both sta- tions (Figures S1 and S2). Highest surface abundances of Prochlorococcus cells generally occurred in fall (except at EOS in 2016, where Prochlorococcus was found in spring), whereas maxima of Synechococcus occurred in spring. 3.2. Variation of TEP, CSP, POC, and PON over the time series The ranges and averages of surface TEP concentrations were similar at both coastal stations. At BBMO, surface TEP concentrations ranged from 8.4 to 80.9 mg XG eq L–1 (average + SD of 36.7 + 21.5 mg XG eq L–1; Figure 2), while at EOS, they ranged from 5.8 to 126 mg XG eq L–1, with an average of 36.6 + 28.3 mg XG eq L–1 (Table 1, Figure 3). Regarding seasonal patterns in surface waters, higher TEP concentrations were observed recurrently in early summer (Figures 2 and 3), which was confirmed by harmonic analyses for both BBMO (P ¼ 0.03) and EOS (P ¼ 0.04; Figure S4). The exception to this general pattern was the sample from December 2016, when a peak of TEP concentration at the surface coincided with a sharp decrease in salinity at both stations. At BBMO, this salinity drop was accompanied with higher nitrate and Chla con- centrations; at EOS, it came along with an increase in phosphate (Figures S1 and S2). The CSP size distributions followed an exponential shape with higher abundances of the smaller particles in the 4 samples analyzed (Figure 4). The slopes of the linear regressions between average particle diameter and TEP abundance (log-transformed) were –1.34, –1.42, –1.52, and –1.34 for November, April, August, and October, respectively. 3.3. TEP and CSP size distributions The TEP size distributions, determined by microscopy and image analysis in surface waters of BBMO in 4 dif- ferent months (November 2016 and April, August and October 2017), followed an exponential distribution, with the smallest particles (<1.25 mm), being the most abundant in 3 of the 4 samples. In April 2017, a unimodal distribution was observed, with particles ranging from 2.5 to 3.5 mm being the most abundant, followed by the smallest ones (Figure 4). The slopes of the linear regres- sions between average particle diameter and TEP abun- dance (log-transformed) were –1.53, –1.83, –1.84, and – 0.93 for November, April, August, and October, respectively. 3.1. Variation of the main physical and chemical variables in the time series (BBMO) and 22.6C + 1.4C (EOS) in summer (Table 1, Figures S1 and S2). In summer, high heat fluxes led to higher surface temperatures and the development of a shallow stratified layer (average MLD of 6.5 + 5.7 m in BBMO and 4.6 + 4.7 m in EOS; Table 1). Water trans- parency ranged from 5 to 20 m in BBMO and 5 to 24 m in EOS, with the highest values in summer at both stations. Dissolved nitrate and silicate concentrations in the surface were lowest in summer at both BBMO and EOS (Table 1). However, surface phosphate concentrations (average of 0.04 + 0.02 mmol L–1 in BBMO and 0.04 + 0.06 mmol L–1 in EOS) did not exhibit marked seasonal variations but were low throughout the year at both stations (Table 1). Dissolved inorganic nutrients at EOS usually increased with depth in summer months, while they were homoge- neously distributed across depth in the other seasons, with a few exceptions (Figure S3). 3.1. Variation of the main physical and chemical variables in the time series 3.1. Variation of the main physical and chemical variables in the time series BBMO and EOS are 2 sites relatively unaffected by human impacts or river discharges (Guadayol et al., 2009a; Ros and Gili, 2015), where oceanographic variables present the typical seasonal cycle of temperate coastal systems. SST changed from an average +SD of 13.4C + 0.4C (BBMO) and 13.2C + 0.5C (EOS) in winter to 22.9C + 2.2C Art. 9(1) page 6 of 23 Zamanillo et al: Seasonality of TEP and CSP in the Mediterranean Sea (BBMO) and 22.6C + 1.4C (EOS) in summer (Table 1, Figures S1 and S2). In summer, high heat fluxes led to higher surface temperatures and the development of a shallow stratified layer (average MLD of 6.5 + 5.7 m in BBMO and 4.6 + 4.7 m in EOS; Table 1). Water trans- parency ranged from 5 to 20 m in BBMO and 5 to 24 m in EOS, with the highest values in summer at both stations. Dissolved nitrate and silicate concentrations in the surface were lowest in summer at both BBMO and EOS (Table 1). However, surface phosphate concentrations (average of 0.04 + 0.02 mmol L–1 in BBMO and 0.04 + 0.06 mmol L–1 in EOS) did not exhibit marked seasonal variations but were low throughout the year at both stations (Table 1). Dissolved inorganic nutrients at EOS usually increased with depth in summer months, while they were homoge- neously distributed across depth in the other seasons, with a few exceptions (Figure S3). were detected (harmonic analysis, P > 0.05). At BBMO, surface POC ranged from 5.82 to 33.16 mmol L–1 (average + SD of 12.1 + 6.0 mmol L–1; Figure 2); at EOS, it ranged from 4.33 to 18.02 mmol L–1 (average of 9.9 + 4.1 mmol L–1; Figure 3). Surface PON concentrations averaged 1.8 + 1.0 mmol L–1 at BBMO and 1.5 + 1.7 mmol L–1 at EOS. At both BBMO and EOS, TEP and POC dynamics were significantly correlated (r ¼ 0.41, P ¼ 0.028, n ¼ 29 at BBMO and r ¼ 0.50, P ¼ 0.029, n ¼ 19 at EOS; Table 2). CSP was significantly correlated to POC (r ¼ 0.58, P ¼ 0.021, n ¼ 16) and PON (r ¼ 0.57, P ¼ 0.020, n ¼ 16) only at EOS. TEP and CSP were not correlated at any of the stations (P > 0.05; Table 2). 3.4. Variation of biological variables over the time series Pi- coeukaryotes and nanoeukaryotes were generally more abundant during the first 4 months of each year. The highest diatom biomasses were found in late fall. POC and PON concentrations were also similar in both coastal stations (Table 1), but no clear seasonal patterns ble 1. Averages + standard deviation of the main physical and biological variables in the sea surface for each season at the BBMO and EOS time series, between June 2015 and vember 2017. DOI: https://doi.org/10.1525/elementa.2020.00165.t1 ablea BBMOb EOSb Winter (n ¼ 6)c Spring (n ¼ 6) Summer (n ¼ 11) Fall (n ¼ 7) Winter (n ¼ 5) Spring (n ¼ 4) Summer (n ¼ 6) Fall (n ¼ 6) mperature (C) 13.4 + 0.4A 16.8 + 3.0B, C 22.9 + 2.2D 17.9 + 2.7C 13.2 + 0.5A 15.7 + 1.7B, C 22.6 + 1.4D 17.2 + 1.8C nity 38.2 + 0.1A 38.1 + 0.1B. 3.4. Variation of biological variables over the time series ates mixed layer depth; Chla, chlorophyll a; POC, particulate organic carbon; PON, particulate organic nitrogen; PHA, prokaryotic heterotrophic abundance; TEP, transparent exopolymer particles; assie stainable particles; C, carbon in phytoplankton biomass; and daily solar radiation dose, average previous 24 h. ted capital letters denote significantly different (P < 0.05) groups after nonparametric analysis of varaince followed by Mann Whitney tests. eses is the sample number (n) whenever it differed from the one reported in the column heading. Variablea BBMOb EOSb Winter (n ¼ 6)c Spring (n ¼ 6) Summer (n ¼ 11) Fall (n ¼ 7) Winter (n ¼ 5) Spring (n ¼ 4) Summer (n ¼ 6) Fall (n ¼ 6) C: Chla (mg mg –1) 37.5 + 26.8A 99.6 + 21.3B 89.1 + 16.2B 43.3 + 17.6A 31.2 + 20.1A 47.4 + 15.8A, B 74.0 + 24.3B (5) 55.2 + 52.0a, b (5) POC: Chla (mg mg –1) 154 + 73A, C 452 + 231B, C(5) 712 + 427B 300 + 184A, C 204 + 49A (3) 565 + 297A, B 992 + 413B (5) 429 + 253a, b (5) BBMO ¼ Blanes Bay Microbial Observatory; EOS ¼ L’Estartit Oceanographic Station; XG ¼ Xanthan Gum. aMLD indicates mixed layer depth; Chla, chlorophyll a; POC, particulate organic carbon; PON, particulate organic nitrogen; PHA, prokaryotic heterotrophic abundance; TEP, transparent exopolymer particles; CSP, Coomassie stainable particles; C, carbon in phytoplankton biomass; and daily solar radiation dose, average previous 24 h. bSuperscripted capital letters denote significantly different (P < 0.05) groups after nonparametric analysis of varaince followed by Mann Whitney tests. cIn parentheses is the sample number (n) whenever it differed from the one reported in the column heading. ( ) Variablea BBMOb EOSb Winter (n ¼ 6)c Spring (n ¼ 6) Summer (n ¼ 11) Fall (n ¼ 7) Winter (n ¼ 5) Spring (n ¼ 4) Summer (n ¼ 6) Fall (n ¼ 6) C: Chla (mg mg –1) 37.5 + 26.8A 99.6 + 21.3B 89.1 + 16.2B 43.3 + 17.6A 31.2 + 20.1A 47.4 + 15.8A, B 74.0 + 24.3B (5) 55.2 + 52.0a, b (5) POC: Chla (mg mg –1) 154 + 73A, C 452 + 231B, C(5) 712 + 427B 300 + 184A, C 204 + 49A (3) 565 + 297A, B 992 + 413B (5) 429 + 253a, b (5) BBMO ¼ Blanes Bay Microbial Observatory; EOS ¼ L’Estartit Oceanographic Station; XG ¼ Xanthan Gum. 3.4. Variation of biological variables over the time series D 37.9 +0.1B, C (10) 37.9 + 0.7A, D 38.0 + 0.4A 37.8 + 0.3A, B (3) 37.8 + 0.2B 37.9 + 0.4b tification index (C) 0.04 + 0.06A, C 1.24 + 1.35B, C 2.01 + 1.73B(10) 0.49 + 1.63C, A –0.15 + 0.38A, C 8.70 + 8.19B, C 10.18 + 5.98B 2.08 + 2.10a, c er transparency (m) 11.0 + 4.0A 16.5 + 3.1A, C 17.5 + 3.0B, C 15.3 + 3.5B, C 11.2 + 2.9A 12.8 + 3.6A, B 17.7 + 3.5B 14.2 + 6.7a, b D (m) 18.0 + 3.2A 8.6 + 6.8B, C 6.5 + 5.7C (10) 13.9 + 5.4A, B 80.2 + 28.7A 6.0 + 5.7A, B (2) 4.6 + 4.7B (5) 52.2 + 38.8b r radiation dose (Wm–2) 40.2 + 25.7C 179 + 103A, B 220 + 72B (10) 61.1 + 30.8A, C 10.8 + 5.7A (4) 221 + 99.5A, B (2) 235 + 80.6B (5) 36.9 + 56.6b ate (mmol L–1) 0.89 + 0.47A 0.34 + 0.29A, B 0.22 + 0.18B 0.68 + 0.65B 1.67 + 0.20A (4) 0.77 + 0.58A, B 0.27 + 0.41B 1.36 + 1.23b sphate (mmol L–1) 0.06 + 0.04A 0.03 + 0.01A 0.04 + 0.02A 0.04 + 0.01A 0.02 + 0.01A (4) 0.03 + 0.02A 0.03 + 0.01A 0.08 + 0.10a ate: Phosphate 15.7 + 8.5A 10.9 + 6.4A, B 5.3 + 2.9B 17.7 + 17.2A, B 81.5 + 27.7A (4) 42.5 + 41.2A 8.3 + 8.7B 37.0 + 54.0a, b ate (mmol L–1) 1.35 + 0.38A 1.04 + 0.50A, B 0.69 + 0.47B 0.91 + 0.50A, B 1.53 + 0.33A (4) 1.29 + 0.42A 0.48 + 0.17B 1.03 + 0.80a, b a (mg L–1) 1.07 + 0.36A 0.32 + 0.13B 0.23 + 0.11B 0.57 + 0.33A 0.50 + 0.20A 0.27 + 0.12A, B 0.14 + 0.03B (5) 0.33 + 0.19a, b (5) C (mmol L–1) 12.4 + 4.5A 11.0 + 3.7A (5) 11.8 + 5.4A 13.1 + 9.4A 8.8 + 2.1A (3) 10.8 + 3.4A 10.2 + 4.8A 9.7 + 5.1a N (mmol L–1) 1.7 + 0.7A 1.6 + 0.6A (5) 1.7 + 1.0A 2.0 + 1.6A 1.0 + 0.3A (3) 1.3 + 0.6A 1.1 + 0.6A 2.3 + 3.0a A (105 cells mL–1) 9.04 + 3.09A 10.21 + 4.04A 8.35 + 1.64A 9.69 + 2.51A 0.17 + 0.10A 0.28 + 0.10A, B 0.30 + 0.07A, B (5) 0.43 + 0.17b (5) (mg XG eq L–1) 32.2 + 9.9A 34.5 + 22.0A 42.3 + 25.2A 33.9 + 24.5A 25.8 + 20.2A (4) 29.2 + 8.7A 57.2 + 38.3A 28.2 + 23.6a (mg BSA eq L–1) 8.8 + 3.8A (5) 15.5 + 6.7A 11.0 + 4.6A (7) 12.1 + 8.0A (6) 11.0 + 6.6A, B (4) 19.8 + 2.5A 12.8 + 4.3A, B (4) 9.3 + 4.6b (5) : Chla (mg XG eq mg –1) 37.0 + 26.3A 126 + 76.9A, B 202 + 123B 60.2 + 24.8B 48.8 + 39.9A (4) 136 + 98.2A, B 444 + 314B (5) 94.7 + 86.1b (5) : Chla (mg BSA eq mg –1) 9.0 + 3.8A (5) 53.2 + 23.5B 59.0 + 18.0B (7) 23.2 + 16.4A (6) 20.5 + 13.7A (4) 87.8 + 51.5B 86.2 + 25.8A, B (3) 42.9 + 47.0a, b (4) : C (mg XG eq mg –1) 1.09 + 0.28A 1.27 + 0.83A 2.26 + 1.31A 1.57 + 0.93A 1.60 + 0.40A (4) 2.69 + 1.30B 5.84 + 3.55B (5) 1.93 + 1.44a, b (5) : C (mg BSA eq mg –1) 0.39 + 0.27A (5) 0.53 + 0.22A 0.62 + 0.24A (7) 0.54 + 0.28A (6) 0.78 + 0.28A, B (4) 1.86 + 0.72A 1.23 + 0.41A, B (3) 0.70 + 0.06b (4) : POC (mg XG eq mg –1) 0.2 + 0.1A 0.2 + 0.1A (5) 0.4 + 0.3A 2.8 + 1.5A 3.6 +1.5A, B (3) 2.7 +0.5B 5.5 + 2.2A 3.1 + 2.0b : POC (mg BSA eq mg –1) 0.8 + 0.4A (5) 1.6 + 1.0A (5) 1.0 + 0.8A (7) 1.1 + 0.6A (6) 1.4 +0.4A (3) 2.0 +0.7A 1.8 +0.9A (4) 1.1 +0.5a (5) BBMOb EOSb Winter (n ¼ 6)c Spring (n ¼ 6) Summer (n ¼ 11) Fall (n ¼ 7) Winter (n ¼ 5) Spring (n ¼ 4) Summer (n ¼ 6) Fall (n ¼ 6) mg –1) 37.5 + 26.8A 99.6 + 21.3B 89.1 + 16.2B 43.3 + 17.6A 31.2 + 20.1A 47.4 + 15.8A, B 74.0 + 24.3B (5) 55.2 + 52.0a, b (5) (mg mg –1) 154 + 73A, C 452 + 231B, C(5) 712 + 427B 300 + 184A, C 204 + 49A (3) 565 + 297A, B 992 + 413B (5) 429 + 253a, b (5) lanes Bay Microbial Observatory; EOS ¼ L’Estartit Oceanographic Station; XG ¼ Xanthan Gum. 3.4. Variation of biological variables over the time series aMLD indicates mixed layer depth; Chla, chlorophyll a; POC, particulate organic carbon; PON, particulate organic nitrogen; PHA, prokaryotic heterotrophic abundance; TEP, transparent exopolymer particles; CSP, Coomassie stainable particles; C, carbon in phytoplankton biomass; and daily solar radiation dose, average previous 24 h. bSuperscripted capital letters denote significantly different (P < 0.05) groups after nonparametric analysis of varaince followed by Mann Whitney tests. cIn parentheses is the sample number (n) whenever it differed from the one reported in the column heading. Downloaded from http://online.ucpress.edu/elementa/article-pdf/9/1/00165/485853/elementa.2020.00165.pdf by guest on 24 January 2022 Zamanillo et al: Seasonality of TEP and CSP in the Mediterranean Sea Art. 9(1) page 9 of 23 llates had maxima in late spring, but coccolitho- Regarding the entire sampled water colum . Surface values of chemical and biological variables in the Blanes Bay Microbial Obs trations of (a) transparent exopolymer particles and Coomassie stainable particles, (b) particula and chlorophyll a, and (c) phytoplankton and prokaryotic heterotrophic biomasses. Each tick on es the monthly sampling date starting in June 2015 (Jl indicates July). Error bars represent standard e n ¼ 2). DOI: https://doi.org/10.1525/elementa.2020.00165.f2 Downloaded from http://online.ucpress.edu/elementa/article-pdf/9/1/00165/485853/elementa.2020.00165.pdf by guest on 24 Figure 2. Surface values of chemical and biological variables in the Blanes Bay Microbial Observatory. Concentrations of (a) transparent exopolymer particles and Coomassie stainable particles, (b) particulate organic carbon and chlorophyll a, and (c) phytoplankton and prokaryotic heterotrophic biomasses. Each tick on the x-axis indicates the monthly sampling date starting in June 2015 (Jl indicates July). Error bars represent standard error of the mean (n ¼ 2). DOI: https://doi.org/10.1525/elementa.2020.00165.f2 Dinoflagellates had maxima in late spring, but coccolitho- phores did not present clear seasonal variations at either of the stations (Figures S1 and S2). Regarding the entire sampled water column at EOS, Prochlorococcus biomass presented high values at 50 m in the summers of 2015 and 2017. Synechococcus showed Zamanillo et al: Seasonality of TEP and CSP in the Mediterranean Sea Art. 9(1) page 10 of 23 . Surface values of chemical and biological variables in L’Estartit Oceanographic rations of (a) transparent exopolymer particles and Coomassie stainable particles, (b) particulat and chlorophyll a, and (c) phytoplankton and prokaryotic heterotrophic biomasses. Each tick on s the monthly sampling date starting in June 2015 (Jl indicates July). Error bars represent standard er n ¼ 2). DOI: https://doi.org/10.1525/elementa.2020.00165.f3 Downloaded from http://online.ucpress.edu/elementa/article-pdf/9/1/00165/485853/elementa.2020.00165.pdf by guest on 24 Figure 3. 3.4. Variation of biological variables over the time series BBMO ¼ Blanes Bay Microbial Observatory; EOS ¼ L’Estartit Oceanographic Station; XG ¼ Xanthan Gum. BBMO ¼ Blanes Bay Microbial Observatory; EOS ¼ L’Estartit Oceanographic Station; XG ¼ Xanthan Gum. maximum concentrations at 20 m and/or 40 m in spring and summer. Diatom biomass was very high in the upper 50 m in November 2015 (Figure S3). 1). The POC: Chla ratios were also higher in summer and lower in winter at both stations (Table 1). The TEP: POC ratios peaked in summer, while the CSP: POC ratios peaked in spring (Table 1). The estimated contribution of TEP-C to the POC pool at BBMO and EOS (surface) ranged between 4% and 44% (average + SD of 14% + 9%) and between 3% and 37% (average of 16% + 9%), respectively.TEP-C was within the ranges of phytoplankton and prokaryotic heterotroph biomass, and TEP-C contributed the most to the nondetrital particulate carbon pool in summer at EOS (Figure 6). 3.5. Variations of TEP and CSP in relation to Chla, biomass, and POC The average surface TEP: Chla ratio was highest in summer at both stations, as was the ratio of TEP to phytoplankton carbon biomass (TEP: C; Table 1, Figure 5). The average surface CSP: Chla ratio was higher in spring and summer, whereas the CSP: C ratio was higher in summer at BBMO and in spring at EOS (Table 1). The minimum values of TEP: Chla and CSP: Chla occurred in winter at both sites (Table 1). The ratio of carbon biomass to Chla (C: Chla) showed a clear seasonal cycle, with the lowest values in winter and fall (generally below 50) and the highest in spring and summer (generally above 50; Figure 5, Table 3.4. Variation of biological variables over the time series Surface values of chemical and biological variables in L’Estartit Oceanographic Station. Concentrations of (a) transparent exopolymer particles and Coomassie stainable particles, (b) particulate organic carbon and chlorophyll a, and (c) phytoplankton and prokaryotic heterotrophic biomasses. Each tick on the x-axis indicates the monthly sampling date starting in June 2015 (Jl indicates July). Error bars represent standard error of the mean (n ¼ 2). DOI: https://doi.org/10.1525/elementa.2020.00165.f3 Art. 9(1) page 11 of 23 Zamanillo et al: Seasonality of TEP and CSP in the Mediterranean Sea Table 2. Spearman correlations between transparent exopolymer particle (TEP) concentration and other environmental and biological variables in the sea surface at BBMO and EOS during the 2015–2017 period. DOI: https://doi.org/ 10.1525/elementa.2020.00165.t2 Dependent Variable Independent Variablea BBMOb EOSb r P n r P n TEP (mg XG eq L–1) Temperature (C) nsc ns 30 ns ns 20 Salinity –0.46 0.012 29 –0.48 0.039 19 Stratification index (C) ns ns 29 ns ns 20 Water transparency (m) ns ns 30 ns ns 20 Daily solar radiation dose (Wm–2) 0.36 0.056 29 0.58 0.020 16 Nitrate (mmol L–1) ns ns 30 ns ns 20 Phosphate (mmol L–1) ns ns 30 ns ns 20 Silicate (mmol L–1) ns ns 30 –0.47 0.036 20 CSP (mg BSA eq L–1) ns ns 27 ns ns 17 Chla (mg L–1) ns ns 30 –0.47 0.047 18 Prochlorococcus (mg C L–1) –0.51 0.004 30 –0.61 0.008 18 Synechococcus (mg C L–1) –0.36 0.051 30 ns ns 18 Picoeukaryotes (mg C L–1) ns ns 30 ns ns 18 Nanoeukaryotes (mg C L–1) 0.40 0.028 30 ns ns 18 Dinoflagellates (mg C L–1) 0.50 0.005 30 ns ns 18 Diatoms (mg C L–1) 0.44 0.015 30 ns ns 18 Coccolithophores (mg C L–1) ns ns 30 ns ns 18 Phytoplankton biomass (mg C L–1) ns ns 30 ns ns 18 POC (mmol L–1) 0.41 0.028 29 0.50 0.029 19 PON (mmol L–1) ns ns 29 ns ns 19 PHA (x103 cells mL–1) ns ns 30 ns ns 18 BBMO ¼ Blanes Bay Microbial Observatory; EOS ¼ L’Estartit Oceanographic Station; XG ¼ Xanthan Gum. aCSP indicates Coomassie stainable particles; Chla, chlorophyll a; POC, particulate organic carbon; PON, particulate organic nitrogen; PHA, prokaryotic heterotrophic abundance. br is Spearman’s correlation coefficient, where bold font indicates significance at P < 0.05; P, level of significance; n, sample size. cnot significant. 3.6. Environmental and biological variables as potential predictors of seasonal TEP and CSP dynamics In April 2017, they averaged 41 + 12 and 42 + 10 mm2 L–1, respectively; in August 2017, 25 + 7 and 19 + 4 mm2 L–1, respectively; and in October 2017, 6 + 3 and 16 + 1 mm2 L–1 respectively Error bars represent standard error of the mean (n ¼ 2) Note log scale for axes Downloaded from http://online.ucpress.edu/elementa/article-pdf/9/1/00165/485853/elementa.2020.00165.pdf by guest on 24 January 2022 oaded from http://online.ucpress.edu/elementa/article-pdf/9/1/00165/485853/elementa.2020.00165.pdf by guest on 24 January 2022 Figure 4. Size distributions of transparent exopolymer particles (TEP; top panel) and Coomassie stainable particles (CSP; bottom panel). Size distributions (number mL–1) were obtained with the microscopic method for 4 samplings at the Blanes Bay Microbial Observatory, 1 in November of 2016 and the others in April, August, and October of 2017. Particle numbers are binned by their mean diameter: those ranging from 0 to 1.3 mm are grouped as 1.0 mm; 1.3 to 1.8 mm, as 1.5 mm; 1.8 to 2.5 mm, as 2.1 mm; 2.5 to 3.5 mm, as 3.0 mm; 3.5 to 5.0 mm, as 4.2 mm; 5.0 to 7.0 mm, as 6.0 mm; 7.0 to 10.0 mm, as 8.5 mm; and 10.0 to 14.0 mm, as 12.0 mm. In November 2016, the total area of TEP and CSP averaged 14 + 12 and 38 + 5 mm2 L–1, respectively. In April 2017, they averaged 41 + 12 and 42 + 10 mm2 L–1, respectively; in August 2017, 25 + 7 and 19 + 4 mm2 L–1, respectively; and in October 2017, 6 + 3 and 16 + 1 mm2 L–1, respectively. Error bars represent standard error of the mean (n ¼ 2). Note log scale for axes. DOI: https://doi.org/10.1525/elementa.2020.00165.f4 negatively with Chla (r ¼ –0.47, P ¼ 0.047, n ¼ 18), Prochlorococcus (r ¼ –0.61, P ¼ 0.008, n ¼ 18), and silicate (r ¼ –0.47, P ¼ 0.036, n ¼ 18). CSP, among all studied variables, was only correlated, positively, with total phyto- plankton biomass (r ¼ 0.53, P < 0.044, n ¼ 15). n ¼ 30), followed by diatoms and nanoeukaryotes (Table 2). However, there was no significant relationship between TEP and Chla or total phytoplankton biomass. On the other hand, Prochlorochoccus showed a significant negative correlation with TEP (Table 2). CSP were not significantly correlated with any of the studied biological and environmental variables. 3.6. Environmental and biological variables as potential predictors of seasonal TEP and CSP dynamics At BBMO, the variable that best correlated to TEP variabil- ity was dinoflagellate abundance (r ¼ 0.50, P ¼ 0.005, Art. 9(1) page 12 of 23 Zamanillo et al: Seasonality of TEP and CSP in the Mediterranean Sea Figure 4. Size distributions of transparent exopolymer particles (TEP; top panel) and Coomassie stainable particles (CSP; bottom panel). Size distributions (number mL–1) were obtained with the microscopic method for 4 samplings at the Blanes Bay Microbial Observatory, 1 in November of 2016 and the others in April, August, and October of 2017. Particle numbers are binned by their mean diameter: those ranging from 0 to 1.3 mm are grouped as 1.0 mm; 1.3 to 1.8 mm, as 1.5 mm; 1.8 to 2.5 mm, as 2.1 mm; 2.5 to 3.5 mm, as 3.0 mm; 3.5 to 5.0 mm, as 4.2 mm; 5.0 to 7.0 mm, as 6.0 mm; 7.0 to 10.0 mm, as 8.5 mm; and 10.0 to 14.0 mm, as 12.0 mm. In November 2016, the total area of TEP and CSP averaged 14 + 12 and 38 + 5 mm2 L–1, respectively. In April 2017, they averaged 41 + 12 and 42 + 10 mm2 L–1, respectively; in August 2017, 25 + 7 and 19 + 4 mm2 L–1, respectively; and in October 2017, 6 + 3 and 16 + 1 mm2 L–1, respectively. Error bars represent standard error of the mean (n ¼ 2). Note log scale for axes. DOI: https://doi.org/10.1525/elementa.2020.00165.f4 Figure 4. Size distributions of transparent exopolymer particles (TEP; top panel) and Coomassie stainable particles (CSP; bottom panel). Size distributions (number mL–1) were obtained with the microscopic method for 4 samplings at the Blanes Bay Microbial Observatory, 1 in November of 2016 and the others in April, August, and October of 2017. Particle numbers are binned by their mean diameter: those ranging from 0 to 1.3 mm are grouped as 1.0 mm; 1.3 to 1.8 mm, as 1.5 mm; 1.8 to 2.5 mm, as 2.1 mm; 2.5 to 3.5 mm, as 3.0 mm; 3.5 to 5.0 mm, as 4.2 mm; 5.0 to 7.0 mm, as 6.0 mm; 7.0 to 10.0 mm, as 8.5 mm; and 10.0 to 14.0 mm, as 12.0 mm. In November 2016, the total area of TEP and CSP averaged 14 + 12 and 38 + 5 mm2 L–1, respectively. 3.6. Environmental and biological variables as potential predictors of seasonal TEP and CSP dynamics p ( ) Unlike TEP concentrations, the ratios of TEP to Chla and phytoplankton biomass correlated positively with solar radiation dose at both stations and negatively with nitrate, except for TEP: C at BBMO (Table 3). Similarly, CSP At EOS, TEP correlated positively only with the daily solar radiation dose (r ¼ 0.58, P ¼ 0.020, n ¼ 16) and Zamanillo et al: Seasonality of TEP and CSP in the Mediterranean Sea Art. 9(1) page 13 of 23 re 5. Ratios of TEP: C, TEP: Chla, and C: Chla over the BBMO (top) and EOS (bottom) time series. TEP icates transparent exopolymer particles (n ¼ 2); C, carbon in phytoplankton biomass (n ¼ 1); Chla, chlorophyll a ¼ 2); BBMO, Blanes Bay Microbial Observatory; and EOS, L’Estartit Oceanographic Station. Each tick on the x-axis icates the monthly sampling date starting in June 2015 (Jl indicates July). Error bars represent standard error of the an. DOI: https://doi.org/10.1525/elementa.2020.00165.f5 Downloaded from http://online.ucpress.edu/elementa/article-pdf/9/1/00165/485853/elementa.2020.00165.pdf by guest on 24 January 2022 Downloaded from http://online.ucpress.edu/elementa/article-pdf/9/1/00165/485853/elementa.2020.00165.pdf by guest on 24 Figure 5. Ratios of TEP: C, TEP: Chla, and C: Chla over the BBMO (top) and EOS (bottom) time series. TEP indicates transparent exopolymer particles (n ¼ 2); C, carbon in phytoplankton biomass (n ¼ 1); Chla, chlorophyll a (n ¼ 2); BBMO, Blanes Bay Microbial Observatory; and EOS, L’Estartit Oceanographic Station. Each tick on the x-axis indicates the monthly sampling date starting in June 2015 (Jl indicates July). Error bars represent standard error of the mean. DOI: https://doi.org/10.1525/elementa.2020.00165.f5 3.7. TEP and CSP distribution along the coast-to- open sea transect 3.7. TEP and CSP distribution along the coast-to- open sea transect Percentage carbon contribution of TEP (TEP-C), phytoplankton biomass (Phyto-C), and prokaryotic heterotroph biomass (PH-C) to the POC pool across seasons in surface waters of the Blanes Bay Microbial Observatory (BBMO, top panel) and L’Estartit Oceanographic Station (EOS, bottom panel). Symbols represent seasonal means; error bars represent standard deviations. From Winter to Fall samplings (left to right), n values were 6, 6, 11, and 7 at BBMO and 3, 4, 5–6, and 5–6 at EOS, where n ¼ 5 for Phyto-C and PH-C and n ¼ 6 for TEP-C at EOS. DOI: https://doi.org/ 10.1525/elementa.2020.00165.f6 with Chla (r ¼ 0.93, P ¼ 0.003, n ¼ 7) and nanoeukar- yotes (r ¼ 0.86, P ¼ 0.024, n ¼ 7). organic particles is unknown and depends on their sources and degradation processes (Passow, 2012). Particles prob- ably consist of a mixture of TEP and CSP, along with other organic substances, inorganic molecules, and microbes. For instance, differential activities of extracellular en- zymes or exoenzymes (e.g., protease or aminopeptidase vs. glucosidase activities) on marine aggregates (Smith et al., 1992; Piontek et al., 2009) could lead to changes in the relative proportion of TEP and CSP. In recent years, at- tempts have been made to elucidate whether these stained particle types (TEP and CSP) are in fact the same particles or not (Cisternas-Novoa et al., 2015; Thornton et al., 2016; Thornton and Chen, 2017), and the results have suggested that they mostly comprise different particulate substances. Our findings also support that TEP and CSP TEP and CSP concentrations were generally lower at the depth of the DCM (average of 11.4 + 3.6 mg XG eq L–1 for TEP and 2.9 + 0.6 mg BSA eq L–1 for CSP) than at surface, despite higher Chla levels in the DCM (Figure 7). 3.7. TEP and CSP distribution along the coast-to- open sea transect to Chla ratios correlated positively with solar radiation dose (r ¼ 0.75, P < 0.001, n ¼ 23 at BBMO and r ¼ 0.83, P ¼ 0.003, n ¼ 13 at EOS) and negatively with nitrate (r ¼ –0.62, P ¼ 0.001, n ¼ 23 at BBMO and r ¼ –0.78, P < 0.001, n ¼ 13), but CSP to phytoplankton biomass ratios correlated only with nitrate at EOS (r ¼ –0.59, P ¼ 0.024, n ¼ 13). Surface TEP concentrations ranged from 21.5 to 36.5 mg XG eq L–1 (average + SD of 30.7 + 5.4 mg XG eq L–1) and increased from the coast to the open ocean (Figure 7). Surface CSP concentrations, which varied between 2.3 and 8.7 mg BSA eq L–1 (average of 5.2 + 2.1 mg BSA eq L–1), decreased from the coast to the slope and beyond (Figure 7), that is, opposite to TEP. Among the variables measured at surface, TEP concentrations were not correlated with any, whereas CSP correlated significantly and positively Surface TEP concentrations ranged from 21.5 to 36.5 mg XG eq L–1 (average + SD of 30.7 + 5.4 mg XG eq L–1) and increased from the coast to the open ocean (Figure 7). Surface CSP concentrations, which varied between 2.3 and 8.7 mg BSA eq L–1 (average of 5.2 + 2.1 mg BSA eq L–1), decreased from the coast to the slope and beyond (Figure 7), that is, opposite to TEP. Among the variables measured at surface, TEP concentrations were not correlated with any, whereas CSP correlated significantly and positively The vertical distribution of TEP coincided with that of dinoflagellate abundance, whereas CSP peaks coin- cided with relatively high Synechococcus abundance (Figure S3). Art. 9(1) page 14 of 23 Zamanillo et al: Seasonality of TEP and CSP in the Mediterranean Sea Figure 6. Contribution of transparent exopolymer particles (TEP) to particulate organic carbon (POC). Percentage carbon contribution of TEP (TEP-C), phytoplankton biomass (Phyto-C), and prokaryotic heterotroph biomass (PH-C) to the POC pool across seasons in surface waters of the Blanes Bay Microbial Observatory (BBMO, Downloaded from http://online.ucpress.edu/elementa/article-pdf/9/1/00165/485853/elementa.2020.00165.pdf by guest on 24 Figure 6. Contribution of transparent exopolymer particles (TEP) to particulate organic carbon (POC). 4. Discussion Aside from this exceptional sampling time, the maximum TEP concentrations found in early summer at both stations (Figures 2, 3, and S4) are in accordance with previous studies in the Mediterranean Sea (Beauvais et al., 2003; Ortega-Retuerta et al., 2018). Ortega-Retuerta et al. (2018) suggested that TEP maxima in summer could be due to the increase of TEP production under nutrient limitation, the presence of specific phytoplankton groups, and/or the accumulation of TEP due to positive buoyancy during water stratification. This study expands the previous tem- poral study conducted at the BBMO (Ortega-Retuerta et al., 2018) by incorporating information about phytoplank- ton biomass and solar radiation dose, which appear to be relevant factors. with a sharp decrease of salinity and an increase of nitrate at BBMO and phosphate at EOS. The BBMO is known to receive inputs of nutrients and terrestrial carbon sporad- ically during storm periods (Guadayol et al., 2009b). Aside from this exceptional sampling time, the maximum TEP concentrations found in early summer at both stations (Figures 2, 3, and S4) are in accordance with previous studies in the Mediterranean Sea (Beauvais et al., 2003; Ortega-Retuerta et al., 2018). Ortega-Retuerta et al. (2018) suggested that TEP maxima in summer could be due to the increase of TEP production under nutrient limitation, the presence of specific phytoplankton groups, and/or the accumulation of TEP due to positive buoyancy during water stratification. This study expands the previous tem- poral study conducted at the BBMO (Ortega-Retuerta et al., 2018) by incorporating information about phytoplank- ton biomass and solar radiation dose, which appear to be relevant factors. correspond to different types of particles, as they pre- sented uncorrelated temporal dynamics over seasonal cy- cles at the 2 studied coastal sites (Figures 2 and 3, Table 2) and also different vertical distributions (Figure S3). These differences also suggest that they are likely pro- duced by different organisms and/or subject to different aggregation and degradation processes, as suggested in previous studies (Cisternas-Novoa et al., 2015; Thornton et al., 2016; Thornton and Chen, 2017). The absence of parallel temporal patterns of TEP and CSP, however, does not totally preclude that both particles are the same, at least in some cases. In fact, marine organic particles have heterogeneous chemical compositions, and both carbohydrate-rich and proteinaceous regions can coexist in the same aggregate (Busch et al., 2017). 4. Discussion For instance, Dreshchinskii and Engel (2017) observed that TEP and CSP dynamics were coupled during 2 seasonal cycles in the Baltic Sea, both in the SML and in SSW, which contrasts with our results. However, visual examination of the particles with a FlowCAM (Cisternas-Novoa et al. 2015) or through microscopic examination (Thornton et al., 2016) led to the conclusion that Alcian Blue and CBB generally stain different particle types. In our case, we can conclude that, if both types of particles coexisted in the same aggregates, their TEP and the CSP contents in those aggregates varied independently: A large proportion of the pelagic aggregates we sampled contained Alcian- Blue stainable substances but no CBB stainable sub- stances, as well as the reverse. The range of our TEP concentrations across the 2 sta- tions (5.8–127 mg XG eq L–1) was similar to that found by Ortega-Retuerta et al. (2018) at the BBMO during the 3 previous years (11.3–289 mg XG eq L–1) and by Iuculano et al. (2017a) in a rocky shore site of the Balearic Sea (4.6– 90.6 mg XG eq L–1), while it was generally lower than the range found in a coastal site accumulating Posidonia ocea- nica leaf litter in the Balearic Sea (26.8–1,880 mg XG eq L–1; Iuculano et al., 2017a). CSP concentrations at both stations (4.5–24.8 mg BSA eq L–1) were within the range of the few previous pub- lished studies (Table 4). The highest CSP concentrations were found in spring; however, according to the har- monic analysis, the seasonal pattern was only significant at EOS (Figures 2, 3, and S4). Cisternas-Novoa et al. (2015) found steady CSP concentrations throughout the 4.1. Dynamics of TEP and CSP over the seasonal cycle in the NW Mediterranean Sea 4. Discussion We present the first simultaneous measurements of the distribution of TEP and CSP concentrations over 2 seasonal cycles in the Mediterranean Sea. The methods used to quantify these particles are based on the capability of different dyes to stain acidic polysaccharides and proteins, respectively (Passow and Alldredge, 1995; Cisternas-Novoa et al., 2014). However, the actual composition of the Art. 9(1) page 15 of 23 Zamanillo et al: Seasonality of TEP and CSP in the Mediterranean Sea Table 3. Spearman correlations between selected ratios (along with chlorophyll a concentration) and the daily solar radiation dose (Wm–2) and nitrate concentration (mmol L–1). DOI: https://doi.org/10.1525/elementa.2020.00165.t3 ( ) (m ) p g Dependent Variablea Independent Variable BBMOb EOSb r P n r P n TEP: Chla Daily solar radiation dose 0.74 <0.001 30 0.96 <0.001 18 TEP: C Daily solar radiation dose 0.41 0.027 30 0.78 0.001 18 CSP: Chla Daily solar radiation dose 0.75 <0.001 23 0.83 0.003 13 CSP: C Daily solar radiation dose nsc ns 24 ns ns 13 Chla Daily solar radiation dose –0.72 <0.001 30 –0.80 <0.001 18 C: Chla Daily solar radiation dose 0.66 <0.001 30 0.87 <0.001 18 TEP: Chla Nitrate –0.62 <0.001 30 –0.80 <0.001 18 TEP: C Nitrate ns ns 30 –0.63 0.005 18 CSP: Chla Nitrate –0.62 0.001 24 –0.78 <0.001 15 CSP: C Nitrate ns ns 24 –0.59 0.024 15 aTEP indicates transparent exopolymer particles; Chla, chlorophyll a; C, carbon in phytoplankton biomass; and CSP, Coomassie stainable particles. br is Spearman’s correlation coefficient, where bold font indicates significance at P < 0.05; p, level of significance; n, sample size. cNot significant. aTEP indicates transparent exopolymer particles; Chla, chlorophyll a; C, carbon in phytoplankton biomass; and CSP, Coomassie stainable particles. br is Spearman’s correlation coefficient, where bold font indicates significance at P < 0.05; p, level of significance; n, sample size. cNot significant br is Spearman’s correlation coefficient, where bold font indicates significance at P < 0.05; p, level of significance; n, sample size. cNot significant. with a sharp decrease of salinity and an increase of nitrate at BBMO and phosphate at EOS. The BBMO is known to receive inputs of nutrients and terrestrial carbon sporad- ically during storm periods (Guadayol et al., 2009b). 4.1. Dynamics of TEP and CSP over the seasonal cycle in the NW Mediterranean Sea The peak of TEP found in December 2016 at both stations was likely related to freshwater discharges, as it coincided Art. 9(1) page 16 of 23 Zamanillo et al: Seasonality of TEP and CSP in the Mediterranean Sea the upper 100 m of the Sargasso Sea, while Dresh- i and Engel (2017) observed the highest CSP abun- 4.2. Main drivers of TEP and CSP dynamics We first examined Chla, typically used as an i 7. Concentrations of transparent exopolymer particles (TEP) and Coomassie stainable parti g a coastal–offshore transect. Concentrations of TEP (circles), CSP (triangles), and chlorophyll a ( closed symbols) and the deep chlorophyll maximum (DCM, open symbols) as a function of distance to that DCM was only present at the deeper (farther offshore) stations. Error bars represent standard e (n ¼ 2). DOI: https://doi.org/10.1525/elementa.2020.00165.f7 Figure 7. Concentrations of transparent exopolymer particles (TEP) and Coomassie stainable particles (CSP) along a coastal–offshore transect. Concentrations of TEP (circles), CSP (triangles), and chlorophyll a (squares) at 5 m (closed symbols) and the deep chlorophyll maximum (DCM, open symbols) as a function of distance to the shore. Note that DCM was only present at the deeper (farther offshore) stations. Error bars represent standard error of the mean (n ¼ 2). DOI: https://doi.org/10.1525/elementa.2020.00165.f7 Downloaded from http://online.ucpress.edu/elementa/article-pdf/9/1/00165/485853/elementa.2020.00165.pdf by guest on 24 J Figure 7. Concentrations of transparent exopolymer particles (TEP) and Coomassie stainable particles (CSP) along a coastal–offshore transect. Concentrations of TEP (circles), CSP (triangles), and chlorophyll a (squares) at 5 m (closed symbols) and the deep chlorophyll maximum (DCM, open symbols) as a function of distance to the shore. Note that DCM was only present at the deeper (farther offshore) stations. Error bars represent standard error of the mean (n ¼ 2). DOI: https://doi.org/10.1525/elementa.2020.00165.f7 4.2. Main drivers of TEP and CSP dynamics We first examined Chla, typically used as an indicator of phytoplankton biomass, as a potential driver of TEP and CSP dynamics, but Chla did not correlate positively with TEP or CSP at either of our sampling sites, and correlated 4.2. Main drivers of TEP and CSP dynamics year in the upper 100 m of the Sargasso Sea, while Dresh- chinskii and Engel (2017) observed the highest CSP abun- dance during the winter and summer periods in the Baltic Sea, with frequent oscillations in the total area of CSP. 4.1. Dynamics of TEP and CSP over the seasonal cycle in the NW Mediterranean Sea Consequently, Chla cannot be con- sidered a good indicator of phytoplankton biomass across seasons, and instead we used biomass estimates based on cell abundances, size or species-specific biovolumes, and volume-to-carbon conversion factors. While CSP did not correlate with measured variables other than phytoplankton biomass (except for POC and PON at EOS), TEP correlated positively with the daily solar radiation dose at EOS (Table 2), and TEP: C correlated positively with the solar radiation dose at both stations, and negatively with nitrate at EOS (Table 2). These corre- lations could be indicative of either the stimulation of microbial TEP release by light stress, as suggested by Iu- culano et al. (2017b), Agustı´ and Llabre´s (2007), and Za- manillo et al. (2019a), or the enhancement of abiotic self- assembly of dissolved exopolymers into TEP (Shammi et al., 2017). The negative relationship of TEP: C with nitrate (Table 2) could be due to the stimulation of phytoplank- ton TEP production under nutrient limitation in summer, as suggested by Ortega-Retuerta et al. (2018). CSP: C was also negatively related with nitrate at EOS, but more research is needed to determine whether nutrient limita- tion stimulates CSP production. The findings that CSP correlated positively with total phytoplankton biomass at EOS, while TEP did not show this correlation at either of our sampling sites, support our initial hypothesis that CSP temporal variations, at least at the sea surface, are more closely related to phytoplankton biomass than those of TEP. Although primary production data are not available for our study years, the highest CSP concentrations of the time series (from late winter to early summer) coincide with the period of maximum primary production recorded in a 12-year study (2003–2014) at BBMO (Gasol et al., 2016). Gasol et al. (2016) also reported that Chla-specific primary production (C production per unit of Chla), measured with the 14C method, was highest in summer and lowest in winter, likely due to higher photosynthetic efficiency of summer phytoplankton. Based on our observations, the higher Chla-specific pri- mary production in summer could be partially explained by enhanced production of TEP and CSP, as a portion of these particles would be retained on the filters used for the 14C-PP measurements and thus taken as phytoplank- ton biomass produced de novo. Along these same lines, Gasol et al. 4.1. Dynamics of TEP and CSP over the seasonal cycle in the NW Mediterranean Sea We first examined Chla, typically used as an indicator of phytoplankton biomass, as a potential driver of TEP and CSP dynamics, but Chla did not correlate positively with TEP or CSP at either of our sampling sites, and correlated Art. 9(1) page 17 of 23 Zamanillo et al: Seasonality of TEP and CSP in the Mediterranean Sea Table 4. Compilation of published Coomassie stainable particle (CSP) concentrations (mean + SD, range), chlorophyll a (Chla) concentrations (mean + SE, range; mg L–1), and CSP: Chla ratios (mean + SE ranges) and CSP: Chla ratios (mean + SE, ranges). DOI: https://doi.org/10.1525/elementa.2020.00165.t4 Location Time Frame Depth (m) CSP (mg BSA eq L–1) Chla (mg L–1) CSP: Chla Reference Sargasso Sea February, May, August, and November 2012; May 2013 0–100 3.2 + 0.7 to 22.4 + 0.4 0.25–0.75a —b Cisternas-Novoa et al. (2015) Baltic Sea June 3–19, 2015 1 and 10 15–56 a 1.2 to 1.7 — Cisternas-Novoa et al. (2019) Northwestern Mediterranean Sea (BBMO and EOS) October 2015–October 2017 (time-series study) Surface 12.4 + 6.0, 4.5–24.8 0.4 + 0.3, 0.13–1.52 45.6 + 35.7, 4.8–163 This study aExtracted from graphs. bNo reported values. Alonso-Sa´ez et al. (2008) found that DOC release ac- counted for up to 45% of total primary production at BBMO, with a lower proportion in winter. Because DOC can self-assemble to form TEP, this release of primary pro- duction as DOC could have further contributed to higher TEP concentrations in spring and summer. negatively with TEP at EOS. Ortega-Retuerta et al. (2018) also found a negative correlation of TEP with Chla (at BBMO). Some time-series studies have reported covaria- tion between TEP and Chla (Beauvais et al., 2003; Scoullos et al., 2006; Engel et al., 2017; Parinos et al., 2017), while this covariation was not found by others (Bhaskar and Bhosle, 2006; Taylor et al., 2014) or was only observed during certain periods of the year (Dreshchinskii and En- gel, 2017). In our study, the C: Chla ratio of phytoplankton changed throughout the year at both BBMO and EOS, in accordance with Gasol et al. (2016) and Gutie´rrez-Rodrı´- guez et al. (2010) for BBMO. Earlier work has shown that the ratio was usually higher in spring and summer driven by the high irradiance and low nutrients in these seasons (Geider et al., 1998). 4.1. Dynamics of TEP and CSP over the seasonal cycle in the NW Mediterranean Sea (2016) found relatively high saturating irra- diances (high values for the light saturation parameter) all year round at BBMO, which is a phytoplankton feature typical of regions receiving relatively high light intensities. In winter, the observed light saturation values were close to the surface irradiance at the time of sampling, whereas in summer, they were close to the irradiance values observed at 5-m depth. This depth differential indicates that in summer, phytoplankton cells living above 5 m receive irradiances in excess of their saturating values, which may favor the production of TEP (Agustı´ and Lla- bre´s, 2007). Prokaryotic heterotrophs can influence TEP and CSP dynamics in the ocean not only through particle coloni- zation and enzymatic degradation (Passow and Alldredge, 1994; Long and Azam, 1996) but also by producing TEP Art. 9(1) page 18 of 23 Zamanillo et al: Seasonality of TEP and CSP in the Mediterranean Sea (Radic et al., 2006; Ortega-Retuerta et al., 2019) or enhanc- ing TEP and CSP production by phytoplankton (Ga¨rdes et al., 2011). We showed previously that TEP dynamics at BBMO are correlated to exoenzyme activities (esterase, alkaline phosphatase, and beta-glucosidase; Ortega- Retuerta et al., 2018), but not to prokaryotic heterotrophic abundance. Our findings are consistent with their study, in that neither TEP nor CSP correlated with prokaryotic het- erotrophic abundance, and phytoplankton abundance was a stronger predictor of the temporal variation of these 2 particle types. (Radic et al., 2006; Ortega-Retuerta et al., 2019) or enhanc- ing TEP and CSP production by phytoplankton (Ga¨rdes et al., 2011). We showed previously that TEP dynamics at BBMO are correlated to exoenzyme activities (esterase, alkaline phosphatase, and beta-glucosidase; Ortega- Retuerta et al., 2018), but not to prokaryotic heterotrophic abundance. Our findings are consistent with their study, in that neither TEP nor CSP correlated with prokaryotic het- erotrophic abundance, and phytoplankton abundance was a stronger predictor of the temporal variation of these 2 particle types. (Table 1), suggesting a higher TEP contribution to the POC pool in this season. A higher contribution might affect particle density: Particles with relatively higher TEP would be of lower density (Engel and Schartau, 1999) and, consequently, would present a lower sinking velocity or could even ascend (Azetsu-Scott and Passow, 2004; Mari et al., 2007) and accumulate at or near the surface. 4.3. Contribution of TEP, phytoplankton, and prokaryotic heterotrophs to POC p y p TEP contributed remarkably to the POC pool (overall range of 3%–44%), in similar proportions to those measured in the northeastern Atlantic Ocean (1.5%–68%; Harlay et al., 2009, 2010), but lower than many of the percentages reported across the Atlantic Ocean (28%–110% by Zama- nillo et al., 2019b), the Mediterranean Sea (60% to >100% by Bar-Zeev et al., 2011; 14% to >100% by Parinos et al., 2017; 4% to >100% by Ortega-Retuerta et al., 2019) and the western Arctic Ocean (135%–179% by Yamada et al., 2015). This disparity across studies, where unrealistic TEP contributions higher than 100% abound, points to the uncertainty in the TEP-to-C conversion factor across sys- tems and seasons. In alignment with Bar-Zeev et al. (2011), Parinos et al. (2017), and Zamanillo et al. (2019b), we used a conservative value (0.51 mg TEP-C mg XG eq–1) from a range of conversion factors determined experimentally with cultured and natural marine diatoms (Engel and Pas- sow, 2001). Other authors (Harlay et al., 2010; Yamada et al., 2015; Ortega-Retuerta et al., 2019) used higher values from the same seminal study (0.63–0.75 mg TEP-C mg XG eq–1). In any case, the C content of TEP is expected to vary with their monomeric composition, which in turn de- pends on TEP origin (Engel and Passow, 2001); with this information lacking, comparisons of TEP carbon between oceanic studies is problematic. Another reason for the frequent oversizing of the TEP contribution to POC is merely methodological: TEP are measured on 0.4-mm pore-size filters because they are not retained quantita- tively on the GF/F filters used for POC (Passow and All- dredge, 1995; Engel and Passow, 2001) and, therefore, an unknown fraction of TEP may be lost in the measured POC pool. 4.4. TEP and CSP size distribution The microscopic examination provided evidence that, even though TEP were generally dominated by small par- ticles as in previous studies (Passow and Alldredge, 1994; Mari and Burd, 1998; Harlay et al., 2009), their size distri- bution changed over time (Figure 4). The modal distribu- tion of the April sample could be indicative of freshly produced TEP from a particular source, maybe a specific phytoplankton population, or to aggregation processes. The steeper TEP size distribution slope in August with respect to October could be related to solar radiation, which could either inhibit TEP aggregation into larger sizes or break up larger aggregates into small TEP, or both. By contrast, CSP size distributions were similar throughout the year, which we suggest hints at a lower aggregation potential of CSP compared to TEP. 4.5. Distribution of TEP and CSP in the coast-to- offshore transect The different distribution patterns of TEP and CSP along the coast-to-offshore transect (Figure 7) indicate that the 2 particle types are also uncoupled on the spatial scale. Like the observations over the temporal scale, the positive correlation of CSP with Chla illustrates that CSP variations are more directly linked to those of phytoplankton than TEP variations. Supporting that hypothesis, Arin et al. (1999) found higher concentrations of particulate protein and higher percentages of detrital protein in surface waters of the shore-most station than in the open sea along a coast-to-offshore transect in the NW Mediterra- nean Sea. 4.1. Dynamics of TEP and CSP over the seasonal cycle in the NW Mediterranean Sea We hypothesize that the lower density of TEP-rich particles in summer would have also contributed to their observed accumulation in the sea surface (i.e., above 20 m at EOS), in conjunction with higher production rates under high light and nutrient limitation. Significant correlations between TEP or CSP dynamics and those of specific phytoplankton groups provide hints about the main producers of these particles at our study sites: TEP mainly correlated with dinoflagellates, diatoms, and nanoeukaryotes over time at the BBMO (Table 2), whereas CSP did not correlate with any group over time at either station, although at the vertical scale, CSP was best related to Synechococcus, as previously observed by Cisternas-Novoa et al. (2015). 5. Conclusions According to our results, TEP and CSP are most likely independent particle types as they presented uncoupled temporal dynamics at 2 coastal sites and uncoupled spa- tial distributions along a coast-to-offshore transect in the NW Mediterranean Sea. Taxonomy-segregated phytoplank- ton biomass was the main driver of both TEP and CSP concentrations: diatoms, dinoflagellates, and nanoeukar- yotes each explained 40%–50% of TEP distribution at surface, with dinoflagellates covarying best with TEP over depth and Synechococcus abundances best matching CSP vertical profiles. While CSP are best explained by phyto- plankton biomass, nutrient limitation and the release of phytoplankton carbon under saturating irradiance are sug- gested as additional drivers of the distribution and Despite these uncertainties and the use of a conserva- tive conversion factor, in summer the estimated carbon in the form of TEP was higher than that of phytoplankton and prokaryotic heterotrophs, at least at EOS (Figure 6). TEP: POC also tended to be higher in summer in this site Art. 9(1) page 19 of 23 Art. 9(1) page 19 of 23 Zamanillo et al: Seasonality of TEP and CSP in the Mediterranean Sea Provided accompanying physical, chemical, and biolog- ical data: CM, JMG, JP, CP. temporal dynamics of TEP. The finding that 2 geographi- cally close coastal sites exhibit similar temporal patterns of TEP concentration suggests that TEP variability indeed reflects a response to the environmental seasonality char- acteristic of this temperate oligotrophic region. Helped with data contextualization: AE, CCN. Wrote this article with the help of all coauthors: MZ, EOR, RS. Wrote this article with the help of all coauthors: MZ, EOR, RS. Approved the submission: All authors. Approved the submission: All authors. Competing interests Bar-Zeev, E, Berman, T, Rahav, E, Dishon, G, Herut, B, Berman-Frank, I. 2011. Transparent exopolymer particle (TEP) dynamics in the eastern Mediterra- nean Sea. Marine Ecology Progress Series 431: 107–118. The authors declare that they have no conflict of interest. Supplemental files Supplemental files The supplemental files for this article can be found as follows: Alldredge, AL, Passow, U, Logan, BE. 1993. The abun- dance and significance of a class of large, transpar- ent organic particles in the ocean. Deep Sea Research Part I: Oceanographic Research Papers 40: 1131– 1140. Figures S1–S4. PDF Figures S1–S4. PDF Funding Thi This research was funded by the Spanish Ministry of Econ- omy and Competitiveness through projects PEGASO (CTM2012–37615), BIOGAPS (CTM2016–81008–R), RE- MEI (CTM2015-70340-R), and DOREMI (CTM12-34294) and the Generalitat de Catalunya, through funding to the Research Group on Marine Biogeochemistry and Global Change (2017 SGR1011) and that of Microbial Diversity and Function in Aquatic Systems (2017SGR/1568) and with the institutional support of the Severo Ochoa Center of Excellence accreditation (CEX2019-000928-S). MZ was supported by a FPU predoctoral fellowship (FPU13/ 04630) from the Spanish Ministry of Education and Cul- ture. The EOS Monitoring Program is supported by the Institut de Cie`ncies del Mar (ICM-CSIC) and the Parc Nat- ural del Montgrı´, les Illes Medes i el Baix Ter (Generalitat de Catalunya). Arin, L, Berdalet, E, Marrase´, C, Estrada, M, Guixa- Boixereu, N, Dolan, J. 1999. 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We also want to thank Maria Castellvı´ and Idaira Santos for helping with transparent exopolymer and Coomassie stainable particle measurements, and Jon Roa, Alexandra Loginova, Sonja Endres, Judith Piontek, Katja Lass, Ruth Flerus, Tania Klu¨- ver, Marie Massmig, and Birthe Za¨ncker for their technical support. We thank the scientists and technicians on board the RV Garcı´a del Cid during the MIFASOL II cruise for providing environmental and logistic support. We also thank two anonymous reviewers for their constructive comments. Aller, JY, Kuznetsova, MR, Jahns, CJ, Kemp, PF. 2005. The sea surface microlayer as a source of viral and bacterial enrichment in marine aerosols. Journal of Aerosol Science 36: 801–812. Aller, JY, Radway, JC, Kilthau, WP, Bothe, DW, Wilson, TW, Vaillancourt, RD, Quinn, PK, Coffman, DJ, Murray, BJ, Knopf, DA. 2017. Size-resolved charac- terization of the polysaccharidic and proteinaceous components of sea spray aerosol. Atmospheric Envi- ronment 154: 331–347. 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https://openalex.org/W4283378327	https://f1000research.com/articles/9-511/v2/pdf	English	null	How to build phylogenetic species trees with OMA	F1000Research	2,022	cc-by	14,023	* Equal contributors Open Peer Review Approval Status 1 2 version 2 (revision) 28 Feb 2022 view view version 1 04 Jun 2020 view view Jianbo Xie, National Engineering Laboratory for Tree Breeding, College of Biological Sciences and Technology, Beijing Forestry University, Beijing, China 1. Denis Baurain , University of Liège, Liège, Belgium 2. Any reports and responses or comments on the article can be found at the end of the article. First published: 04 Jun 2020, 9:511 https://doi.org/10.12688/f1000research.23790.1 Latest published: 28 Feb 2022, 9:511 https://doi.org/10.12688/f1000research.23790.2 v2 Abstract Knowledge of species phylogeny is critical to many fields of biology. In an era of genome data availability, the most common way to make a phylogenetic species tree is by using multiple protein-coding genes, conserved in multiple species. This methodology is composed of several steps: orthology inference, multiple sequence alignment and inference of the phylogeny with dedicated tools. This can be a difficult task, and orthology inference, in particular, is usually computationally intensive and error prone if done ad hoc. This tutorial provides protocols to make use of OMA Orthologous Groups, a set of genes all orthologous to each other, to infer a phylogenetic species tree. It is designed to be user-friendly and computationally inexpensive, by providing two options: (1) Using only precomputed groups with species available on the OMA Browser, or (2) Computing orthologs using OMA Standalone for additional species, with the option of using precomputed orthology relations for those present in OMA. A protocol for downstream analyses is provided as well, including creating a supermatrix, tree inference, and visualization. All protocols use publicly available software, and we provide scripts and code snippets to facilitate data handling. The protocols are accompanied with practical examples. Jianbo Xie, National Engineering Laboratory for Tree Breeding, College of Biological Sciences and Technology, Beijing Forestry University, Beijing, China 1. Any reports and responses or comments on the article can be found at the end of the article. F1000Research 2022, 9:511 Last updated: 12 MAR 2024 METHOD ARTICLE How to build phylogenetic species trees with OMA [version 2; peer review: 2 approved] David Dylus1-3, Yannis Nevers1-3, Adrian M. Altenhoff 1,4, Antoine Gürtler2,3, Christophe Dessimoz 1-3,5,6, Natasha M. Glover 1-3 1Department of Computational Biology, University of Lausanne, Lausanne, 1015, Switzerland 2Center for Integrative Genomics, University of Lausanne, Lausanne, 1015, Switzerland 3Swiss Institute of Bioinformatics, Lausanne, 1015, Switzerland 4Department of Computer Science, ETH Zurich, Zurich, 8092, Switzerland 5Department of Computer Science, University College London, London, WC1E 6BT, UK 6Department of Genetics, Evolution and Environment, University College London, London, WC1E 6BT, UK * Equal contributors First published: 04 Jun 2020, 9:511 https://doi.org/10.12688/f1000research.23790.1 Latest published: 28 Feb 2022, 9:511 https://doi.org/10.12688/f1000research.23790.2 v2 Abstract Knowledge of species phylogeny is critical to many fields of biology. In an era of genome data availability, the most common way to make a phylogenetic species tree is by using multiple protein-coding genes, conserved in multiple species. This methodology is composed of several steps: orthology inference, multiple sequence alignment and inference of the phylogeny with dedicated tools. This can be a difficult task, and orthology inference, in particular, is usually computationally intensive and error prone if done ad hoc. This tutorial provides protocols to make use of OMA Orthologous Groups, a set of genes all orthologous to each other, to infer a phylogenetic species tree. It is designed to be user-friendly and computationally inexpensive, by providing two options: (1) Using only precomputed groups with species available on the OMA Browser, or (2) Computing orthologs using OMA Standalone for additional species, with the option of using precomputed orthology relations for those present in OMA. A protocol for downstream analyses is provided as well, including creating a supermatrix, tree inference, and visualization. All protocols use publicly available software, and we provide scripts and code snippets to facilitate data handling. The protocols are accompanied with practical examples. Keywords Open Peer Review Approval Status 1 2 version 2 (revision) 28 Feb 2022 view view version 1 04 Jun 2020 view view Jianbo Xie, National Engineering Laboratory for Tree Breeding, College of Biological Sciences and Technology, Beijing Forestry University, Beijing, China 1. Denis Baurain , University of Liège, Liège, Belgium 2. Any reports and responses or comments on the article can be found at the end of the article. This article is included in the Bioinformatics gateway. This article is included in the Bioinformatics gateway. This article is included in the The OMA collection collection. Corresponding author: Natasha M. Glover (natasha.glover@sib.swiss) Author roles: Dylus D: Conceptualization, Formal Analysis, Investigation, Methodology, Project Administration, Software, Visualization, Writing – Original Draft Preparation; Nevers Y: Data Curation, Formal Analysis, Investigation, Methodology, Validation, Writing – Original Draft Preparation, Writing – Review & Editing; Altenhoff AM: Conceptualization, Data Curation, Methodology, Resources, Software, Writing – Review & Editing; Gürtler A: Formal Analysis, Investigation, Methodology; Dessimoz C: Funding Acquisition, Project Administration, Supervision, Writing – Review & Editing; Glover NM: Conceptualization, Project Administration, Supervision, Validation, Writing – Original Draft Preparation, Writing – Review & Editing Competing interests: No competing interests were disclosed. Grant information: This work was funded by a Service and Infrastructure grant from the Swiss Institute of Bioinformatics and a Swiss National Science Foundation Professorship grant (Grant 183723). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Copyright: © 2022 Dylus D 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 work is properly cited. How to cite this article: Dylus D, Nevers Y, Altenhoff AM et al. How to build phylogenetic species trees with OMA [version 2; peer review: 2 approved] F1000Research 2022, 9:511 https://doi.org/10.12688/f1000research.23790.2 First published: 04 Jun 2020, 9:511 https://doi.org/10.12688/f1000research.23790.1 Keywords y hylogenetics, phylogenomics, species tree, OMA, Orthologous Matrix Page 1 of 25 F1000Research 2022, 9:511 Last updated: 12 MAR 2024 This article is included in the Genomics and Genetics gateway. Introduction I f i Inferring accurate and complete species phylogenies is a fundamental problem in biology1. Traditionally, spe- cies trees have been inferred using ubiquitous marker genes such as the small subunit ribosomal RNA (SSU rRNA 16S/18S) gene2. However, as there are fewer sites to sample, using only one gene per species limits the resolution of the inference; the phylogeny of the gene may not necessarily reflect the evolutionary history of the entire species (due to, for example, incomplete lineage sorting, “hidden” duplications followed by loss of one copy, horizontal gene transfer, etc.3). Additionally, phylogenies based on one gene are not always sufficient to obtain statistical support for difficult nodes due to their limited number of characters. For this reason, species phylogenies are now overwhelmingly inferred from multiple genes4. As long as one takes the necessary precau- tions, notably selecting true orthologs for their comparisons (see 4,5 for common pitfalls in phylogenomics), multilocus phylogenies are better resolved and more robust6. Recently, multiple protein-coding genes were used to infer a tree comprising ~3000 species, but this was still limited to a small number of concatenated ribosomal genes7. With the rise of next-generation sequencing, many hundreds of genes can now be considered when building species trees, which tremendously increases the available information for the inference. To make use of all available genes, one needs to identify groups of genes that emerged from a common ances- tral gene solely through speciation. These sets of genes, in which all pairs of genes are orthologs8, are com- monly referred to as Orthologous Groups (OGs). Another term used for these types of groups are marker genes, or phylogenetic marker genes. The inference of OGs is non-trivial due to additional evolutionary events such as gene duplications, gene losses or horizontal gene transfers9. Furthermore, there are numerous algorithms for inferring orthology which can result in different OG composition, further complicating matters. In this tutorial, we focus on OGs obtained from Orthologous MAtrix (OMA). Alternatively called “OMA Groups,” they are stringently computed orthologs, they make use of all the available species in OMA, and are specifically designed for species tree inference. OMA Groups are defined as gene families that contain genes which are all orthologous to each other, with a maximum of one gene per species10. If recently diverged in-paralogs are inferred (i.e., co-orthologs), only one of the copies will be selected for the OG. Corresponding author: Natasha M. Glover (natasha.glover@sib.swiss) Author roles: Dylus D: Conceptualization, Formal Analysis, Investigation, Methodology, Project Administration, Software, Visualization, Writing – Original Draft Preparation; Nevers Y: Data Curation, Formal Analysis, Investigation, Methodology, Validation, Writing – Original Draft Preparation, Writing – Review & Editing; Altenhoff AM: Conceptualization, Data Curation, Methodology, Resources, Software, Writing – Review & Editing; Gürtler A: Formal Analysis, Investigation, Methodology; Dessimoz C: Funding Acquisition, Project Administration, Supervision, Writing – Review & Editing; Glover NM: Conceptualization, Project Administration, Supervision, Validation, Writing – Original Draft Preparation, Writing – Review & Editing Page 2 of 25 F1000Research 2022, 9:511 Last updated: 12 MAR 2024 Amendments from Version 1 REVISED There are no major differences between this version and the previous version. We mainly clarified the points in the text (as suggested by the reviewers), and updated Figure 3 and Figure 4 to reflect bootstrap values. We added a list of the species used in this analysis in the figshare archive, and updated the python scripts to make it compatible with the latest version of its dependency. Any further responses from the reviewers can be found at the end of the article There are no major differences between this version and the previous version. We mainly clarified the points in the text (as suggested by the reviewers), and updated Figure 3 and Figure 4 to reflect bootstrap values. We added a list of the species used in this analysis in the figshare archive, and updated the python scripts to make it compatible with the latest version of its dependency. Any further responses from the reviewers can be found at the end of the article Introduction I f i Thus, all members of the OG are still orthologous to each other. This type of Orthologous Group is provided by only a few orthology data- bases such as BUSCO11 and OMA12,13 and, to our knowledge, only OMA allows for use of both precomputed and user-computed OGs. Moreover, OMA Groups have repeatedly been shown to produce reliable trees12 and its underlying algorithm was shown to accurately infer OGs in a large-scale benchmark14. It is sometimes pos- sible to rely on existing marker genes used in large-scale studies (for example15, but they are generally available only for a subset of species and do not include newly sequenced species. The OMA algorithm is freely available as an open source software tool (OMA Standalone12) that integrates well with the public OMA Browser (https://omabrowser.org), which is a database that provides orthology information among more than 2400 genomes across the tree of life, selected to maximize taxon coverage and users’ needs16. To date (December 2021 release) there are 1719 Bacteria, 155 Archaea, and 622 Eukaryotes. In this protocol, we show how to leverage the publicly available orthology data to infer phylogenetic species trees. First, we set up the prerequisites and explain how to search for precomputed OGs for species of interest in the OMA database. Then, we show how to infer a species tree under two scenarios: (1) using only species that are present in the public OMA database, or (2) using species in OMA in addition to other proteomes not avail- able in the database, e.g. a proteome obtained from sequencing a new species. By proteome we mean the protein sequences of all protein-coding genes annotated in a genome. Finally, we show how to do downstream processing and tree inference. Each of these steps is illustrated by an example on real data. Page 3 of 25 Page 3 of 25 F1000Research 2022, 9:511 Last updated: 12 MAR 2024 F1000Research 2022, 9:511 Last updated: 12 MAR 2024 Materials The tools needed for this tutorial can be found in Table 1. All commands can be run from the command line and/or with python scripts. We reference four tree inference software tools, but there are many other alternatives (see List of phylogenetics software). Two examples will be used in this tutorial, one to illustrate Protocol 1, and another to illustrate Protocol 2. Both examples can be downloaded from FigShare17. Instructions on how to obtain the data from the OMA Browser for Protocol 1 are described in the next section, so it is not required to download anything to complete this protocol. However, Protocol 2 demonstrates how to add external proteomes. For this example we chose two proteomes available in the OMA Browser, but we set them aside after downloading the data. We then re-add them as external data (FOMPI.fa and YEAST.fa). For reproducibility, these proteomes can be found in the /data/ AddedGenomes subdirectory of Protocol 2. The rest of the data included in the example tarball are OGs and alignment files needed to compute the trees, which are also included as results. The tree computations on these data have been performed using both RAxML 8.2.12 and IQ-TREE 1.7.beta17, as specified in the PDF accompanying the examples. Protocols ylogenetic tree inference using OMA is done in three steps: getting OGs data, aligning all sequences of e G and combining them into a supermatrix, and finally, using tree inference tools on the supermatrix. Depen Table 1. Computational tools needed for making a phylogenetic species tree using OMA. Note that four phylogenetic tree inference software packages are given, but only one (user preference) is needed to complete this tutorial. Tool Use case How to get it Command line Mandatory to run the commands written in this tutorial Installed by default on Unix and Mac Python 3 Python language interpreter. Mandatory for running the scripts used in this paper https://www.python.org/downloads/ OMA Browser Needed to import orthology relations used for tree inference https://omabrowser.org OMA standalone Needed to infer orthology for data not available in the OMA Browser https://omabrowser.org/standalone/#downloads MAFFT Multiple sequence alignment software https://mafft.cbrc.jp/alignment/software/ High Performance Computing (HPC) Needed if a high amount of computation is involved Institutional infrastructure IQ-Tree Phylogenetic tree software http://www.iqtree.org/#download RAxML Phylogenetic tree software https://cme.h-its.org/exelixis/web/software/raxml/index.html Phylobayes Phylogenetic tree software http://www.atgc-montpellier.fr/phylobayes/ PhyML Phylogenetic tree software http://www.atgc-montpellier.fr/phyml/ Phylo.io Phylogenetic tree visualization website http://phylo.io Table 1. Computational tools needed for making a phylogenetic species tree using OMA. Note that four phylogenetic tree inference software packages are given, but only one (user preference) is needed to complete this tutorial. Table 1. Computational tools needed for making a phylogenetic species tree using OMA. Note that four phylogenetic tree inference software packages are given, but only one (user preference) is needed to complete this tutorial. Page 4 of 25 Page 4 of 25 Page 4 of 25 F1000Research 2022, 9:511 Last updated: 12 MAR 2024 F1000Research 2022, 9:511 Last updated: 12 MAR 2024 on the species requirement, two options are available to obtain OG data, they are detailed in Protocol 1 and 2 subsections. Protocol 1 is the fastest and can be used if all species of interest are available on the OMA Browser. Alternatively, Protocol 2 is for the cases when new proteomes must be added, or when solely using data computed by OMA Standalone. Later steps are the same for both cases and are addressed in Protocol 3. on the species requirement, two options are available to obtain OG data, they are detailed in Protocol 1 and 2 subsections. Protocol 1 is the fastest and can be used if all species of interest are available on the OMA Browser. Protocols Alternatively, Protocol 2 is for the cases when new proteomes must be added, or when solely using data computed by OMA Standalone. Later steps are the same for both cases and are addressed in Protocol 3. Protocol 1: Export marker genes to make a phylogeny of species found in OMA This method is the quickest way to obtain data to build a phylogenetic species tree, but is only useful if one is interested in making a tree from species already in the OMA database. To do this, the Export marker genes func- tion in the browser takes advantage of the precomputed OMA Groups. As mentioned in the Introduction, OMA Groups are a specific type of OG which contain sets of genes that are all orthologous to one another. This implies that there is at most one gene from each species in a group. Finding species of interest in OMA. The OMA public database and all related information are accessed through the OMA browser (https://omabrowser.org/). One can search for species of interest in the OMA database by browsing through the available data in OMA using the release info page (from the menu in the upper right corner: Explore -> Release information). Two browsing options are available, the default one is through an inter- active tree, with colors indicating domains of life: bacteria are blue, archaea are green and eukaryotes are red. The other option is a table viewer featuring a search bar and both can be accessed through the Select species browser widget icons in the top right of the Species Information visualization. Export the relevant data from OMA. The way to obtain OGs with only species present in the OMA data- base is by using the Download -> Export marker genes option (Figure 1A) in the top right menu. This will open a page which allows the user to select species. Species can be searched by name or clade. A whole clade can be selected by clicking on the node (select all species). A single species can be selected by clicking on the leaf (select species). All selected species will be displayed in the right box with additional species information (release info, taxon id, etc.) (Figure 1B). Specifying the Minimum Species Coverage and Maximum Number of Markers parameters. Protocol 2: Export precomputed OMA all-against-all data as a backbone to add your own genomes and use with OMA standalonei Protocol 2: Export precomputed OMA all-against-all data as a backbone to add your own genomes and use with OMA standalonei Orthology computation first starts with an all-against-all alignment phase一comparing all proteins in every species of interest to each other. If genomes to be included in the species phylogeny are not present in OMA (hereafter referred to as “added genomes”), it is necessary to first compute orthology predictions for the combined set of species (those in OMA plus the added genomes). This approach is computationally more expensive and requires that computations are performed on a local machine or high performance computing cluster (HPC). However, by using the OMA Browser’s Download -> Export All/All option, one can take advantage of the precomputed all-against-all data for those species in OMA, saving time. The following protocol describes how to make use of this data and run OMA Standalone, the software for running the OMA algorithm on added genomes. In the case where the user wants to only use genomes unavailable in OMA, skip to the “Running OMA Standalone” section. Export the all-against-all from OMA. Choose species which you want to combine with your own genomes by choosing Download -> Export All/All from the top right menu (Figure 1A). This will lead to an interactive species tree of all the species in OMA, for which you can choose your species of interest to export (Figure 1B). After selecting species and clicking submit, the OMA Browser will export a tarball (described in Figure 2) which contains: • The all-against-all alignments of the selected species, found in the folder “Cache.” • All exported genomes, in the format of protein fasta files, found in the folder “DB.” • The full OMA standalone software tool. No need to download it separately. Combining the added genomes with exported OMA data. Next, the added genomes data must be combined with the OMA data. For this procedure, the added genomes data must fulfill certain conditions: Combining the added genomes with exported OMA data. Next, the added genomes data must be combined with the OMA data. For this procedure, the added genomes data must fulfill certain conditions: • Each additional dataset is in the form of a fasta file, containing protein sequences of all coding genes in the corresponding genome. Protocols After species selec- tion, exported OGs will depend on the minimum fraction of covered species and the maximum number of markers parameters: • Minimum species coverage: the lowest acceptable proportion of selected species that are present in any given OG in order to be exported. • Minimum species coverage: the lowest acceptable proportion of selected species that are present in any given OG in order to be exported. Figure 1. Exporting data from OMA for building a species tree. A) Choose which type of data to export from the Download tab on the right hand side of the home page. B) Select your proteomes from those in the OMA database by using the interactive species tree, which is based on the NCBI taxonomy. Figure 1. Exporting data from OMA for building a species tree. A) Choose which type of data to export from the Download tab on the right hand side of the home page. B) Select your proteomes from those in the OMA database by using the interactive species tree, which is based on the NCBI taxonomy. Page 5 of 25 F1000Research 2022, 9:511 Last updated: 12 MAR 2024 F1000Research 2022, 9:511 Last updated: 12 MAR 2024 A more permissive (lower) minimum species coverage will result in a higher number of exported groups. Choosing this parameter depends on the number of and how closely related are the selected species. For instance, consider the Drosophila clade versus chordates clade (20 and 116 species in the January 2020 release, respectively). If one selects the 20 Drosophila genomes and sets the minimum species coverage to 0.5, only OGs with at least 10 Drosophila species will be exported. In the January 2020 release, this results in 11,855 OGs which meet this criteria. If using the same 0.5 minimum species coverage for the chordates, it results in 14,357 OGs exported. On the other hand, for a 0.8 minimum species coverage, 7,886 and 6,329 OGs are exported for Drosophila and chordates clades, respectively. • Maximum number of markers: the maximum number of OGs/marker genes to return. To consider as much information as possible in the tree inference, remove any limit by setting this parameter to -1, in which case all OGs fulfilling the minimum species coverage parameter will be returned. To speed up the tree inference, set this value to below 1000 genes. Protocols When the number of markers is limited in this way, OGs with the highest coverage will be prioritized. • Maximum number of markers: the maximum number of OGs/marker genes to return. To consider as much information as possible in the tree inference, remove any limit by setting this parameter to -1, in which case all OGs fulfilling the minimum species coverage parameter will be returned. To speed up the tree inference, set this value to below 1000 genes. When the number of markers is limited in this way, OGs with the highest coverage will be prioritized. After filling in the parameters and submitting the request, the browser will return a compressed archive (“tarball”) that contains a fasta file with unaligned sequences for each OG. Depending on the size of the request, it may take a few minutes for this operation to complete. As an example for Protocol 1, we performed an analysis on 20 yeast species, using only OGs shared by all species (Minimum species coverage : 1) and no limit to the number of OGs retrieved (Maximum number of markers : -1). We obtained 169 OGs with this query. The corresponding data, including a list of the 20 species used, can be found at FigShare17, in the Protocol_1 folder. Upon exporting the marker genes, i.e. OGs, from OMA, the data can be used to make a phylogenetic tree (skip to Protocol 3: Downstream processing and tree inference). Protocol 2: Export precomputed OMA all-against-all data as a backbone to add your own genomes and use with OMA standalonei Please note that OMA Standalone can work on nucleic coding sequences when starting from scratch, however for compatibility with pre-computed OMA data, only protein sequences may be used when combining new and exported data. Page 6 of 25 Page 6 of 25 F1000Research 2022, 9:511 Last updated: 12 MAR 2024 Figure 2. Tree organization of the tarball downloaded through the OMA Browser after exporting an all- Figure 2. Tree organization of the tarball downloaded through the OMA Browser after exporting an all- against-all of selected species. The important files and folders are colored. In green, the executable files mentioned in the course of the tutorial. In blue are the files and folder that will need to be modified. Other files and folders (in black) will not be used in the course of the tutorial. Files and folders not shown are represented by three dots. • The name of the fasta file should identify the species clearly and uniquely. The exported genomes from OMA use for example UniProt’s mnemonic five-letter species codes. The filename must end with a “.fa” suffix and must not contain any whitespace characters. The filename without the “.fa” suffix is used as the species name throughout the process and result files. • The name of the fasta file should identify the species clearly and uniquely. The exported genomes from OMA use for example UniProt’s mnemonic five-letter species codes. The filename must end with a “.fa” suffix and must not contain any whitespace characters. The filename without the “.fa” suffix is used as the species name throughout the process and result files. • Each sequence in the fasta file has a clear and unique identifier. We suggest not to use special characters such as brackets, dots, or a pipe character. The reason is that many programs use them for special purposes, e.g. brackets are used in the newick format for tree representation, and the pipe character is often used to separate ids and annotations. • Each sequence in the fasta file has a clear and unique identifier. We suggest not to use special characters such as brackets, dots, or a pipe character. The reason is that many programs use them for special purposes, e.g. brackets are used in the newick format for tree representation, and the pipe character is often used to separate ids and annotations. Protocol 2: Export precomputed OMA all-against-all data as a backbone to add your own genomes and use with OMA standalonei If these conditions are fulfilled, these fasta files must be put into the DB folder with the other exported OMA genomes (Figure 2), where they will be considered as a unique dataset for the following steps. Setting the parameters for OMA standalone. Before starting the computation, it is wise to adjust the parameters file, called “parameters.drw” (Figure 2), which can be edited with any text editor. If the goal is to only generate a dataset for species phylogeny inference (and not to keep other unrelated orthology inferences, such as Hierarchical Orthologous Groups10, which better represent individual genes’ evolutionary histories but take time to compute), one can avoid doing computations and generating output files that are not needed by the following: • Uncomment (remove the # from) all the lines starting with WriteOutput EXCEPT #WriteOut- put_OrthologousGroupsFasta := false. By keeping that one commented, OMA standalone will produce one fasta file for each inferred OG. • Uncomment (remove the # from) all the lines starting with WriteOutput EXCEPT #WriteOut- put_OrthologousGroupsFasta := false. By keeping that one commented, OMA standalone will produce one fasta file for each inferred OG. Page 7 of 25 Page 7 of 25 F1000Research 2022, 9:511 Last updated: 12 MAR 2024 • Deactivate the Hierarchical Orthologous Group inference, which is not needed here, by setting DoHierarchicalGroups := false; • Deactivate the Hierarchical Orthologous Group inference, which is not needed here, by setting DoHierarchicalGroups := false; • Likewise, deactivate the gene function prediction by setting DoGroupFunctionPrediction := false; • Likewise, deactivate the gene function prediction by setting DoGroupFunctionPrediction := false; • Tip: do not omit a semicolon at the end of each uncommented statement. • Tip: do not omit a semicolon at the end of each uncommented statement. Running OMA standalone. To run OMA standalone, one needs to be aware that the OMA pipeline can be split into two parts: all-against-all alignments for homology inference and orthology calling. Because OMA can compute Smith-Waterman alignments in parallel for all species which were not exported from OMA (see Export the all-against-all from OMA), it is beneficial to perform the computations on a computer cluster. However, if the dataset is small (e.g. 2–3 additional genomes), the computations can be run locally on a standard computer. To run OMA standalone on a small dataset locally: To run OMA standalone on a small dataset locally: 1. Within the extracted tarball folder you can start the computation with the command line: 1. Within the extracted tarball folder you can start the computation with the command line: $: bin/oma -n NR_PROCESSES $: bin/oma -n NR_PROCESSES NR_PROCESSES should not be higher than the number of CPUs you have available on your machine. larger dataset, we recommend the use of an HPC cluster. We recommend to break up the computation wo parts: first the all-against-all part, then the orthology inference part: 1. Create a submission script for your cluster. Examples of submission scripts are provided at https://omabrowser.org/standalone/#schedulers and 18. 1. Create a submission script for your cluster. Examples of submission scripts are provided at https://omabrowser.org/standalone/#schedulers and 18. 2. Make sure that the submission script enters the folder into which the tarball was extracted, by either running the script from inside that directory or using the cd command appropriately. 2. Make sure that the submission script enters the folder into which the tarball was extracted, by either running the script from inside that directory or using the cd command appropriately. 3. The line to start the OMA all-against-all computation in the submission scripts is: $: bin/oma -s $:bin/oma-cleanup $: python filter_groups.py --min-nr-species <MIN_NR_SPECIES> --input Output/ OrthologousGroupFasta/ --output <destination/directory> For example, we performed an analysis adding two yeast proteomes hypothetically not available in OMA and 18 available yeast proteomes. As a first step, we downloaded the precomputed data for the 18 proteomes from the OMA Browser and launched the computation after adding two separate proteomes. Once the com- putation finished, we selected 880 OGs that included at least 90% of the 20 species 一 18 一 as a dataset to construct a tree. The data used in this example is available at FigShare17 in the Protocol_2 folder. $: bin/oma Once the computation finishes, all results will be stored in the newly-created “Output” folder. In this folder there will be an “EstimatedSpeciesTree.nwk” file that contains a phylogenetic tree that can be visualized using a tree visualization tool such as Phylo.io19. This is a distance tree based on the weighted average of the pairwise distances between sequences within the most complete OMA groups. This species tree is a rough esti- mate that is computed on the fly, and is not the final tree. It can be used as control to identify prob- lems in the dataset but will not be as reliable as the tree inferred using the generated OGs later in this protocol. Therefore, it is recommended to use the OGs to compute your own tree with external software. The OGs (OMA Groups)20 can be found in the “OrthologousGroupsFasta” folder, with each OG containing at least two species. Usually for the construction of phylogenetic trees, one would select only OGs that contain at least X% of spe- cies, as described above with the parameter Minimum Species Coverage. The python script filter_groups.py from the git repository associated to this publication (https://doi.org/10.5281/zenodo.603751621) can be used to filter the OMA groups that contain at least X MIN_NR_SPECIES (replace <MIN_NR_SPECIES> and <destination/directory> with your own values): $: python filter_groups.py --min-nr-species <MIN_NR_SPECIES> --input Output/ OrthologousGroupFasta/ --output <destination/directory> $: bin/oma -s The -s option means stop after the all-against-all phase. Since this part can be parallelized, we recommend using job-arrays. For this you need to set the number of parallel jobs as an environment variable (export NR_PROCESSES=100) and use the job-array syntax in the submission script (e.g. in LSF: bsub -J oma[1-$NR_PROCESSES] bin/oma -s). OMA Standalone auto- matically partitions the work chunks in a static and deterministic way among the specified number of workers. Progress of the entire computation can be checked with the OMA Status command (see below). For environments with limited runtimes/walltimes see https://omabrowser.org/standalone/ #advanced%20optimisations. 4. Check whether the all-against-all computation is finished using: $: bin/oma-status -i 4. Check whether the all-against-all computation is finished using: $: bin/oma-status -i This command will output a file formatted as: Summary of OMA standalone All-vs-All computations: -------------------------------------------------- Nr chunks started: A (D%) Nr chunks finished: B (E%) Nr chunks finished w/o exported genomes: C (F%) Where the letters A, B, C, D, E and F represent numbers. Once the computations are completed, D should be equal to 0.0%, and both E and F to 100.0% 5. In the case where the jobs are finished but the all-against-all computation is still not complete, use the oma-cleanup and oma-compact commands before re-submitting. $:bin/oma-cleanup $: bin/oma-compact P This command will output a file formatted as: Nr chunks started: A (D%) Nr chunks finished: B (E%) Where the letters A, B, C, D, E and F represent numbers. Once the computations are completed, D should be equal to 0.0%, and both E and F to 100.0% 5. In the case where the jobs are finished but the all-against-all computation is still not complete, use the oma-cleanup and oma-compact commands before re-submitting. $:bin/oma-cleanup $: bin/oma-compact Page 8 of 25 F1000Research 2022, 9:511 Last updated: 12 MAR 2024 F1000Research 2022, 9:511 Last updated: 12 MAR 2024 These commands remove partially finished output files in the Cache/AllAll folder and zip all partial computations that are finished to one file, respectively. 6. Once the all-against-all computation has finished, the final step is the orthology calling. This step is more memory intensive, requires a single process, and can be called with: $: bin/oma Protocol 3: Downstream processing and tree inferencei Once all selected OGs are obtained from either of the first two protocols, the next step is to align all sequences within each OG. This can be done with any Multiple Sequence Alignment (MSA) tools, in this example we use MAFFT22. To run it, navigate to the folder containing the selected OGs and execute the following command, which runs mafft on each fasta file: $: for i in $(ls -1 .fa); do mafft --maxiterate 1000 --localpair $i > $i.aln; done This command sequentially generates an MSA file (.aln) for each OG. Depending on the number of OGs and species in your dataset, executing it may take a prohibitive amount of time. If it is the case, we recommend using job-arrays to execute the alignments in parallel. In order to infer the phylogeny of the species from these alignments, they have to be concatenated in a single alignment commonly referred to as supermatrix. We provide a python script to automate this, concat_alignment.py, available on https://doi.org/10.5281/zenodo.603751621. The --format-output option allows for choosing the output format of this concatenation, either fasta or phylip format (some phylogenetic software requires a specific format as input). Once the python script is downloaded or cloned, ensure that all alignments are in the same folder, and launch using the following command: $: python concat_alignments.py <path>/<to>/<alignments>/aln --format-output [fasta/phylip] > output After computing the supermatrix, the phylogenetic tree can be inferred using any number of available software. We recommend choosing from the tools in Table 2, sorted by computing time and increasing precision. Tree visualization. Most of the current phylogenetic inference tools provide trees in Newick format as out- put. In order to visualize such a tree, one can use the web-based viewer phylo.io (http://phylo.io) or other tree Page 9 of 25 F1000Research 2022, 9:511 Last updated: 12 MAR 2024 F1000Research 2022, 9:511 Last updated: 12 MAR 2024 visualization tools (e.g. FigTree, phylogeny.io, etc). Displaying bootstrap values for internal nodes is recommended to evaluate the confidence of the inferred tree topology. In our examples, we inferred trees by aligning the sequences with MAFFT, concatenated the alignments using the aforementioned concat_alignments.py, and ran both IQ-TREE and RAxML (Figure 3 and Figure 4). The data used for and the results from the computations can be found on FigShare17 (alignments in the “data/Alignments” folder, and trees in the “tree” folder). The exact code used for these examples is on21. Discussion and conclusion With the wealth of genomic data available in an era of high-throughput sequencing, there is much to gain by making phylogenies from concatenations of multiple genes rather than from one single gene. This can bet- ter represent the evolutionary history of a clade, because the evolutionary history of a single gene can be Figure 3. Comparison of phylogenetic trees computed by IQ-TREE, using an LG substitution model (left), and RAxML, using an LG substitution model, a discrete Gamma model of rate heterogeneity with 8 categories, and empirical amino-acid frequencies (right). Trees were computed with 20 yeast species present in OMA. The leaves of the trees are the UniProt 5-letter species codes. The following export options were used: Minimum species coverage: 1, Maximum nr of markers: -1 (uncapped). 168 marker genes were exported. Visualization was done with phylo.io; different shades of blue show variations in topology. Bootstrap values are reported in red for each bipartition with a bootstrap <100 Table 2. Recommended software and example commands for computing a phylogenetic tree. Parameters, such as memory or threads, may vary based on size of dataset. Software for making phylogenetic trees Example command IQ-Tree iqtree -s alignment.phy -m LG -T 20 --mem 20G -seed 12345 -bb 1000 RaML raxml-ng --threads 20 --model LG+G8+F --seed 15826 --msa alignment.phy --all --bs-trees 100 PhyloBayes pb_mpi -dc -gtr -cat -dgam 4 -x 10 1000 -d alignment.phy alignment.chain1 Table 2. Recommended software and example commands for computing a phylogenetic tree. Parameters, such as memory or threads, may vary based on size of dataset. Table 2. Recommended software and example commands for computing a phylogenetic tree. Parameters, such as memory or threads, may vary based on size of dataset. Software for making phylogenetic trees Example command IQ-Tree iqtree -s alignment.phy -m LG -T 20 --mem 20G -seed 12345 -bb 1000 RaML raxml-ng --threads 20 --model LG+G8+F --seed 15826 --msa alignment.phy --all --bs-trees 100 PhyloBayes pb_mpi -dc -gtr -cat -dgam 4 -x 10 1000 -d alignment.phy alignment.chain1 Figure 3. Comparison of phylogenetic trees computed by IQ-TREE, using an LG substitution model (left), and RAxML, using an LG substitution model, a discrete Gamma model of rate heterogeneity with 8 categories, and empirical amino-acid frequencies (right). Trees were computed with 20 yeast species present in OMA. The leaves of the trees are the UniProt 5-letter species codes. Discussion and conclusion Comparison of phylogenetic trees, using additional species, computed by IQ-TREE, under a LG substitution model (left), and RAxML, under a LG substitution model, a discrete Gamma model of rate heterogeneity with 8 categories and empirical amino-acid frequencies (right). Trees were computed with 18 yeast species present in OMA, plus two additional proteomes (YEAST and FOMPI). The leaves of the trees are the UniProt 5-letter species codes. Genes used to compute the tree had to be shared by at least 90% of the species (minimum species coverage: 0.9, maximum number markers: -1). This represents 880 OGs. Visualization was done with phylo.io; different shades of blue show variations in topology (in this case both trees have identical topology). Bootstrap values are reported in red for each bipartition with a bootstrap <100. misrepresentative of a species evolutionary history. The principle of a supermatrix approach is that by combin- ing multiple genes in one single phylogeny, one can combine the phylogenetic signal of multiple genes. One has to be careful however, to not combine “phylogenetic noise”. Orthologs selection is particularly impor- tant in this regard4,23, because errors in orthology inference could add genes that are not true orthologs, but rather paralogs descending from the ancestral genes by a duplication event. Thus, they would have a different evolutionary history than the sought species phylogeny. misrepresentative of a species evolutionary history. The principle of a supermatrix approach is that by combin- ing multiple genes in one single phylogeny, one can combine the phylogenetic signal of multiple genes. One has to be careful however, to not combine “phylogenetic noise”. Orthologs selection is particularly impor- tant in this regard4,23, because errors in orthology inference could add genes that are not true orthologs, but rather paralogs descending from the ancestral genes by a duplication event. Thus, they would have a different evolutionary history than the sought species phylogeny. OMA Groups (or Orthologous Groups) are a well-suited set of orthologs for this kind of analysis, as the cri- teria used to compute these orthologs are stringent. They require that all genes are reciprocally closest genes in their respective species to all the other genes of the group and do not allow more than one gene in a species, thus excluding paralogs. In the Quest for Orthologs Benchmark14, the community benchmark for orthology infer- ence, OMA Groups are consistently the most specific inference, although lacking in recall. Discussion and conclusion The following export options were used: Minimum species coverage: 1, Maximum nr of markers: -1 (uncapped). 168 marker genes were exported. Visualization was done with phylo.io; different shades of blue show variations in topology. Bootstrap values are reported in red for each bipartition Figure 3. Comparison of phylogenetic trees computed by IQ-TREE, using an LG substitution model (left), and RAxML, using an LG substitution model, a discrete Gamma model of rate heterogeneity with 8 categories, and empirical amino-acid frequencies (right). Trees were computed with 20 yeast species present in OMA. The leaves of the trees are the UniProt 5-letter species codes. The following export options were used: Minimum species coverage: 1, Maximum nr of markers: -1 (uncapped). 168 marker genes were exported. Visualization was done with phylo.io; different shades of blue show variations in topology. Bootstrap values are reported in red for each bipartition with a bootstrap <100. Figure 3. Comparison of phylogenetic trees computed by IQ-TREE, using an LG substitution model (left), and RAxML, using an LG substitution model, a discrete Gamma model of rate heterogeneity with 8 categories, and empirical amino-acid frequencies (right). Trees were computed with 20 yeast species present in OMA. The leaves of the trees are the UniProt 5-letter species codes. The following export options were used: Minimum species coverage: 1, Maximum nr of markers: -1 (uncapped). 168 marker genes were exported. Visualization was done with phylo.io; different shades of blue show variations in topology. Bootstrap values are reported in red for each bipartition with a bootstrap <100. Page 10 of 25 F1000Research 2022, 9:511 Last updated: 12 MAR 2024 Figure 4. Comparison of phylogenetic trees, using additional species, computed by IQ-TREE, under a LG substitution model (left), and RAxML, under a LG substitution model, a discrete Gamma model of rate heterogeneity with 8 categories and empirical amino-acid frequencies (right). Trees were computed with 18 yeast species present in OMA, plus two additional proteomes (YEAST and FOMPI). The leaves of the trees are the UniProt 5-letter species codes. Genes used to compute the tree had to be shared by at least 90% of the species (minimum species coverage: 0.9, maximum number markers: -1). This represents 880 OGs. Visualization was done with phylo.io; different shades of blue show variations in topology (in this case both trees have identical topology). Bootstrap values are reported in red for each bipartition with a bootstrap <100. Figure 4. Discussion and conclusion As potentially missing genes are less detrimental to phylogenetic determination than false predictions are24, this is an appro- priate choice of orthology inference method for this tutorial. Several phylogenies have already been published using OMA standalone or data from the OMA Browser, including those for archaea, sharks, spiders, worms, and insects, among others25–29. This tutorial demonstrated how to carry out these different steps to infer a phylogenetic tree: orthology determina- tion, sequence alignments, supermatrix construction, and phylogeny inference. It is designed to allow users to leverage the state of the art orthology inference provided by OMA Groups while reducing the necessary compu- tation from their side, namely by relying on precomputed all-against-all alignments provided by the OMA Browser. We include code snippets and scripts that automate the whole process, and ensure reproducibility of all phylogenetic analyses following this protocol. The tutorial is accompanied by practical examples with all data available on GitHub and Figshare. Page 11 of 25 F1000Research 2022, 9:511 Last updated: 12 MAR 2024 This tutorial is designed to help users generate a species tree phylogeny on reliable data, by relying on the least amount of computation. Nevertheless, we advise care in interpretation of the obtained species tree. In particular, even with accurate selection of orthologs, non-phylogenetic noise may persist in the data, making some branches hard to resolve. It is exemplified in this tutorial by a few differences between the species tree produced by the two different protocols, likely due to difference in the number of genes used. To avoid misinterpretation of the data, it is wise to compute and report measures of bipartition consistency, like bootstrap support values30, while generating a species tree. A low bootstrap value will flag bipartitions that are subject to phylogenetic noise and that cannot be asserted with confidence. In our examples, bipartitions that differ between protocols have relatively low bootstrap values in the species tree. For more information about the theory behind phylogenomics and the different methods, we refer the reader to recent reviews31–33. In the context of this tutorial, we used well-established MSA and phylogenetic tree infer- ence tools. For the more difficult cases however, it advised to carefully choose which tool to use, including some tools which are not mentioned here. For more information about existing tools the readers are invited to turn to the relevant literature32,34. Discussion and conclusion The protocols described here can be adapted to suit any other software compatible with standard data formats. Software availability OMA Browser available at: https://omabrowser.org/. Source code for OMA Standalone available from: https://github.com/DessimozLab/OmaStandalone/tree/v2.4.0 Archived source code of OMA StandAlone at time of publication: https://doi.org/10.5281/zenodo.355559513. OMA Browser license: Mozilla Public License version 2. Acknowledgments We would like to thank Natalia Zajac, Marion Brechet, Katharina Pfaller, and the reviewers Jianbo Xie and Denis Baurain for their useful feedback on the tutorial. Data availability The imported OG data and the OMA standalone software can be obtained from the OMA Browser (https://omabrowser.org), following instructions in this tutorial. Figshare: Phylogenetic Tree Tutorial Example Data, https://doi.org/10.6084/m9.figshare.10780820.v617 Data are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication). Additional python scripts (filter_groups.py and concat_alignments.py) are publicly available: https://github.com/ DessimozLab/f1000_PhylogeneticTree Archived scripts as at time of publication: https://zenodo.org/record/6037516#.YgVAju6ZP0s21 License for scripts: MIT license 2. Lane DJ, Pace B, Olsen GJ, et al.: Rapid Determination of 16S ribosomal RNA Sequences for Phylogenetic Analyses. Proc Natl References 1. Hinchliff CE, Smith SA, Allman JF, et al.: Synthesis of Phylogeny and Taxonomy into a Comprehensive Tree of Life. Proc Natl Acad Sci U S A. 2015; 112(41): 12764–9. PubMed Abstract \| Publisher Full Text \| Free Full Text 2. Lane DJ, Pace B, Olsen GJ, et al.: Rapid Determination of 16S ribosomal RNA Sequences for Phylogenetic Analyses. Proc Natl Acad Sci U S A. 1985; 82(20): 6955–9. PubMed Abstract \| Publisher Full Text \| Free Full Text 3. Maddison WP: Gene Trees in Species Trees. Syst Biol. 1997; 46(3): 523–36. Publisher Full Text 4. Philippe H, Brinkmann H, Lavrov DV, et al.: Resolving Difficult Page 12 of 25 Page 12 of 25 Page 12 of 25 F1000Research 2022, 9:511 Last updated: 12 MAR 2024 5. Philippe H, de Vienne DM, Ranwez V, et al.: Pitfalls in supermatrix phylogenomics. EJT. 2017; (283). Publisher Full Text 22. Katoh K, Standley DM: MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol. 2013; 30(4): 772–80. PubMed Abstract \| Publisher Full Text \| Free Full Text 6. Gadagkar SR, Rosenberg MS, Kumar S: Inferring Species Phylogenies From Multiple Genes: Concatenated Sequence Tree Versus Consensus Gene Tree. J Exp Zool B Mol Dev Evol. 2005; 304(1): 64–74. 23. Fernández R, Gabaldon T, Dessimoz C: Orthology: Definitions, prediction, and impact on species phylogeny inference. Phylogenetics in the Genomic Era. 2020; 2–4. Reference Source ( ) PubMed Abstract \| Publisher Full Text 7. Hug LA, Baker BJ, Anantharaman K, et al.: A New View of the Tree of Life. Nat Microbiol. 2016; 1: 16048. PubMed Abstract \| Publisher Full Text 24. Baurain D, Philippe H: Current Approaches to Phylogenomic Reconstruction. In: Caetano-Anollés G, editor. Evolutionary Genomics and Systems Biology. Hoboken, NJ, USA: John Wiley & Sons, Inc.; 2010; 17–41. Publisher Full Text 8. Fitch WM: Distinguishing homologous from analogous proteins. Syst Zool. 1970; 19(2): 99–113. PubMed Abstract \| Publisher Full Text 9. Altenhoff AM, Glover NM, Dessimoz C: Inferring Orthology and Paralogy. Methods Mol Biol. In Anisimova M, editor. Evolutionary Genomics: Statistical and Computational Methods. New York, NY: Springer New York; 2019; 1910: 149–75. PubMed Abstract \| Publisher Full Text 25. Williams TA, Szöllősi GJ, Spang A, et al.: Integrative modeling of gene and genome evolution roots the archaeal tree of life. Proc Natl Acad Sci U S A. 2017; 114(23): E4602–11. PubMed Abstract \| Publisher Full Text \| Free Full Text p g PubMed Abstract \| Publisher Full Text 10. Zahn-Zabal M, Dessimoz C, Glover NM: Identifying orthologs with OMA: A primer [version 1; peer review: 2 approved]. F1000Res. 2020; 9(27): 27. PubMed Abstract \| Publisher Full Text \| Free Full Text 26. Hara Y, Yamaguchi K, Onimaru K, et al.: Shark genomes provide insights into elasmobranch evolution and the origin of vertebrates. Nat Ecol Evol. 2018; 2(11): 1761–71. PubMed Abstract \| Publisher Full Text 11. Simão FA, Waterhouse RM, Ioannidis P, et al.: BUSCO: Assessing Genome Assembly and Annotation Completeness With Single- Copy Orthologs. Bioinformatics. 2015; 31(19): 3210–2. PubMed Abstract \| Publisher Full Text 27. Wood HM, González VL, Lloyd M, et al.: Next-generation museum genomics: Phylogenetic relationships among palpimanoid spiders using sequence capture techniques (Araneae: Palpimanoidea). Mol Phylogenet Evol. 2018; 127: 907–18. PubMed Abstract \| Publisher Full Text 12. Altenhoff AM, Levy J, Zarowiecki M, et al.: OMA Standalone: Orthology Inference Among Public and Custom Genomes and Transcriptomes. Genome Res. 2019; 29(7): 1152–63. PubMed Abstract \| Publisher Full Text \| Free Full Text 28. Philippe H, Poustka AJ, Chiodin M, et al.: Mitigating Anticipated Effects of Systematic Errors Supports Sister-Group Relationship between Xenacoelomorpha and Ambulacraria. Curr Biol. 2019; 29(11): 1818–26.e6. PubMed Abstract \| Publisher Full Text 13. Altenhoff AM, Glover NM, Train CM, et al.: The OMA orthology database in 2018: retrieving evolutionary relationships among all domains of life through richer web and programmatic interfaces. Nucleic Acids Res. 2018; 46(D1): D477–85. PubMed Abstract \| Publisher Full Text \| Free Full Text 29. Dikow RB, Frandsen PB, Turcatel M, et al.: Genomic and transcriptomic resources for assassin flies including the complete genome sequence of Proctacanthus coquilletti (Insecta: Diptera: Asilidae) and 16 representative transcriptomes. PeerJ. 2017; 5: e2951. PubMed Abstract \| Publisher Full Text \| Free Full Text 14. Altenhoff AM, Boeckmann B, Capella-Gutierrez S, et al.: Standardized benchmarking in the quest for orthologs. Nat Methods. 2016; 13(5): 425–30. PubMed Abstract \| Publisher Full Text \| Free Full Text 30. Felsenstein J: CONFIDENCE LIMITS ON PHYLOGENIES: AN APPROACH USING THE BOOTSTRAP. Evolution. 1985; 39(4): 783–91. PubMed Abstract \| Publisher Full Text 15. Zhu Q, Mai U, Pfeiffer W, et al.: Phylogenomics of 10,575 genomes reveals evolutionary proximity between domains Bacteria and Archaea. Nat Commun. 2019; 10(1): 5477. PubMed Abstract \| Publisher Full Text \| Free Full Text f Competing Interests: No competing interests were disclosed. Reviewer Expertise: Phylogenomics, comparative genomics, software engineering PubMed Abstract \| Publisher Full Text 31. Yang Z, Rannala B: Molecular phylogenetics: principles and practice. Nat Rev Genet. 2012; 13(5): 303–14. PubMed Abstract \| Publisher Full Text 16. Altenhoff AM, Train CM, Gilbert KJ, et al.: OMA orthology in 2021: website overhaul, conserved isoforms, ancestral gene order and more. Nucleic Acids Res. 2021; 49(D1): D373–9. PubMed Abstract \| Publisher Full Text \| Free Full Text 32. Patané JSL, Martins J Jr, Setubal JC: Phylogenomics. Methods Mol Biol. 2018; 1704: 103–87. PubMed Abstract \| Publisher Full Text 17. Nevers Y: Phylogenetic Tree Tutorial Example Data. 2022. https://figshare.com/articles/dataset/Example_Data/10780820/6 33. Simion P, Delsuc F, Philippe H: To What Extent Current Limits of Phylogenomics Can Be Overcome? No commercial publisher\| Authors open access book; 2020. Reference Source 18. Glover N: OMA standalone cheat sheet. 2021; [cited 2022 Feb 8]. Publisher Full Text 19. Robinson O, Dylus D, Dessimoz C: Phylo.io: Interactive Viewing and Comparison of Large Phylogenetic Trees on the Web. Mol Biol Evol. 2016; 33(8): 2163–6. PubMed Abstract \| Publisher Full Text \| Free Full Text 34. Scornavacca C, Delsuc F, Galtier N: Phylogenetics in the Genomic Era. No commercial publisher\| Authors open access book; 2020. Reference Source 20. Roth AC, Gonnet GH, Dessimoz C: Algorithm of OMA for Page 13 of 25 Open Peer Review Current Peer Review Status: I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Reviewer Report 14 March 2022 https://doi.org/10.5256/f1000research.121232.r125602 Version 2 BioS - PhytoSYSTEMS, Eukaryotic Phylogenomics, University of Liège, Liège, Belgium Thank you for the revised version, which adequately addresses all my comments. I apologize for the delay in endorsing it. Thank you for the revised version, which adequately addresses all my comments. I apologize for the delay in endorsing it. ompeting Interests: No competing interests were disclosed. F1000Research 2022, 9:511 Last updated: 12 MAR 2024 Open Peer Review https://doi.org/10.5256/f1000research.121232.r125602 © 2022 Xie J. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Jianbo Xie National Engineering Laboratory for Tree Breeding, College of Biological Sciences and Technology, Beijing Forestry University, Beijing, China All my concerns have been solved. Competing Interests: No competing interests were disclosed. Reviewer Expertise: Genetics; evolution All my concerns have been solved. Competing Interests: No competing interests were disclosed. Reviewer Expertise: Genetics; evolution Page 14 of 25 F1000Research 2022, 9:511 Last updated: 12 MAR 2024 I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. https://doi.org/10.5256/f1000research.26251.r84387 "using species in OMA in addition to other genomes not available in the database": It is not clear if OMA Standalone requires conceptual translations of the sequences (i.e., proteins) or if it can translate DNA sequences on the fly; if it cannot, please specify that one needs Page 15 of 25 F1000Research 2022, 9:511 Last updated: 12 MAR 2024 protein sequences and not genomes to make use of OMA. "phylogenetic tree inference softwares": I am not a native English speaker, but "software" is uncountable; maybe you mean "software packages"? ○ IQtree, RaxML, Phylobayes, Figtree etc: Please try to use the correct mixture of uppercase and lowercase letters in each name across the manuscript: IQ-TREE, RAxML, PhyloBayes, FigTree. ○ A better download address for PhyloBayes would be: http://www.atgc- montpellier.fr/phylobayes/. ○ The interface of OMA Browser has slightly changed since the manuscript has been written. Two details that would need updating: "in the upper left of the home screen" is incorrect [for "Explore"] and "Compute" has now become "Download". ○ The interface of OMA Browser has slightly changed since the manuscript has been written. Two details that would need updating: "in the upper left of the home screen" is incorrect [for "Explore"] and "Compute" has now become "Download". ○ "To speed up the tree inference, set this value to below 1000 genes": One wonders how these <1000 genes would be prioritized and selected; this should be mentioned here. ○ "The name of the fasta file is either the species name or the 5-letter UniProt identifier...": This is not specific enough; what about whitespace, dots in strain names, etc? Are they allowed? Similarly, you should better define "other special characters" just below. "The name of the fasta file is either the species name or the 5-letter UniProt identifier...": This is not specific enough; what about whitespace, dots in strain names, etc? Are they allowed? Similarly, you should better define "other special characters" just below. "...and not keeping other unrelated orthology inferences": This is a bit mysterious. I am aware that OMA can do more than what you demonstrate here, but this sentence should probably be slightly expanded with some examples. "...use the job-array syntax in the submission script": This is the part where reproducibility is not straightforward. In particular, is there a master OMA instance controlling the parallelization or is this left to the user? https://doi.org/10.5256/f1000research.26251.r84387 © 2021 Baurain D. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Denis Baurain 1 InBioS - PhytoSYSTEMS, Eukaryotic Phylogenomics, University of Liège, Liège, Belgium 2 InBioS - PhytoSYSTEMS, Eukaryotic Phylogenomics, University of Liège, Liège, Belgium This tutorial illustrates how to use the online OMA Browser and OMA Standalone package to assemble datasets of single-copy orthologous genes for phylogenomic inference. It covers two use cases: 1) all species of interest are present in the OMA Browser and 2) some species are not available and must thus be added by the user using OMA Standalone. Generally speaking, the structure and writing are clear, the figures and tables are appropriate, and the data and code underlying the examples are well described and made available in two public repositories (Figshare and Zenodo). Therefore, I do not have major comments. However, I think that it is possible to easily improve a number of minor points in the manuscript. I provide a list below, simply sorted by order of occurrence. There are no line numbers, so I try to provide some context when needed. "16 rRNA ribosomal genes" should rather be "small subunit ribosomal RNA (SSU rRNA 16S/18S) gene" (no plural) to encompass the eukaryotic gene as well. ○ "see 3 for common pitfalls in phylogenomics": Don't you mean Philippe et al. (2017) "Pitfalls in supermatrix phylogenomics1" instead? ○ "see 3 for common pitfalls in phylogenomics": Don't you mean Philippe et al. (2017) "Pitfalls in supermatrix phylogenomics1" instead? ○ "OGs can have at most one representative gene per species": You should probably say a word about recently diverged in-paralogs (which do not harm species phylogenies) because exactly single-copy genes barely exist. ○ "among more than 2300 genomes across the tree of life": You might provide a few numbers here: bacteria, archaea, non-animals, non-plants - or at least recall the rationale for genome inclusion in OMA Browser because it is quite incomplete in terms of species diversity. https://doi.org/10.5256/f1000research.26251.r84387 In other words, it is a pity that this use case does not provide a clear phylogenetic solution for the selected genomes. ○ "in this case both trees have identical topology" [Figure 4]: This is correct, but this topology is widely different from the two topologies of Figure 3. This shows that phylogenetic artefacts are at play (which is well known for yeasts...) and this might worth mentioning in the sentences about the caveats of phylogenomics. In other words, it is a pity that this use case does not provide a clear phylogenetic solution for the selected genomes. ○ https://doi.org/10.5256/f1000research.26251.r84387 From the code snippet provided, it is not clear, albeit the output of the oma-status command suggests that there is some orchestrator freeing the user from this chore. Please explain a bit more. "we selected 880 OGs with at least 18 species as a dataset": Considering Figure 4, I understand your point: 90% of 20 species is 18. However, since you start with 18 genomes and add two "new" ones, the re-use of the number "18" is confusing to the user. As the analyses are done, I would not suggest to change this number, but rather to explain directly the 90% rationale in the text. "This command generates a MSA file (.aln) for each OG.": This "for" loop is a very fine candidate for parallelization. Please remind the user that they could use a job array for this. Otherwise, they may wait a lot of time staring at the shell. "there is much to gain by making phylogenies from multiple gene families": Here, I am strongly opposed to the words "gene families". The whole point of OMA is to identify single- copy (orthologous) genes, not gene families (i.e., including paralogues). Please use instead "from concatenations of multiple genes, rather than from one single gene" (or something similar). Page 16 of 25 F1000Research 2022, 9:511 Last updated: 12 MAR 2024 "because the evolutionary history of a single gene can be misrepresentative of a species evolutionary history": This is said twice in the manuscript (and this is true) but not backed up by specific reasons. Please enumerate a few of them between parentheses. ○ In the legend of Figure 3 and 4, please provide the evolutionary model used. I know it is in Table 2, but somewhat hidden in the command line arguments of the software packages. ○ In the legend of Figure 3 and 4, please provide the evolutionary model used. I know it is in Table 2, but somewhat hidden in the command line arguments of the software packages. ○ "in this case both trees have identical topology" [Figure 4]: This is correct, but this topology is widely different from the two topologies of Figure 3. This shows that phylogenetic artefacts are at play (which is well known for yeasts...) and this might worth mentioning in the sentences about the caveats of phylogenomics. Is the description of the method technically sound? Yes Are sufficient details provided to allow replication of the method development and its use by others? Partly If any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes Competing Interests: No competing interests were disclosed. Reviewer Expertise: Phylogenomics, comparative genomics, software engineering I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. References 1. Philippe H, Vienne D, Ranwez V, Roure B, et al.: Pitfalls in supermatrix phylogenomics. European Journal of Taxonomy. 2017. Publisher Full Text Are sufficient details provided to allow replication of the method development and its use by others? Partly Partly If any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions about the method and its performance adequately supported by the Are the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes Competing Interests: No competing interests were disclosed. Reviewer Expertise: Phylogenomics, comparative genomics, software engineering I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Author Response 10 Feb 2022 Page 17 of 25 F1000Research 2022, 9:511 Last updated: 12 MAR 2024 Done. 2. "see 3 for common pitfalls in phylogenomics": Don't you mean Philippe et al. (2017) "Pitfalls in supermatrix phylogenomics1" instead? he previous reference addressed most of the same issues as the one the reviewe uggests, but the latter is more recent and comprehensive. We now cite both. Please note that OMA Standalone can work on nucleic coding sequences when starting from scratch, however for compatibility with the pre-computed OMA data, only protein sequences may be used when combining new and exported data. ” 6. "phylogenetic tree inference softwares": I am not a native English speaker, but "software" is uncountable; maybe you mean "software packages"? he previous reference addressed most of the same issues as the one the reviewe uggests, but the latter is more recent and comprehensive. We now cite both. Please note that OMA Standalone can work on nucleic coding sequences when starting from scratch, however for compatibility with the pre-computed OMA data, only protein sequence may be used when combining new and exported data. ” 6. "phylogenetic tree inference softwares": I am not a native English speaker, but "software" is uncountable; maybe you mean "software packages"? We corrected this error, following the reviewer's suggestion. 7. IQtree, RaxML, Phylobayes, Figtree etc: Please try to use the correct mixture of uppercase an lowercase letters in each name across the manuscript: IQ-TREE, RAxML, PhyloBayes, FigTree. We thank the reviewer for pointing out this oversight. Now all the mentioned software names have been corrected. 8. A better download address for PhyloBayes would be: http://www.atgc- montpellier.fr/phylobayes/. Thanks, corrected. 9. The interface of OMA Browser has slightly changed since the manuscript has been written. T details that would need updating: "in the upper left of the home screen" is incorrect [for "Explore"] and "Compute" has now become "Download". We changed the text to be adapted to the latest version of the browser. We update Figure 1 as well to reflect the new menus and design. 10. "To speed up the tree inference, set this value to below 1000 genes": One wonders how the <1000 genes would be prioritized and selected; this should be mentioned here. When the number of markers is limited, Orthologous Groups are selected in decreasing order of species representation. This ensures the genes with representatives in most of the selected species will be prioritized. We adapted the t to reflect this. 11. "The name of the fasta file is either the species name or the 5-letter UniProt identifier...": Th is not specific enough; what about whitespace, dots in strain names, etc? Are they allowed? Similarly, you should better define "other special characters" just below. We agree, this could be clarified. We now changed the text to say: “The added genomes data must fulfill certain conditions: -The name of the fasta file should identify the species clearly and uniquely. The data” to read: “For this procedure, the added genomes data must fulfil certain conditions: Each additional dataset is in the form of a fasta file, containing protein sequences of all coding genes in the corresponding genome. 8. A better download address for PhyloBayes would be: http://www.atgc- montpellier.fr/phylobayes/. 8. A better download address for PhyloBayes would be: http://www.atgc- montpellier.fr/phylobayes/. We corrected this error, following the reviewer's suggestion. 7. IQtree, RaxML, Phylobayes, Figtree etc: Please try to use the correct mixture of uppercase and lowercase letters in each name across the manuscript: IQ-TREE, RAxML, PhyloBayes, FigTree. We thank the reviewer for pointing out this oversight. Now all the mentioned software names have been corrected. 8. A better download address for PhyloBayes would be: http://www.atgc- montpellier.fr/phylobayes/. he previous reference addressed most of the same issues as the one the reviewe uggests, but the latter is more recent and comprehensive. We now cite both. 3. "OGs can have at most one representative gene per species": You should probably say a word about recently diverged in-paralogs (which do not harm species phylogenies) because exactly single-copy genes barely exist. We have now updated the text to describe how in-paralogs are handled: “If recently diverged in-paralogs are inferred (i.e., co-orthologs), only one of the copies will be selected for the OG. Thus, all members of the OG are still orthologous to each other.” 4. "among more than 2300 genomes across the tree of life": You might provide a few numbers here: bacteria, archaea, non-animals, non-plants - or at least recall the rationale for genome inclusion in OMA Browser because it is quite incomplete in terms of species diversity. We added more details on species selection and species breakdown on major clades. We also updated the numbers to be in line with the latest release of OMA: “...provides orthology information among more than 2400 genomes across the tree of life, selected to maximize taxon coverage and users’ needs (Altenhoff et al. 2021); to date, there are 1710 Bacteria, 153 Archaea, and 561 Eukaryotes.” 5. "using species in OMA in addition to other genomes not available in the database": It is not clear if OMA Standalone requires conceptual translations of the sequences (i.e., proteins) or if it can translate DNA sequences on the fly; if it cannot, please specify that one needs protein sequences and not genomes to make use of OMA. We added precision on the input format specification for Protocol 2. Since it needs to be compatible with precomputed OMA data, only protein sequence may be used for this Protocol. We updated the text at the end of the Introduction to say “using species in OMA in addition to other proteomes not available in the database, e.g. a proteome obtained from sequencing a new species. By proteome we mean all protein or nucleic acid sequences of protein-coding genes annotated in a genome.” We also changed the later text in “Combining the added genomes with exported OMA Page 18 of 25 F1000Research 2022, 9:511 Last updated: 12 MAR 2024 data” to read: “For this procedure, the added genomes data must fulfil certain conditions: Each additional dataset is in the form of a fasta file, containing protein sequences of all coding genes in the corresponding genome. Thanks, corrected. 9. The interface of OMA Browser has slightly changed since the manuscript has been written. Two details that would need updating: "in the upper left of the home screen" is incorrect [for "Explore"] and "Compute" has now become "Download". Page 19 of 25 F1000Research 2022, 9:511 Last updated: 12 MAR 2024 exported genomes from OMA use for example UniProt’s mnemonic five-letter species codes. The filename must end with a “.fa” suffix and must not contain any whitespace characters. The filename without the “.fa” suffix is used as the species name throughout the process and result files. -Each sequence in the fasta file has a clear and unique identifier. We suggest not to use special characters such as brackets, dots, or a pipe character. The reason is that many programs use them for special purposes, e.g. brackets are used in the newick format for tree representation, and the pipe character is often used to separate ids and annotations.” 12. "...and not keeping other unrelated orthology inferences": This is a bit mysterious. I am aware that OMA can do more than what you demonstrate here, but this sentence should probably be slightly expanded with some examples. We added more details concerning Hierarchical Orthologous Group, which is the main thing we referred to here. We changed the text to say: “If the goal is to only generate a dataset for species phylogeny inference (and not to keep other unrelated orthology inferences, such as Hierarchical Orthologous Groups (Zahn-Zabal et al. 2020), which are better representation of individual genes’ evolutionary histories but take time to compute), one can avoid doing computations and generating output files that are not needed by the following:...” 13. "...use the job-array syntax in the submission script": This is the part where reproducibility is not straightforward. In particular, is there a master OMA instance controlling the parallelization or is this left to the user? From the code snippet provided, it is not clear, albeit the output of the oma-status command suggests that there is some orchestrator freeing the user from this chore. Please explain a bit more. We try to make this more clear by now saying: “OMA Standalone automatically partitions the work chunks in a static and deterministic way among the specified number of workers. Progress of the entire computation can be checked with the OMA Status command (see below).” We try to make this more clear by now saying: “OMA Standalone automatically partitions the work chunks in a static and deterministic way among the specified number of workers. Progress of the entire computation can be checked with the OMA Status command (see below).” 14. "we selected 880 OGs with at least 18 species as a dataset": Considering Figure 4, I understand your point: 90% of 20 species is 18. However, since you start with 18 genomes and add two "new" ones, the re-use of the number "18" is confusing to the user. As the analyses are done, I would not suggest to change this number, but rather to explain directly the 90% rationale in the text. We reformulated a bit to make the reasoning more explicit. We now say: “Once the computation finished, we selected 880 OGs that included at least 90% of the 20 species --18 -- as a dataset to construct a tree.” 15. "This command generates a MSA file (.aln) for each OG.": This "for" loop is a very fine candidate for parallelization. Please remind the user that they could use a job array for this. Otherwise, they may wait a lot of time staring at the shell. Page 20 of 25 F1000Research 2022, 9:511 Last updated: 12 MAR 2024 We now add a reminder that using job arrays is a recommended solution with big datasets. We changed the text to say: “This command sequentially generates a MSA file (.aln) for each OG. Depending on the number of OG and species in your dataset, executing it may take a prohibitive amount of time. If it is the case, we recommend using job-arrays to execute the alignments in parallel.” 16. "there is much to gain by making phylogenies from multiple gene families": Here, I am strongly opposed to the words "gene families". The whole point of OMA is to identify single-copy (orthologous) genes, not gene families (i.e., including paralogues). Please use instead "from concatenations of multiple genes, rather than from one single gene" (or something similar). We agree this phrase is misleading because even if OMA also provides orthology at the level of whole gene families (HOGs) we are using OGs here - single-copy orthologs. We changed the text as suggested by the reviewer. 17. We try to make this more clear by now saying: “OMA Standalone automatically partitions the work chunks in a static and deterministic way among the specified number of workers. Progress of the entire computation can be checked with the OMA Status command (see below).” "because the evolutionary history of a single gene can be misrepresentative of a species evolutionary history": This is said twice in the manuscript (and this is true) but not backed up by specific reasons. Please enumerate a few of them between parentheses. We made two major change to address this comment: We changed Figures 3 and 4 to show the least statistically bipartitions in the species tree. ○ https://doi.org/10.5256/f1000research.26251.r84386 © 2021 Xie J. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Jianbo Xie J 1 National Engineering Laboratory for Tree Breeding, College of Biological Sciences and Technology, Beijing Forestry University, Beijing, China 2 National Engineering Laboratory for Tree Breeding, College of Biological Sciences and Technology, Beijing Forestry University, Beijing, China J 1 National Engineering Laboratory for Tree Breeding, College of Biological Sciences and Technology, Beijing Forestry University, Beijing, China 2 National Engineering Laboratory for Tree Breeding, College of Biological Sciences and Technology, Beijing Forestry University, Beijing, China 1 National Engineering Laboratory for Tree Breeding, College of Biological Sciences and Technology, Beijing Forestry University, Beijing, China 2 National Engineering Laboratory for Tree Breeding, College of Biological Sciences and Technology, Beijing Forestry University, Beijing, China The manuscript provides useful tools to make use of OMA Orthologous Groups to infer a phylogenetic species tree. This manuscript is an interesting study, but I found some shortcomings that the authors should be improved before the manuscript can be considered for indexing. Some marker gene databases, such as Qiyun Zhu et al. (2019)1, have been published - could you integrate the database into your tool? Or describe the advantage of your tool? We added a paragraph in the discussion pointing out the difference between the species tree and discussing the importance of accounting for statistical support when constructing a species phylogeny. ○ Competing Interests: No competing interests were disclosed. Reviewer Report 25 May 2021 We now enumerate some reasons and link to a reference on the first time this is mentioned, in the introduction. 18. In the legend of Figure 3 and 4, please provide the evolutionary model used. I know it is in Table 2, but somewhat hidden in the command line arguments of the software packages. We now provide the evolutionary models used in both figures. The legends now say “Comparison of phylogenetic trees computed by IQ-TREE, using an LG substitution model (left), and RAxML, using an LG substitution model, a discrete Gamma model of rate heterogeneity with 8 categories, and empirical amino-acid frequencies (right)...” 19. "in this case both trees have identical topology" [Figure 4]: This is correct, but this topology is widely different from the two topologies of Figure 3. This shows that phylogenetic artefacts are at play (which is well known for yeasts...) and this might worth mentioning in the sentences about the caveats of phylogenomics. In other words, it is a pity that this use case does not provide a clear phylogenetic solution for the selected genomes. We thank the reviewer for pointing this out. It is true that there are some differences between the species tree obtained from the two protocols and that it must be addressed. The difference corresponds to bipartitions that are not well supported in either species tree but this was not easily apparent in the first version of the manuscript. We changed Figures 3 and 4 to show the least statistically bipartitions in the species tree. ○ Page 21 of 25 F1000Research 2022, 9:511 Last updated: 12 MAR 2024 We added a paragraph in the discussion pointing out the difference between the species tree and discussing the importance of accounting for statistical support when constructing a species phylogeny. ○ Minor: 1. The introduction section should include some similar tools such as PGAP, Orthomcl. 2. Please discuss the advantage of OMA compared with other tools. 3. Could you integrate the steps of analyses to one steps with multiple parameters? References 1. Zhu Q, Mai U, Pfeiffer W, Janssen S, et al.: Phylogenomics of 10,575 genomes reveals evolutionary proximity between domains Bacteria and Archaea. Nature Communications. 2019; 10 (1). Publisher Full Text Is the rationale for developing the new method (or application) clearly explained? Yes Is the rationale for developing the new method (or application) clearly explained? Yes Page 22 of 25 F1000Research 2022, 9:511 Last updated: 12 MAR 2024 Yes Yes Are sufficient details provided to allow replication of the method development and its use by others? Yes If any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes Competing Interests: No competing interests were disclosed. Reviewer Expertise: Genetics; evolution I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Author Response 10 Feb 2022 2. Please discuss the advantage of OMA compared with other tools. Please discuss the advantage of OMA compared with other tools. We added a line indicating the main advantage of OMA compared to other methods: it is possible to use a precomputed dataset, and also to mix it with local genomic data, which makes it usable in numerous contexts. We now state: “This type of Orthologous Group is provided by only a few orthology databases such as BUSCO (8) and Orthologous MAtrix (OMA) (Altenhoff et al. 2019; Altenhoff et al. 2018) and, to our knowledge, only OMA allows for use of both precomputed and user- computed OGs.” 3. Could you integrate the steps of analyses to one steps with multiple parameters? As the scope of the paper is to provide a tutorial, we do not plan on integrating the steps into only one command line script. We thank the reviewer for their comments, which are listed below (in italics) with our responses (in bold): We thank the reviewer for their comments, which are listed below (in italics) with our responses (in bold): The manuscript provides useful tools to make use of OMA Orthologous Groups to infer a phylogenetic species tree. This manuscript is an interesting study, but I found some shortcomings that the authors should be improved before the manuscript can be considered for indexing. Some marker gene databases, such as Qiyun Zhu et al. (2019)1, have been published - could you integrate the database into your tool? Or describe the advantage of your tool? We now mention the marker gene database as the reviewer suggests. This tutorial’s aim is mainly to indicate how to use OMA for inferring marker genes from orthology relationships and then construct a species tree. We do not think it is desirable to integrate other tools into our protocol. One of the clear advantages in using OMA is a clear procedure which can work for any subset of species. We now make it more clear in the text, and state: “It is sometimes possible to rely on existing marker genes used in large-scale studies (for example (Zhu et al. 2019)), but they are generally available only for a subset of species and do not include newly sequenced species.” Page 23 of 25 F1000Research 2022, 9:511 Last updated: 12 MAR 2024 1. The introduction section should include some similar tools such as PGAP, Orthomcl. In the introduction, we chose to mention only OMA and BUSCO, because they are to our knowledge the only maintained software that provides single-copy orthologs (does not include any paralogs). This is a prerequisite to use the supermatrix approach for species tree inference. As the scope of the paper is to provide a tutorial, we do not plan on integrating the steps into only one command line script. One of the main reasons to not integrate it into a single command line is to leave more choice to the user regarding the non-OMA tools they may use, as well as their parameters. Given the high number of possible parameters at any step, it would make designing and using such a script highly complicated. A second reason is that such an implementation will need to make technical choices (e.g regarding parallelization) which would make it incompatible with some computing infrastructures. Competing Interests: No competing interests were disclosed. Competing Interests: No competing interests were disclosed. Page 24 of 25 F1000Research 2022, 9:511 Last updated: 12 MAR 2024 The benefits of publishing with F1000Research: Your article is published within days, with no editorial bias • You can publish traditional articles, null/negative results, case reports, data notes and more • The peer review process is transparent and collaborative • Your article is indexed in PubMed after passing peer review • Dedicated customer support at every stage • For pre-submission enquiries, contact research@f1000.com The benefits of publishing with F1000Research: Your article is published within days, with no editorial bias • You can publish traditional articles, null/negative results, case reports, data notes and more • The peer review process is transparent and collaborative • Your article is indexed in PubMed after passing peer review • Dedicated customer support at every stage • For pre-submission enquiries, contact research@f1000.com The benefits of publishing with F1000Research: The benefits of publishing with F1000Research: Page 25 of 25
https://openalex.org/W3193029008	https://genomemedicine.biomedcentral.com/track/pdf/10.1186/s13073-021-00941-8	English	null	Using single-nucleus RNA-sequencing to interrogate transcriptomic profiles of archived human pancreatic islets	Genome medicine	2,021	cc-by	12,258	© The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. METHOD Open Access Giorgio Basile1†, Sevim Kahraman1†, Ercument Dirice1,2†, Hui Pan3, Jonathan M. Dreyfuss3 and Rohit N. Kulkarni1,4,5* Giorgio Basile1†, Sevim Kahraman1†, Ercument Dirice1,2†, Hui Pan3, Jonathan M. Dreyfuss3 and Rohit N. Kulkarni1,4,5* Rohit N. Kulkarni1,4,5* Abstract Background: Human pancreatic islets are a central focus of research in metabolic studies. Transcriptomics is frequently used to interrogate alterations in cultured human islet cells using single-cell RNA-sequencing (scRNA- seq). We introduce single-nucleus RNA-sequencing (snRNA-seq) as an alternative approach for investigating transplanted human islets. Methods: The Nuclei EZ protocol was used to obtain nuclear preparations from fresh and frozen human islet cells. Such preparations were first used to generate snRNA-seq datasets and compared to scRNA-seq output obtained from cells from the same donor. Finally, we employed snRNA-seq to obtain the transcriptomic profile of archived human islets engrafted in immunodeficient animals. Results: We observed virtually complete concordance in identifying cell types and gene proportions as well as a strong association of global and islet cell type gene signatures between scRNA-seq and snRNA-seq applied to fresh and frozen cultured or transplanted human islet samples. Conclusions: We propose snRNA-seq as a reliable strategy to probe transcriptomic profiles of freshly harvested or frozen sources of transplanted human islet cells especially when scRNA-seq is not ideal. Keywords: Transplanted human islets, Single-nucleus RNA-sequencing, Single-cell RNA-sequencing, Human β-cells, Frozen samples, Archived tissue, Diabetes Correspondence: Rohit.Kulkarni@joslin.harvard.edu †Giorgio Basile, Sevim Kahraman and Ercument Dirice contributed equally to this work. * Correspondence: Rohit.Kulkarni@joslin.harvard.edu †Giorgio Basile, Sevim Kahraman and Ercument Dirice contributed equally to this work. 1Section of Islet Cell and Regenerative Biology, Joslin Diabetes Center and Harvard Medical School, Boston, MA 02215, USA 4Department of Medicine, Brigham and Women’s Hospital, Boston, MA, USA Full list of author information is available at the end of the article Background transplantation are promising strategies to resolve dia- betes in patients. While the identification of factors able to stimulate β-cell proliferation [3–11] and the improve- ment of differentiation protocols to generate functional β-like cells [12–17] continue to evolve, gaining insights into dynamic changes in the global transcriptome of β- cells, especially following manipulation in an in vivo en- vironment (e.g., in transplanted islets in humanized mouse models) is worth exploring. Type 1 diabetes (T1D) and type 2 diabetes (T2D) are both characterized by a progressive reduction of func- tional mass of insulin-producing β-cells [1, 2]. Therefore, restoring physiological numbers of endogenous β-cells, improving β-cell functionality, or generating insulin- producing β-like cells derived from stem cells for One challenge when investigating the biology of pan- creatic islets is the presence of at least five different hormone-secreting endocrine cell types and non- endocrine cells such as endothelial cells, glia, fibroblasts, 1Section of Islet Cell and Regenerative Biology, Joslin Diabetes Center and Harvard Medical School, Boston, MA 02215, USA 4Department of Medicine, Brigham and Women’s Hospital, Boston, MA, USA Full list of author information is available at the end of the article Basile et al. Genome Medicine (2021) 13:128 https://doi.org/10.1186/s13073-021-00941-8 Basile et al. Genome Medicine (2021) 13:128 https://doi.org/10.1186/s13073-021-00941-8 Animal studies Healthy female 8–12-week-old non-obese diabetic (NOD)/ severe combined immunodeficiency (SCID)-ɣ (NSG) mice were used as human islet transplant recipients. We used 4 animals in total, where a single animal received human islet preparation from a single donor. Animals were housed in the Animal Care Facilities at Joslin Diabetes Center on a 12- h light/12-h dark cycle with water and food ad libitum. Stud- ies and protocols were approved by the Institutional Animal Care and Use Committee of the Joslin Diabetes Center (IACUC #05-01). Single-nucleus isolation Frozen or freshly cultured or engrafted human islet sam- ples were transferred to Dounce tissue grinder tubes (D8938; Sigma) containing 0.5 ml ice-cold Nuclei EZ lysis buffer (NUC-101; Sigma) and homogenized with pestles A and B for 1 min each on ice. Samples were transferred to clean 15 ml tubes and homogenizers were rinsed with 1.5 ml buffer followed by 2 ml buffer and transferred to the same 15 ml tubes to obtain a final vol- ume of 4 ml. The tubes were vortexed briefly at moder- ate speed and kept on ice for 5 min for cell lysis. To separate the nucleus and cytoplasm, the tubes were cen- trifuged at 500×g for 5 min at 4 °C. Supernatants con- taining cytoplasmic components were saved for later analyses. The pellet containing nuclei was resuspended in 0.5 ml cold buffer by vortexing briefly at moderate speed followed by the addition of 3.5 ml cold buffer. The nuclear suspension was mixed by vortexing briefly and set on ice for 5 min. The tubes were centrifuged at 500×g for 5 min, the supernatant was saved for later ana- lysis, and the pellet was resuspended in suspension buf- fer (0.5 ml PBS containing 0.01% non-acetylated bovine serum albumin (BSA); Sigma and 0.1% RNase inhibitor; 2313A from Clontech). The nuclear suspension was pi- petted ten times with a 1 ml tip, filtered through a 30- μm pre-separation filter (130-041-407; Miltenyi Biotech); cell number and cell viability were determined by cell counter using 0.4% trypan blue stain. The average num- ber of total nuclei obtained from one-half graft was approximately 8.5 × 105 nuclei (1.7 × 106 cells/ml) with 5–10 μm size and 93.3 ± 1.1% dead cell rate (n = 32 Human islet transplantations On the day of the experiment, 1000 hand-picked IEQs from 4 separate human islet donors were transplanted under the kidney capsule of 8-to-12-week-old male NSG mice and the animals were followed for 4 weeks. At the end of the follow-up period, mice were sacrificed by cervical dislocation. Human islet grafts were rapidly dis- sected under the microscope, snap-frozen, and stored at −80 °C. To overcome these limitations [31], single-nucleus RNA-sequencing (snRNA-seq) has been employed in studies on tissues composed of diverse cell types, includ- ing the brain [33, 34], kidney [35–37], heart [38], skeletal muscle [39, 40], stria vascularis [41], retina [42], liver [43, 44], lung [45], or white [46, 47] and brown adipose tissue [48, 49] obtained from mice or human donors. Here, we report a side-by-side comparison between snRNA-seq and scRNA-seq to validate the robustness of the former as an alternative sequencing strategy. We propose that snRNA-seq is a reliable approach to inter- rogate the transcriptomic profiles of archived frozen tis- sues that, to our knowledge, has not been applied previously to pancreatic islets or engrafted tissues. Page 2 of 17 Page 2 of 17 Basile et al. Genome Medicine (2021) 13:128 pericytes, and immune cells (tissue-resident macro- phages, mast cells, B cells, cytotoxic T cells) [18]. A mix- ture of such diverse cell types can hinder precise identification of cell-specific transcriptomes or exclusive biological signals when bulk tissue analyses are per- formed. To circumvent this limitation, over the past few years, multiple groups have utilized single-cell RNA-seq (scRNA-seq) methodologies on islet cells isolated from mouse [19, 20] or human pancreas [21–24]. These stud- ies have focused on dissecting the transcriptomic signa- ture of endocrine cell types across different ages [25] and to define differentially expressed genes in type 2 dia- betic β-cells [26–29]. However, single-cell preparations are also known to have limitations, including the need to harvest live cells [30] which may inadvertently induce stress responses [31, 32]. These aspects gain relevance when identifying disease-related transcriptomic signa- tures in tissues obtained from multiple human donors at different time points that require immediate, although usually varied, processing and individual analyses. were approved by the Joslin Diabetes Center’s Commit- tee on Human Studies (CHS#5-05). Upon receipt, hu- man islets were centrifuged, washed, transferred to Petri dishes (2000–3000 human islets equivalents [IEQs]/ plate), and cultured with fresh Miami Media #1A (Cell- gro) overnight at 37 °C and 5% CO2. Human islets from one donor (N = 1) were processed to isolate nuclei and obtain single-cell preparations as a common source for the scRNA-seq and snRNA-seq procedures (detailed below), while human islets from 4 donors (N = 4) were used in the kidney capsule transplantation experiments (detailed below). DAPI/Phase microscopy Fifty thousand dispersed human islet cells (50 μl) or iso- lated nuclei from frozen grafts were mixed with 50 μl of 4′,6-diamidino-2-phenylindole (double-stranded DNA staining, DAPI) solution (D9564; Sigma) diluted 1000 times in DPBS. Stained cells (10 μl) were loaded onto a Hemocytometer slide and imaged under a fluorescent microscope. Digital images were taken at 40× magnifica- tion with AXIO Imager A2 upright microscope equipped with X-Cite series 120Q light source, Axiocam 512 color camera. Bright field and fluorescent images were over- laid using ImageJ Software to determine complete cell lysis and nuclear integrity in isolated nuclear samples. RNA extraction and analysis Cells were lysed in RLT buffer and RNA was extracted using the Qiagen RNeasy kit according to the manufac- turer’s instructions. RNA concentrations were measured by Nanodrop (Thermo). RNA integrity was determined by using Agilent RNA 6000 Nano Kit (5067-1511; Agi- lent) according to the manufacturer’s instructions (Joslin Genomics Core). Western blotting samples across three independent experiments). The number of isolated nuclei was adjusted to 1000 nuclei/μl with suspension buffer and 10,000 nuclei were immedi- ately used for the generation of Gel Beads In-Emulsion (GEMs) and barcoding. Leftover nuclei were saved for subsequent analysis. g Nuclei or cell pellets were lysed in RIPA buffer (pH 7.4) containing 100 mM NaF, 50 mM Hepes, 150 mM NaCl, 10% Glycerol, 1.2% Triton X, 1 mM MgCl2, 1 mM EDTA, 1 mM Na3VO4, protease inhibitor cocktail (P8340; Sigma), phosphatase inhibitor 2 (P5726; Sigma), and phosphatase inhibitor 3 (P0044; Sigma). The super- natants collected after the first and second centrifuge steps of nuclear isolation were used after adding prote- ase and phosphatase inhibitors. Total protein concentra- tion was determined by Pierce BCA Protein Assay Kit (23225; Thermo). Lysates (50 μg protein) were run in 8% SDS-PAGE and transferred to PVDF membrane (Milli- pore). Membranes were blocked for 10 min at room temperature with 5% milk and were incubated overnight at 4 °C with antibodies against Lamin A/C (1:1000, 4777; Cell Signaling Technology) or GAPDH (1:1000, 5174; Cell Signaling Technology). After three washes (10 min), the membranes were incubated for 1 h at RT with anti- bodies against rabbit IgG-HRP conjugate (1:1000, 170- 6515: Bio-Rad) or mouse IgG-HRP conjugate (1:1000, 170-6516, Bio-Rad). After three 10-min washes, signals were visualized via Pierce ECL Western blotting sub- strate (PI32106; Thermo). Single-nucleus and single-cell RNA-sequencing procedures p Gel Bead In-Emulsion (GEMs) were generated using the Chromium 3’ Single Cell Library Kit (v2, 10X Genomics, CA) according to the manufacturer’s instructions and adapting the adjustments for the cDNA and libraries amplification steps, as recommended by 10X genomics for snRNA-seq procedures. Briefly, 10,000 cells or nuclei were combined with Single Cell Master Mix and encap- sulated into the barcoded Gel Beads through the Chro- mium™Controller. After GEM-RT incubation, cDNA samples were recovered, purified, and amplified through a cDNA Amplification Reaction using a 14-cycle setting. Quality controls on the undiluted amplified cDNA sam- ples were performed using a High Sensitivity DNA Kit (Agilent, CA) on a 2100 BioAnalizer (Agilent, CA) plat- form. Libraries were then constructed following Frag- mentation and Adaptor Ligation. Sample Index PCR was performed adjusting the reactions at 15 cycles. Finally, purified libraries were run on 2100 BioAnalizer (Agilent, Human islet studies Human islets were obtained from 5 non-diabetic brain- dead donors. Islet preparations were generated by the Integrated Islet Distribution Program or Prodo labora- tories according to the standard procedures [50] (Add- itional file 1: Table S1). All studies and protocols used Basile et al. Genome Medicine (2021) 13:128 Page 3 of 17 Page 3 of 17 Basile et al. Genome Medicine (2021) 13:128 Human islet dispersion Human islets were dissociated into single cells by Try- pLE (12604-013; Thermo). Briefly, 1 ml TrypLE was added to human islet pellets and incubated at 37 °C for 12 min by mixing the tube every 3–4 min. At the end of incubation, TrypLE was neutralized by adding 9 ml cold Dulbecco’s modified Eagle’s medium with high glucose (DMEM HG, MT 10-017-CV; Corning) containing 10% fetal bovine serum (FBS) (10437028; Gibco). Cells were centrifuged at 1200 rpm for 3 min at 4 °C, supernatant was removed, and cells were resuspended in 0.5 ml Dul- becco’s phosphate-buffered saline (DPBS) (14190250; Gibco). The cell suspension was filtered through 30 μm filter to remove aggregates and counted. A final concen- tration of 1 × 106 cells/ml cells was used for DAPI/Phase microscopy. For single-cell RNA-seq procedures, human islet cell suspensions were filtered using a 30-μm filter to remove any aggregates, and dead cells were excluded using Dead Cell Removal Kit (Miltenyi Biotec). Cells were counted and 10,000 cells were used for the gener- ation of GEMs. Immunohistochemistry Paraffin-embedded human islet graft sections were im- munostained using anti-insulin (1:400, ab7842; abcam), anti-glucagon (1: 10,000, ab92517; abcam), and anti- somatostatin (1:500, ab30788; abcam) antibodies using previously described techniques [6, 51, 52]. Nuclei were labeled using DAPI. Images were acquired using a Zeiss LSM-710 Confocal Microscope and the Zen Black soft- ware (Carl Zeiss). Basile et al. Genome Medicine (2021) 13:128 Page 4 of 17 Page 4 of 17 CA) using a High Sensitivity DNA Kit (Agilent, CA) to evaluate the quality of the ~ 400 bp fragments. CA) using a High Sensitivity DNA Kit (Agilent, CA) to evaluate the quality of the ~ 400 bp fragments. CA) using a High Sensitivity DNA Kit (Agilent, CA) to evaluate the quality of the ~ 400 bp fragments. expression levels between datasets per cell type by evalu- ating the linear regression of one dataset’s mean natural log of counts per gene against the other datasets for the same gene and reporting the R2, which is the square of the Pearson correlation coefficient, and the p value. Single-nucleus and single-cell RNA-seq data analysis Single-nucleus and single-cell RNA-seq data analysis Both the single-nucleus (snRNA-seq) and single-cell (scRNA-seq) RNA-sequencing datasets were produced using cultured human islet samples obtained from the same donor. In particular, we generated single-cell and single-nucleus libraries using 4 technical replicates for each method. Gene counts were obtained by Cell Ranger (10x Genomics, CA) using the human reference genome. To eliminate empty droplets and technical artifacts, we applied Cell Bender [53]. Cell Bender concludes that droplets are empty if the probability that they are empty > 50%, and then estimates ambient RNA among non- empty droplets. We normalized each sample’s data using sctransform [54], part of the Seurat toolkit, and detected and removed doublets with DoubletFinder [55]. To analyze the complete dataset, we combined the 4 sam- ples, including genes that are detected in at least 3 cells and including cells where at least 200 genes are detected. From the combined dataset, we filtered out nuclei that have more than 20% mitochondrial unique molecular identifiers (UMI). For the engrafted snRNA-seq, gene counts were gener- ated by Cell Ranger (10x Genomics, CA) using the human-mouse joint reference genome. To eliminate empty droplets and technical artifacts, we applied Cell Bender [53]. We normalized each sample’s data using sctransform [54], part of the Seurat toolkit, and detected and removed doublets with DoubletFinder [55]. To analyze the complete dataset, we combined the 4 sam- ples, including genes that are detected in at least 3 cells and including cells where at least 200 genes are detected. From the combined dataset, we filtered out nuclei that have more than 20% mitochondrial UMI and more than 25% mouse UMI and removed all mouse genes in the remaining nuclei. For each of them (i.e., the engrafted islet snRNA-seq, the scRNA-seq, or the snRNA-seq from cultured human islets), we normalized the dataset of combined samples with sctransform [54], part of the Seurat toolkit; and then using Seurat, we identified clus- ters and marker genes per cluster and plotted the data as Uniform Manifold Approximation and Projection (UMAP), heatmaps, and violin plots. For all datasets, genes were considered expressed in a cell or nucleus if they had at least one UMI. We evaluated gene Next generation sequencing Single-nucleus and single-cell libraries were sent for se- quencing at GeneWiz or NovoGene laboratories. Sam- ples were run in independent lanes on a HiSeq 4000 platform (Illumina, CA), using a coverage of 500,000 pair-ended reads targeted per cell. Integration with published scRNA-seq datasets Integration with published scRNA-seq datasets The ability of Seurat [56] to integrate single-cell datasets was demonstrated on publicly-available human pancre- atic islet scRNA-seq datasets spanning 27 donors, four laboratories, and five technologies: The datasets that have been used in this article have been deposited in the public Genomic Spatial Event Database (GSE) and in the ArrayExpress Database at the EMBL-EBI. For InDrop, accession number GSE84133 (https://www.ncbi.nlm.nih. gov/geo/query/acc.cgi?acc=GSE84133) [21]; for CelSeq2, accession number GSE85241 (https://www.ncbi.nlm.nih. gov/geo/query/acc.cgi?acc=GSE85241) [23]; for SMART- Seq2, accession number E-MTAB-5061 (https://www. ebi.ac.uk/arrayexpress/experiments/E-MTAB-5061/) [26]; for Fluidigm C1, accession number GSE86469 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc= GSE86469) [27]; and for CelSeq, accession number GSE81076 (https://www.ncbi.nlm.nih.gov/geo/query/acc. cgi?acc=GSE81076) [57] were used. We followed Seurat instructions to normalize these datasets with sctrans- form [54], identify integration anchors (we used dimen- sionality 25, consistent with the dimensionality we applied in Seurat analysis of our datasets), and construct our reference with Seurat function IntegrateData. We then used Seurat to project this universal scRNA-seq dataset, named “reference,” onto our datasets (i.e., the engrafted islet snRNA-seq and the scRNA-seq and snRNA-seq from cultured human islets) to harmonize the data. Integration with published scRNA-seq datasets The ability of Seurat [56] to integrate single-cell datasets was demonstrated on publicly-available human pancre- atic islet scRNA-seq datasets spanning 27 donors, four laboratories, and five technologies: The datasets that have been used in this article have been deposited in the public Genomic Spatial Event Database (GSE) and in the ArrayExpress Database at the EMBL-EBI. For InDrop, accession number GSE84133 (https://www.ncbi.nlm.nih. gov/geo/query/acc.cgi?acc=GSE84133) [21]; for CelSeq2, accession number GSE85241 (https://www.ncbi.nlm.nih. gov/geo/query/acc.cgi?acc=GSE85241) [23]; for SMART- Seq2, accession number E-MTAB-5061 (https://www. ebi.ac.uk/arrayexpress/experiments/E-MTAB-5061/) Isolation of single nuclei from frozen human islets E Yield (μg, left Y-axis, dots) and RNA Integrity Number (RIN, right Y-axis, squares) of RNA isolated from six technical replicates across three independent experiments. Data are represented as mean ± SEM DAPI staining, protein, and RNA evaluation. To pre- clude confounding factors such as the quality of the iso- lation and the purity of the nuclear preparation we also obtained whole islet cell samples by dispersing a fresh sample of non-diabetic human islets, containing 500 IEQs (donor 2, Fig. 1A). expected range, considering that a mammalian cell con- tains 10–20 pg of total RNA of which 20–30% resides in the nucleus [63, 64]. RNA integrity evaluated by measur- ing two major peaks representing 18S and 28S rRNA re- vealed an average RNA integrity number (RIN) of 7.6 ± 0.4 which indicates high quality with minimal degrad- ation [62]. Based on the efficacy of the tested protocol for obtaining pure high-quality nuclei from human islet samples, we decided to use this methodology for subse- quent single-nucleus RNA-seq experiments. First, the quality of the isolation protocol was evalu- ated by visualizing the nuclei samples obtained from fro- zen human islets under phase-contrast microscopy and by assessing cell integrity in comparison to intact cell samples obtained by dissociating fresh human islets, col- lected by a separate donor, into single cells (whole cells), as previously described [60] (Fig. 1B). Phase-contrast im- ages of isolated nuclei samples showed that the cyto- plasm was depleted completely in all the cells, and the nucleus was intact (Fig. 1B). Whereas, as expected, the cytoplasm surrounding the DAPI-stained nucleus was easily distinguished in the dispersed single islet cells (Fig. 1B). Moreover, the lysis of the outer cell mem- branes in the samples processed for nuclei isolation was confirmed by assessing cytoplasmic (GAPDH) and nu- clear (Lamin A/C) proteins in the samples collected dur- ing and after the isolation process (Fig. 1C). Western blot analysis of nuclei (Nuc), cytoplasmic fractions col- lected after the first (Cyt A) and the second (Cyt B) cen- trifuge steps, obtained by processing human islets from donor 1, and the whole cell lysate (WCL) sample, ob- tained by processing human islets from donor 2, showed that the nuclear isolation process efficiently removed the cytoplasm from nuclear samples, and preserved nuclear integrity, consistent with the DAPI staining (Fig. 1B). Isolation of single nuclei from frozen human islets Isolation of single nuclei from frozen human islets To isolate nuclei from human islet preparations, we tested the Nuclei EZ lysis buffer-based protocol. We employed this isolation method because it was previ- ously successfully used to isolate nuclei from frozen compact tissues, such as tumor tissues [58, 59]. In our hands, we validated that this protocol removes cytoplas- mic content quickly and consistently. To test the efficacy of this protocol, we used a frozen sample of non-diabetic human islets (donor 1, Fig. 1A). Briefly, the archived hu- man islet sample, consisting of 500 human islet equiva- lents (IEQs) was subjected to homogenization using the Nuclei EZ buffer. After a short incubation period, the cytoplasmic fraction was removed by centrifugation, and the pellet containing the nuclear fraction was washed to remove cytoplasmic contamination. The nuclei were fil- tered, counted, and tested in control assays including Basile et al. Genome Medicine (2021) 13:128 Page 5 of 17 Basile et al. Genome Medicine Fig. 1 (See legend on next page.) Fig. 1 (See legend on next page.) Page 6 of 17 Basile et al. Genome Medicine (2021) 13:128 (See figure on previous page.) Fig. 1 Single nuclei isolation from frozen human islet samples. A Experimental workflow of isolation of nuclei from frozen cultured human islets (donor 1) and human islet dispersion into single cells (donor 2). B Isolated nuclei from frozen human islets (donor 1) and dissociated human islet cells from fresh intact islets (donor 2, whole cells) were imaged by DAPI/Phase microscopy. Left image: Intact nuclei stained in blue without cytoplasm surrounding the isolated nucleus . Right image: Nuclei stained in blue surrounded by cytoplasm of human islet cells. Scale bar is 25 μm. C Western blot analysis of human pancreatic islet cells (whole cell lysate; WCL, obtained from donor 2), supernatant collected after the first centrifuge step (cytosolic fraction A; Cyt A) and second centrifuge step (cytosolic fraction B; Cyt B) of the nuclear isolation protocol, and isolated nuclei (nuclear fraction; Nuc, obtained from Donor 1). LaminA/C is a nuclear marker; GAPDH is a cytoplasmic marker. D RNA was isolated from islet cell nuclei and analyzed by Bioanalyzer. Representative electropherogram showing two major peaks (18S and 28S rRNA) indicates minimal degradation of total RNA. The X-axis indicates the size of RNA fragments (nt: nucleotides) and the Y-axis represents the intensity of the fluorescence signal (FU, fluorescence unit). Isolation of single nuclei from frozen human islets In- deed, we were able to isolate 8.32 ± 1.6 × 105 nuclei from ~ 500 IEQs consistent with previous reports [61]. The amounts of nuclei were adequate to perform snRNA-seq—which required only 10,000 nuclei, as pre- viously reported [48]—as well as for validation experi- ments such as qRT-PCR. Side-by-side comparison of scRNA-seq and snRNA-seq methods in cultured human islets Statistical significance was tested using a Wilcoxon rank-sum test. E, F UMAP plots representing the clustering of the high-quality E cells and F nuclei in the E scRNA-seq and the F snRNA-seq datasets. Y-axis of plot in F is like the plot in E. G Global integrated UMAP plot representing the distribution of high-quality cells (from scRNA-seq, orange dots) and nuclei (from snRNA-seq, blue dots) (endothelial cells, stellate cells, Schwann cells, acinar cells, and ductal cells) cell types previously reported using established scRNA-seq methodologies, suggesting that snRNA-seq protocols did not hinder the characterization of any islet cell type, including the low abundance PP-cell population (Fig. 3A, Additional file 4: Table S3). We then undertook one-to-one comparisons of global and cell type-specific gene expression profiles between (a) the scRNA-seq and the reference datasets and (b) between the scRNA-seq and the snRNA-seq datasets we had gener- ated. We observed an almost total overlap (99.9 %) of the genes detected between the reference (10,497 genes) and our scRNA-seq (11,694 genes) and between the scRNA- seq and the snRNA-seq (11,692 genes) datasets (Fig. 3B, C). We tested the association of gene expression levels be- tween datasets within the major islet cell types (α-cells, β- cells, PP-cells, and δ-cells) and observed positive correla- tions within α-cells, β-cells, PP-cells, and δ-cells between scRNA-seq and the reference (Fig. 3D). In addition, such correlations were driven by cell type-specific genes, such as GCG for α-cells, INS for β-cells, and so forth, an aspect that was absent in all the other correlations between a given islet cell type from the scRNA-seq dataset and a dif- ferent one from the reference output (Additional file 3: Figure S2A). Notably, the positive correlations of the gene signatures were also evident within α-cells, β-cells, PP- cells, and δ-cells in the scRNA-seq and snRNA-seq dataset comparisons (Fig. 3E). obtained estimates of ambient RNA within the cell-/nu- cleus-containing droplets by applying Cell Bender [53], with averages of 5.87 ± 1.76 % and 1.42 ± 0.72 % (mean ± SD) among the four single-cell and four single-nucleus library replicates, respectively (Additional file 2: Table S2, Additional file 3: Figure S1A,B). g We clustered the data with Seurat, shown as UMAP plots of recovered cells (Fig. 2E) and nuclei (Fig. 2F). In particular, we were able to identify 10 clusters of cells and nuclei. Side-by-side comparison of scRNA-seq and snRNA-seq methods in cultured human islets Cells and nuclei expressing high levels of INS gene (natural log of counts > 3) were distributed in clus- ters #3, while GCG-enriched cells/nuclei were distrib- uted in clusters #1 (Fig. 2E, F; Additional file 3: Figure S1D-G). Interestingly, cluster #7 was enriched in low INS-expressing nuclei and GCG-expressing nuclei, but not cells (Fig. 2E, F; Additional file 3: Figure S1D,E). Specimens expressing high levels of SST (natural log of counts > 3) were also found in clusters #3, while cells and nuclei expressing high levels of PPY (natural log of counts > 1.5) were distributed in clusters #1 and #3 (Fig. 2E, F’ Additional file 3: Figure S1H-K). In addition, clus- ter #0 in both the scRNA-seq and snRNA-seq sets com- prised of cells and nuclei expressing low levels of all the endocrine cell marker genes (Fig. 2E, F; Additional file 3: Figure S1D-K). These findings suggest that the snRNA- seq protocol allows the identification of endocrine marker gene-expressing nuclei in a manner that is simi- lar to scRNA-seq methods. We then analyzed the fractional overlap of increas- ing N number of top genes (N = 100, 200, 500, and 1000 top genes) within cell types between the scRNA- seq and the reference and between the snRNA-seq and the scRNA-seq datasets (Fig. 3F). In particular, a significant proportion (∼72%) of the top 100 tran- scripts was shared by the major islet cell types (α- cells, β-cells, and δ-cells) between the reference and the scRNA-seq datasets (Fig. 3F, Additional file 3: Figure S2B-E). Similar levels of fractional overlap of an increasing number of top genes were also observed in the scRNA-seq vs. snRNA-seq comparison (Fig. 3F). Curiously, the δ-cell groups displayed the highest overlap, reaching 95% in the top 100 and 85% in the top 1000 cell type genes (Fig. 3F). These results sup- port the notion that snRNA-seq identifies genes expressed in the least as well as the most abundant Side-by-side comparison of scRNA-seq and snRNA-seq methods in cultured human islets To compare the single-nucleus and single-cell RNA- sequencing procedures (snRNA-seq and scRNA-seq, re- spectively), we obtained human islets from a non- diabetic donor and divided them into two groups: (1) one to generate single-cell suspensions and (2) a second to isolate single nuclei using the protocol described above (Fig. 2A). We generated 4 technical replicates for each group of 10,000 cells or nuclei and loaded them into the 10X genomics Chromium Controller to obtain Gel-beads in Emulsion (GEMs). Following the 10X gen- omics standard protocol, we obtained single-cell and single-nuclei libraries that were then sequenced using next-generation sequencing (NGS). Following the appli- cation of the quality check filters, including removal of doublets and multiplets, we recovered 1277.2 ± 234.2 and 976.2 ± 82.0 (mean ± SD) high-quality cells and nu- clei per sample, respectively (Additional file 2: Table S2). Although the number of reads and genes sequenced per cell/nucleus was slightly higher in the scRNA-seq com- pared to the snRNA-seq method (Fig. 2B, C), the dupli- cation rate, indicating the ratio between usable vs. sequenced reads, as a read-out of sequencing efficiency, were similar between the two transcriptomic procedures (Additional file 3: Figure S1C, Additional file 2: Table S2). As confirmation of the high purity of the single- nucleus preparations, the percentage of mitochondrial genes sequenced in the nucleus-containing droplets was lower than the cell-containing particles (2.7% and 4.2%, respectively) (Fig. 2D, Additional file 2: Table S2). We Total RNA was isolated from the nuclear samples, and the yield and integrity were evaluated using a Bioanaly- zer [62] (Fig. 1D, E). We were able to isolate 3.3 ± 0.7 μg RNA from isolated nuclear samples (n = 6; three inde- pendent experiments). The RNA yield was in the Page 7 of 17 Basile et al. Genome Medicine (2021) 13:128 Basile et al. Genome Medicine (2021) 13:128 Basile et al. Genome Medicine (2021) 13:128 Page 8 of 17 (See figure on previous page.) Fig. 2 Side-by-side comparison of single-cell vs. single-nucleus RNA-sequencing in freshly cultured human islets. A Experimental design of the workflow for generating single-cell RNA-seq (scRNA-seq) and single-nucleus RNA-seq (snRNA-seq) datasets of cultured human islets. B–D Violin plots representing B number of reads (UMI), C number of genes, and D proportion (expressed in %) of mitochondrial genes per cell/nucleus in scRNA-seq (orange violins) and snRNA-seq (blue violins) datasets. Data are expressed in logarithmic scale (Y-axis). Comparison of gene expression signatures of human islet cells using scRNA-seq and snRNA-seq snRNA-seq (lower panels) datasets following harmonization to the reference. G Pie chart representing proportions of biotypes of genes detected with higher confidence in snRNA-seq compared to scRNA-seq (snRNA-seq enriched genes) mice and followed up for 4 weeks. At the end of 4 weeks, grafts were removed carefully and divided into two equiva- lent parts (~ 500 IEQs each) that were subsequently snap fro- zen. On the day of nuclei isolation, frozen graft fractions (~ 500 IEQs) were placed in ice-cold lysis buffer individually and homogenized immediately to obtain pure nuclei as de- scribed above (Fig. 1) and single-nucleus cDNA libraries were generated by using the Chromium Single-Cell 3’ Li- brary Kit (Fig. 4A) [41]. Similar to the analyses of cultured islet snRNA-seq, the engrafted islet snRNA-seq data were initially analyzed using quality check pipelines in order to re- move low-quality nuclei, including those with a total number of reads (UMI) < 1000, a total number of expressed genes < 500, and a proportion of mitochondrial genes > 20%, in line with previous reports [35, 36, 38] (Additional file 2: Table S2, Additional file 3: Figure S3A-C). It is worth noting that the percentage of expression of mitochondrial genes per nucleus was < 2% of the whole transcriptome (Additional file 2: Fig- ure S3C), in line with the in vitro snRNA-seq results, sug- gesting a high efficiency of nuclear isolation method and allowing for a greater confidence to interpret and analyze the snRNA-seq data [36]. In addition, by aligning the recovered reads to the murine genome (GRCm38), we filtered out nu- clei containing > 25% mouse-specific UMI (Additional file 3: Figure S3D). Finally, Cell Bender estimated ambient RNA contamination levels at 4.6 ± 1.3 % (Additional file 2: Table S2; Additional file 3: Figure S3E) within nucleus-containing droplets. With this approach, we recovered 3565 (891.2 ± 409.6 per sample) high-quality nuclei, which is comparable with previous studies using a similar [35] or different plat- forms [35, 42, 66, 67]. endocrine cells, providing confidence that snRNA-seq is a reliable approach for identifying genes expressed in the various islet cell types. To evaluate whether snRNA-seq would allow identification of specific types of genes which are covered with higher con- fidence compared to scRNA-seq, we analyzed the biotypes of the genes detected in the snRNA-seq dataset with a signifi- cantly higher (> 1.5-folds) percentage of detection compared to the scRNA-seq dataset. Comparison of gene expression signatures of human islet cells using scRNA-seq and snRNA-seq To determine whether snRNA-seq would represent a reli- able transcriptomic method to identify human islet cell types, we compared our snRNA-seq and scRNA-seq data- sets with the publicly available scRNA-seq datasets. We chose five published scRNA-seq datasets of human pancreatic islets spanning 27 donors, four laboratories, and five technologies for harmonizing with Seurat [21,23,26,27,57]. We used this harmonized dataset as a “reference” (Fig. 3A) upon which to integrate our data, allowing for correspondence of cells and clusters. Indeed, when we compared the cultured human islet scRNA-seq and snRNA-seq datasets we had generated to the refer- ence, we were able to identify all the major islet endocrine (i.e., α-cells, β-cells, PP-cells, δ-cells) and non-endocrine Page 9 of 17 Basile et al. Genome Medicine (2021) 13:128 Basile et al. Genome Medicine Fig. 3 (See legend on next page.) Fig. 3 (See legend on next page.) Fig. 3 (See legend on next page.) Page 10 of 17 Basile et al. Genome Medicine (2021) 13:128 (See figure on previous page.) Fig. 3 Comparison between snRNA-seq and scRNA-seq datasets following harmonization on reference dataset. A Global UMAP plots and cell type prediction in the scRNA-seq (middle panel) and snRNA-seq (right panel) following harmonization on the reference dataset generated by integrating five published scRNA- seq datasets of cultured human islets [21, 23, 26, 27, 57] (left panel). Y-axis is similar in all three panels. B Venn diagram of detected genes in the reference (pink circle) and in our scRNA-seq dataset (blue circle). C Venn diagram of detected genes in our scRNA-seq (blue circle) and in our snRNA-seq dataset (green circle). D, E Scatter plots of harmonized cell type-specific gene expression in α-cells, β-cells, PP-cells, or δ-cells D between the reference and the scRNA-seq datasets and E between the scRNA-seq and the snRNA-seq datasets. X-axis and Y-axis represent the expression levels in natural log of counts in the indicated datasets. The blue line in each plot represents the regression line, whose fit is indicated by the R2 value (the square of the Pearson correlation coefficient). The P and R values are provided for each correlation. F Fractional overlap expressed in percentages (%, Y-axis) of increasing numbers (100, 200, 500, and 1000, X-axis) of top genes within α-cells (yellow bars), β-cells (green bars), PP-cells (blue bars), or δ-cells (red bars) in reference vs. scRNA-seq (upper panel) and in scRNA-seq vs. Comparison of gene expression signatures of human islet cells using scRNA-seq and snRNA-seq Among the 1896 genes which were covered in the snRNA-seq with a higher confidence compared to the scRNA-seq method, 7.5% accounted for long non-coding RNAs (lncRNAs), whereas 92.1% were pro- tein coding genes (Fig. 3G). Importantly, lncRNA genes were also among the top genes detected in snRNA-seq with higher confidence compared to scRNA-seq protocols. Indeed, the proportions of detection of cytochrome c oxidase assembly factor heme A:farnesyltransferase (COX10) antisense RNA 1 (COX10-AS1) and minichromosome maintenance complex component 3 associated protein (MCM3AP) antisense RNA 1 (MCM3AP-AS1) genes in snRNA-seq were 15.5-fold and 13.7-fold higher than scRNA-seq, respectively (Additional file 5). In addition, nuclear paraspeckle assembly transcript 1 (NEAT1) and maternally expressed 3 (MEG3), also lncRNA genes, were detected in the snRNA-seq dataset with a pro- portion of 70% and 26.9%, respectively, whereas their detec- tion rates in the scRNA-seq dataset were 35.5% and 7.0%, respectively (Additional file 5). These data suggested that the snRNA-seq method allows for detecting nuclei-enriched lncRNA genes with a higher confidence compared to scRNA-seq. The ability of the nuclear transcriptomic analysis to detect genes enriched in non-coding RNAs provides an important resource for studying the epigenetic regulatory mechanisms in human islets. Next, clustering with Seurat yielded 7 major clusters (Fig. 4B). Notably, we observed a cluster of nuclei enriched in α-cell-specific genes, namely cluster 1; a nu- clear cluster enriched in SST and PPY genes, namely cluster 2; and a high INS and MAF bZIP transcription factor A (MAFA)-expressing nuclear cluster (natural log of counts ≥5), namely cluster 3 (Fig. 4B–D). Interest- ingly, other β-cell marker genes, including ATP binding cassette subfamily C member 8 (ABCC8) and solute car- rier family 30 member 8 (SLC30A8), were also expressed in cluster 0, which displayed low levels of INS expression snRNA-seq of transplanted human islets To reveal the transcriptomic signature of frozen engrafted human islets, we undertook snRNA-seq experiments as depicted in Fig. 4A. We used the immunodeficient NSG mouse model which is a widely utilized in vivo model for β- cell regeneration studies [6, 8, 10, 65]. Human islets (1000 IEQs) obtained from 4 different donors were transplanted in- dividually under the kidney capsule of 8-to-12-week-old male Basile et al. Genome Medicine (2021) 13:128 Page 11 of 17 Fig. 4 Single-nucleus RNA-sequencing in human islet grafts. A Experimental scheme of isolation of nuclei and snRNA-seq protocol in transplanted human islets. B Global UMAP plot displaying clustering of high-quality nuclei derived from engrafted human islets. C Heatmap representing standardized gene expression (standardized natural log of counts) of selected endocrine and exocrine gene markers in each nucleus separated by cluster. Each column represents a single nucleus and each row is a different gene (D upper panels) Violin plots representing the expression levels of endocrine cell gene markers including insulin (INS) Fig. 4 Single-nucleus RNA-sequencing in human islet grafts. A Experimental scheme of isolation of nuclei and snRNA-seq protocol in transplanted human islets. B Global UMAP plot displaying clustering of high-quality nuclei derived from engrafted human islets. C Heatmap representing standardized gene expression (standardized natural log of counts) of selected endocrine and exocrine gene markers in each nucleus separated by cluster. Each column represents a single nucleus, and each row is a different gene. (D, upper panels) Violin plots representing the expression levels of endocrine cell gene markers, including insulin (INS), glucagon (GCG), pancreatic polypeptide (PPY), or somatostatin (SST) in each nuclear cluster. (D, lower panels) Violin plots representing the expression levels of β- cell gene markers, such as ABCC8, SLC30A8, MAFA, and endocrine markers, such as PAX6 within the nuclear clusters. Expression levels (Y-axis) are natural log of counts Fig. 4 Single-nucleus RNA-sequencing in human islet grafts. A Experimental scheme of isolation of nuclei and snRNA-seq protocol in transplanted human islets. B Global UMAP plot displaying clustering of high-quality nuclei derived from engrafted human islets. C Heatmap representing standardized gene expression (standardized natural log of counts) of selected endocrine and exocrine gene markers in each nucleus separated by cluster. Each column represents a single nucleus, and each row is a different gene. Discussion (natural log of counts < 5) (Fig. 4B–D). In addition, clus- ter 0 was enriched in endocrine cell-specific genes, such as paired box 6 (PAX6), suggesting that such clusters likely included β-cells in a different maturity state. How- ever, low INS expression levels were also found in nuclei belonging to cluster 2 (Fig. 4B–D). Regarding the exo- crine cell marker gene expression, a cluster of nuclei enriched in ductal cell genes—including CF transmem- brane conductance regulator (CFTR), SRY-box transcrip- tion factor 9 (SOX9), and keratin 19 (KRT19), was identified in cluster 4, whereas the acinar cell-specific genes were virtually absent (Fig. 4B,C). Finally, we har- monized the in vivo snRNA-seq to the reference dataset to evaluate an unsupervised prediction of the cell type identity. Strikingly, all the endocrine and non-endocrine islet cell types were identified following harmonization, indicating the efficacy of the snRNA-seq approach to re- cover virtually all islet cells from transplanted samples (Additional file 4: Table S3, Additional file 3: Figure S3F). Taken together, these data indicate that snRNA- seq of human islet grafts reveals the presence of genes marking all islet cell types, and as expected, a virtual ab- sence of exocrine cell genes. Over the last few decades, the research community is in- creasingly utilizing human islet studies with the long- term goal of developing novel translational strategies to counteract diabetes [69]. Moreover, the establishment of avant-garde technologies for studying biological pro- cesses at the single-cell resolution have provided new tools for exploring human islet cells in physiological conditions and their defects during diabetes disease pro- gression [25–28, 70]. While such studies were commonly conducted on cultured human islets in vitro, the tran- scriptome of frozen human islet cells after transplant- ation in mouse models remains largely unexplored and provides an opportunity to study archived tissues. Ana- lyzing the transcriptomic signatures of human islet cells at the single-cell level represents a state-of-the-art tool for gathering insights into dynamic molecular mecha- nism(s) in response to diverse stimuli, including mito- gens, differentiating factors, or stimulators of hormone secretion. In this context, human β-cell proliferation, dif- ferentiation/transdifferentiation, or neogenesis have all been topics of investigation in the context of physio- logical (e.g., pregnancy) or pathophysiological (e.g., insu- lin resistance/T2D) states. Nevertheless, there continues to be an urgent need for new tools to reliably study ar- chived human or mouse tissues. Discussion Here we present single- nucleus RNA-sequencing (snRNA-seq) for interrogating the human islet transcriptome that is especially relevant for small samples that have been frozen following in vivo manipulation. The presence of nuclei with high SST-expression and low INS-expression in cluster 2 prompted us to under- take immunofluorescence studies to validate polyhormo- nal expression at the protein level. To this end, we immunostained α-cells, β-cells, and δ-cells in graft sec- tions following transplantation of human islets from the same donors used for the in vivo transcriptomic analysis. We observed overlap between SST and INS protein im- munostaining in transplanted human islets, suggesting that, indeed, cell fate transition occurs over the 4-week in vivo engraftment period, as previously observed [51, 52, 68] (Additional file 3: Figure S4). It has been established that the isolation of nuclei has sev- eral advantages over single-cell isolation [35, 67, 71]. First, this method can be simultaneously applied to multiple sam- ples that are collected at different time points, reducing the potential variations introduced by sample handling. The sec- ond advantage is that the nucleus isolation method is effi- cient and requires fewer steps compared to the single-cell protocol. For example, snRNA-seq does not require enzym- atic digestion to dissociate tissues into single cells which often results in decreased viability and cell loss [72]. Finally, rapid isolation of nuclei compared to tissue dissociation and isolation of single cells minimizes changes in the transcrip- tome during the isolation process [31]. Such considerations are important, especially when the volume of tissue available for studies is necessarily limited, such as human islet grafts. snRNA-seq of transplanted human islets (D, upper panels) Violin plots representing the expression levels of endocrine cell gene markers, including insulin (INS), glucagon (GCG), pancreatic polypeptide (PPY), or somatostatin (SST) in each nuclear cluster. (D, lower panels) Violin plots representing the expression levels of β- cell gene markers, such as ABCC8, SLC30A8, MAFA, and endocrine markers, such as PAX6 within the nuclear clusters. Expression levels (Y-axis) are natural log of counts Page 12 of 17 Page 12 of 17 Basile et al. Genome Medicine (2021) 13:128 Basile et al. Genome Medicine (2021) 13:128 Comparison of in vitro vs. in vivo single-nucleus RNA- sequencing methods To determine the reliability of the snRNA-seq dataset generated from transplanted human islets, we compared the transcriptomic profiles of the snRNA-seq output ob- tained from cultured (in vitro) with that from trans- planted (in vivo) human islets. The genes detected in each dataset showed a significant overlap (99.9%; dia- gram Fig. 5A). Testing the association of the islet cell type gene expression profiles revealed a positive correl- ation in α-cells and δ-cells (Fig. 5B, E) that was stronger in β-cells and PP-cells between in vitro and the in vivo snRNA-seq datasets (Fig. 5C, D). These data confirm the similarity of transcriptomic profiles between freshly cul- tured and frozen engrafted human islets and highlight the ability of snRNA-seq procedures to interrogate gene expression of human islet cells. To test the reliability of snRNA-seq in comparison to the well-established scRNA-seq procedures, we under- took a direct comparison between the two methodolo- gies by analyzing the transcriptomics of freshly isolated cells and nuclei obtained from the same human islet donor. Despite the differences in terms of sequenced reads and detected genes per specimen between the two sequencing procedures, likely due to the lower RNA content in the nucleus compared to the cytosol [63], the Basile et al. Genome Medicine (2021) 13:128 Page 13 of 17 Fig. 5 Comparison between in vitro and in vivo snRNA-seq datasets. A Intersection of the total number of detected genes in the snRNA-seq dataset of cultured human islets (in vitro snRNA-seq, green circle) and the snRNA-seq dataset of transplanted human islets (in vivo snRNA-seq, yellow circle). B–D Scatter plots of harmonized cell type-specific gene expression in B α-cells, C β-cells, D PP-cells, and E δ-cells between the in vitro and the in vivo snRNA-seq datasets. X-axis and Y-axis represent the gene expression levels expressed as natural log of counts in the indicated datasets. The blue line in each plot represents the regression line, whose fit is indicated by the R2 value (the square of the Pearson correlation coefficient). The P and R values are provided for each correlation Fig. 5 Comparison between in vitro and in vivo snRNA-seq datasets. A Intersection of the total number of detected genes in the snRNA-seq dataset of cultured human islets (in vitro snRNA-seq, green circle) and the snRNA-seq dataset of transplanted human islets (in vivo snRNA-seq, yellow circle). Comparison of in vitro vs. in vivo single-nucleus RNA- sequencing methods The blue line in each plot represents the regression line, whose fit is indi- cated by the R2 value (the square of the Pearson correlation coefficient). The red circles indicate the islet cell specific marker genes driving the correlation between the same cell type from the two datasets. The red dotted squares highlight the correlation plots used in the main Fig. 3D. (B-E) Fractional overlap expressed in percentages (%, Y-axis) of the (B) top 100, (C) 200, (D) 500, or (E) 1000 genes between the indicated islet cell types from the reference dataset (X-axis) and the α-cells (yellow bars), β- cells (green bars), PP-cells (blue bars), or δ-cells (red bars) from the scRNA-seq dataset. Figure S3. Quality check parameters in the trans- planted human islet snRNA-seq dataset. (A-D) Violin plots of (A) number of genes, (B) number of reads, (C) proportion of mitochondrial genes, and (D) proportion of mouse genes per nucleus across the 4 transplanted human islet samples in the snRNA-seq dataset. (E) Box plot of the levels of ambient RNA contamination in droplets of the in vivo snRNA-seq data- set within each human islet graft. (F) UMAP plot of nuclear cluster distri- bution and cell type prediction of in vivo snRNA-seq dataset following harmonization to the reference dataset. Figure S4. Islet cell type valid- ation in human islet graft sections by immunofluorescence. Representa- tive images of human islet cells identified as α-cells, β-cells or δ-cells according to the glucagon (GCG, green), insulin (INS, red) and somato- statin (SST, white) labeling. Nuclei are stained in blue. Scale bar is: 50 μm. We then applied snRNA-seq to archived human islet graft samples. As demonstrated for the cultured islet samples, single-nucleus preparations from transplanted human islets allowed the identification of all the islet cell types with a coverage that is comparable to single-cell profiling. Notably, the global and the islet cell-specific single-nucleus transcriptomics of cultured versus trans- planted human islet were highly concordant, confirming that snRNA-seq represents a reliable strategy to analyze the gene signature of human islets in vivo at single-cell resolution. Finally, the transcriptome of polyhormonal islet cell clusters, e.g., cluster 2 expressing INS and SST, could be recapitulated at the protein level by immuno- fluorescence. Comparison of in vitro vs. in vivo single-nucleus RNA- sequencing methods B–D Scatter plots of harmonized cell type-specific gene expression in B α-cells, C β-cells, D PP-cells, and E δ-cells between the in vitro and the in vivo snRNA-seq datasets. X-axis and Y-axis represent the gene expression levels expressed as natural log of counts in the indicated datasets. The blue line in each plot represents the regression line, whose fit is indicated by the R2 value (the square of the Pearson correlation coefficient). The P and R values are provided for each correlation sequencing efficiency was similar between scRNA-seq and snRNA-seq procedures, consistent with reports on other metabolic tissues [35, 67, 71]. We also generated a reference dataset by harmonizing 5 previously published scRNA-seq datasets in human islets in order to compare our transcriptomic results in an unsupervised fashion [56]. This approach indicated that snRNA-seq allowed for the identification of all the pancreatic islet cell types, including the least abundant, such as the PP-cells. The data also showed an almost total overlap in global gene expression (99.9%) between the two methodologies highlighted by positive correlations of gene signatures, mainly driven by cell-specific genes, between each of the islet cell types. Furthermore, the ability to identify genes Page 14 of 17 Basile et al. Genome Medicine (2021) 13:128 whose percentage of detection in snRNA-seq was > 1.5- fold higher than scRNA-seq indicated those genes were detected by snRNA-seq with a higher confidence in comparison to scRNA-seq. The fact that ~ 7.5% of the snRNA-seq enriched genes were lncRNAs suggested that snRNA-seq represented a potential tool for identifying non-coding RNAs in human islets that would be useful to examine chromatin remodeling, post-transcriptional modifications, and crosstalk with other RNA species. (blue plots) datasets. Figure S2. Correlation plots and fractional overlap estimation of islet cell types from scRNA-seq dataset with those from the reference dataset. (A) Scatter plots representing correlation of gene ex- pression levels between scRNA-seq-derived (Y-axis) α-cells (first row from top), β-cells (second row from top), PP-cells (third row from top), and δ- cells (fourth row from top) and reference-derived (X-axis) α-cells (first col- umn from left), β-cells (second column from left), PP-cells (third column from left) or δ-cells (fourth column from left). X-axis and Y-axis represent the expression levels in natural log of counts in the indicated datasets. Comparison of in vitro vs. in vivo single-nucleus RNA- sequencing methods Although other in situ hybridization methods, such as RNA-scope, would be more appropri- ate to validate transcriptomic results, the validation at the protein level provides an important functional per- spective to the snRNA-seq data. Additional file 4: Table S3. Number of endocrine and exocrine pancreatic cell types predicted in scRNA-seq or snRNA-seq of cultured or transplanted human islets following harmonization. Additional file 4: Table S3. Number of endocrine and exocrine pancreatic cell types predicted in scRNA-seq or snRNA-seq of cultured or transplanted human islets following harmonization. Additional file 5. List of top 20 snRNA-seq enriched genes ordered by fold change of percentage of detection (upper table) or percentage of detection (lower table) compared to scRNA-seq. Additional file 5. List of top 20 snRNA-seq enriched genes ordered by fold change of percentage of detection (upper table) or percentage of detection (lower table) compared to scRNA-seq. Acknowledgements We thank Michael DeRan PhD (Broad Institute), Qiong Zhou, and Mary- Elizabeth Patti MD (Joslin Genomics Core) for technical assistance with per- forming 10X Genomics experiments and Bridget Wagner PhD (Broad Insti- tute) and Amit Chaudhary PhD (Broad Institute) for advice. We thank Christopher Cahill (Joslin Advanced Microscopy Core) for assistance with im- aging experiments. Funding R N K k R.N.K. acknowledges support from NIH Grants RO1 DK067536, RO1 DK117639, RO1 DK105588, and UC4 DK 116278 and the Joslin DRC Cores including the Molecular Phenotyping and Genotyping, the Advanced Microscopy and Bioinformatics Cores (P30 DK 36836). Additional file 3: Figure S1. Ambient contribution and islet cell gene marker UMAP and violin plots in scRNA-seq and snRNA-seq in cultured human islets. (A,B) Box plots representing the levels of ambient RNA con- tamination in droplets of (A) scRNA-seq and (B) snRNA-seq datasets. (C) Violin plot representing the duplication rate in scRNA-seq (orange plot) and snRNA-seq (blue plot) methodologies. The rate was calculated by normalizing the average number of UMI per cell/nucleus on the average number of reads per cell/nucleus. (D, F, H, J) UMAP plots displaying ex- pression levels of (D) GCG, (F) INS, (H) PPY, and (J) SST within the global distribution in scRNA-seq (left panels) and snRNA-seq (right panels). Ex- pression levels are indicated as natural log of counts and range from 0 (gray) to 5 (purple). (E, G, I, K) Violin plots representing expression levels (natural log of counts, Y-axis) of (E) GCG, (G) INS, (I) PPY, and (K) SST within each cluster (X-axis) identified in scRNA-seq (orange plots) or snRNA-seq Conclusions We propose snRNA-seq as a reliable tool to explore the transcriptomic profile of human islets especially from frozen archived samples which may not be ideal for single-cell procedures. Supplementary Information Th li i i l The online version contains supplementary material available at https://doi. org/10.1186/s13073-021-00941-8. Authors’ contributions G.B., S.K., and E.D. designed the study, performed the experiments, and analyzed the data. G.B. and S.K. wrote the manuscript. H.P. and J.M.D. performed the bioinformatics analyses. R.N.K. designed the study, directed the project and edited the manuscript. All authors edited, read, and approved the final manuscript. Additional file 1: Table S1. Characteristics of human islet donors and human islet preparations used for single-cell or single-nucleus transcrip- tomic analysis. Additional file 1: Table S1. Characteristics of human islet donors and human islet preparations used for single-cell or single-nucleus transcrip- tomic analysis. Additional file 2: Table S2. Number of different types of droplets, reads and genes generated by scRNA-seq and snRNA-seq in cultured or transplanted human islets. Additional file 2: Table S2. Number of different types of droplets, reads and genes generated by scRNA-seq and snRNA-seq in cultured or transplanted human islets. Availability of data and materials All raw and processed snRNA-seq data generated from engrafted human islet tissues have been submitted to the NCBI Gene Expression Omnibus (GEO; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE150212) under accession number GSE150212. The publicly available scRNA-seq data- sets on human islets that were used in this the manuscript to integrate our scRNA-seq and snRNA-seq datasets have been deposited in the public Gen- omic Spatial Event Database (GSE) and in the ArrayExpress Database at the EMBL-EBI. For InDrop, accession number GSE84133 (https://www.ncbi.nlm. nih.gov/geo/query/acc.cgi?acc=GSE84133) [21]; for CelSeq2, accession num- ber GSE85241 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc= All raw and processed snRNA-seq data generated from engrafted human islet tissues have been submitted to the NCBI Gene Expression Omnibus (GEO; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE150212) under accession number GSE150212. The publicly available scRNA-seq data- sets on human islets that were used in this the manuscript to integrate our scRNA-seq and snRNA-seq datasets have been deposited in the public Gen- omic Spatial Event Database (GSE) and in the ArrayExpress Database at the EMBL-EBI. For InDrop, accession number GSE84133 (https://www.ncbi.nlm. nih.gov/geo/query/acc.cgi?acc=GSE84133) [21]; for CelSeq2, accession num- ber GSE85241 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc= Page 15 of 17 Page 15 of 17 Basile et al. Genome Medicine (2021) 13:128 Basile et al. Genome Medicine (2021) 13:128 GSE85241) [23]; for SMART-Seq2, accession number E-MTAB-5061 (https:// www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-5061/) [26]; for Fluidigm C1, accession number GSE86469 (https://www.ncbi.nlm.nih.gov/geo/query/acc. cgi?acc=GSE86469) (27); and for CelSeq, accession number GSE81076 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE81076) [57] were used. GSE85241) [23]; for SMART-Seq2, accession number E-MTAB-5061 (https:// www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-5061/) [26]; for Fluidigm C1, accession number GSE86469 (https://www.ncbi.nlm.nih.gov/geo/query/acc. cgi?acc=GSE86469) (27); and for CelSeq, accession number GSE81076 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE81076) [57] were used. 9. Wang P, Alvarez-Perez JC, Felsenfeld DP, Liu H, Sivendran S, Bender A, et al. A high-throughput chemical screen reveals that harmine-mediated inhibition of DYRK1A increases human pancreatic beta cell replication. 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Author details 1 f l 1Section of Islet Cell and Regenerative Biology, Joslin Diabetes Center and Harvard Medical School, Boston, MA 02215, USA. 2Current Address: Department of Pharmacology, New York Medical College School of Medicine, Valhalla, NY 10595, USA. 3Bioinformatics and Biostatistics Core, Joslin Diabetes Center and Harvard Medical School Boston MA USA 1Section of Islet Cell and Regenerative Biology, Joslin Diabetes Center and Harvard Medical School, Boston, MA 02215, USA. 2Current Address: Department of Pharmacology, New York Medical College School of Medicine, Valhalla, NY 10595, USA. 3Bioinformatics and Biostatistics Core, Joslin Diabetes Center and Harvard Medical School, Boston, MA, USA. 4Department of Medicine, Brigham and Women’s Hospital, Boston, MA, USA 5Harvard Stem Cell Institute, Harvard Medical School, Boston, MA, USA. 20. Zeng C, Mulas F, Sui Y, Guan T, Miller N, Tan Y, et al. Pseudotemporal Ordering of Single Cells Reveals Metabolic Control of Postnatal β Cell Proliferation. Cell Metab. 2017;25(5):1160–1175.e11. 21. Baron M, Veres A, Wolock SL, Faust AL, Gaujoux R, Vetere A, et al. A Single- Cell Transcriptomic Map of the Human and Mouse Pancreas Reveals Inter- and Intra-cell Population Structure. Cell Syst. 2016;3(4):346–60. https://www. ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE84133. https://doi.org/10.1016/j. cels.2016.08.011. 4Department of Medicine, Brigham and Women’s Hospital, Boston, MA, USA. 5 4Department of Medicine, Brigham and Women’s Hospital, Boston, MA, USA. 5Harvard Stem Cell Institute, Harvard Medical School, Boston, MA, USA. 4Department of Medicine, Brigham and Women’s Hospital, Boston, MA, USA. 5Harvard Stem Cell Institute, Harvard Medical School, Boston, MA, USA. 5Harvard Stem Cell Institute, Harvard Medical School, Boston, MA, USA. Received: 17 July 2020 Accepted: 13 July 2021 Received: 17 July 2020 Accepted: 13 July 2021 22. Li J, Klughammer J, Farlik M, Penz T, Spittler A, Barbieux C, et al. Single-cell transcriptomes reveal characteristic features of human pancreatic islet cell types. EMBO Rep. 2016;17(2):178–87. https://doi.org/10.15252/embr.201540946. Declarations 11. Ackeifi C, Wang P, Karakose E, Manning Fox JE, González BJ, Liu H, et al. GLP-1 receptor agonists synergize with DYRK1A inhibitors to potentiate functional human β cell regeneration. Sci Transl Med. 2020;12(530). Consent for publication Not applicable. 18. Mawla AM, Huising MO. Navigating the depths and avoiding the shallows of pancreatic islet cell transcriptomes. Diabetes. 2019;68(7):1380–93. https:// doi.org/10.2337/dbi18-0019. Competing interests The authors declare that they have no competing interests. 19. Qiu WL, Zhang YW, Feng Y, Li LC, Yang L, Xu CR. Deciphering Pancreatic Islet β Cell and α Cell Maturation Pathways and Characteristic Features at the Single-Cell Level. Cell Metab. 2017;25(5):1194–1205.e4. References 1. 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https://openalex.org/W4378714754	https://www.scielo.br/j/aabc/a/FxhsYdzmdpyRsNGTCmxMZkR/?lang=en&format=pdf	English	null	Biological behavior of Chrysomya putoria (Wiedemann, 1819) (Diptera: Calliphoridae) after refrigeration: Logistics for use in Biotherapy	Anais da Academia Brasileira de Ciências	2,023	cc-by	5,669	DANIELE L. DALLAVECCHIA, RENATO G. DA SILVA FILHO, ALEXANDRE S. SILVA & VALÉRIA M. AGUIAR Abstract: The influence of refrigeration on the post-embryonic development of Chrysomya putoria larvae was evaluated, regarding its resistance in the logistics of storage and distribution in biotherapy. Previously sterilized larvae were submitted to four periods of storage under refrigeration (T1=12 h, T2=24 h, T3=48 h and T4=72 h) and control (without sterilization and refrigeration). Newly hatched larvae (0.200 g) were stored between 3 and 9ºC. After refrigeration, 40 neo-larvae (in triplicate) were transferred to 50 g of protein diet and incubated in an acclimatized chamber. There was a significant difference in the larval body mass (T1 and T2) and in the duration of larval, pupal and total development (T3 and T4). The sex ratios found in the four treatments did not differ from what was expected. Normality rates were 100% for all treatments. There was no significant difference between the Control, T1 and T2 treatments for larval, pupal and total viability. There was a significant difference between control (C) and T4 (larval viability), between C, T3 and T4 (pupa) and between C and T4 (total). C. putoria has resistance under refrigeration and storage of up to 56 h, presenting viability above 70% for use in biotherapy. Key words: biotherapy, entomotherapy, storage, logistics. An Acad Bras Cienc (2023) 95(Suppl. 1): e20220578 DOI 10.1590/0001-3765202320220578 Anais da Academia Brasileira de Ciências \| Annals of the Brazilian Academy of Sciences Printed ISSN 0001-3765 I Online ISSN 1678-2690 www.scielo.br/aabc \| www.fb.com/aabcjournal An Acad Bras Cienc (2023) 95(Suppl. 1): e20220578 DOI 10.1590/0001-3765202320220578 Anais da Academia Brasileira de Ciências \| Annals of the Brazilian Academy of Sciences Printed ISSN 0001-3765 I Online ISSN 1678-2690 www.scielo.br/aabc \| www.fb.com/aabcjournal An Acad Bras Cienc (2023) 95(Suppl. 1) Biological behavior of Chrysomya putoria (Wiedemann, 1819) (Diptera: Calliphoridae) after refrigeration: Logistics for use in Biotherapy DANIELE L. DALLAVECCHIA, RENATO G. DA SILVA FILHO, ALEXANDRE S. SILVA & VALÉRIA M. AGUIAR DANIELE L. DALLAVECCHIA, RENATO G. DA SILVA FILHO, ALEXANDRE S. SILVA & MATERIALS AND METHODS several beneficial actions of biotherapy on these lesions: debridement of necrotic tissue (Mumcuoglu 2001), microbial decontamination (Sherman 2014, Nigam et al. 2010), preventing the development of bacterial resistance (Chernysh et al. 2015), immunomodulation of the local inflammatory response (Elkington et al. 2009, Chernysh et al. 2012), anti-inflammatory activity (Van Der Plas et al. 2009), pro-angiogenic activity (Wang et al. 2020, Morgan & Nigam 2013), proliferation and migration of fibroblasts for extracellular matrix remodelling and stimulation of granulation tissue (Prete 1997), and disaggregation action of bacterial biofilms (Van der Plas et al. 2008). The study was conducted at the Laboratório de Estudos de Dípteros (LED) (Diptera Study Laboratory) and at the Laboratório de Microbiologia do Departamento de Microbiologia e Parasitologia do Instituto Biomédico da Universidade Federal do Estado do Rio de Janeiro (UNIRIO). The sterilization of the egg masses (0.200g) was performed with 2% glutaraldehyde, a liquid sterilant commonly used in hospital materials and which has a fast and effective action on gram-positive and gram-negative bacteria, mycobacteria, fungi and viruses (Gomes et al. 2007). Sterile newly hatched larvae from eggs sterilized according to a previously developed protocol (Dallavecchia et al. 2014) were placed in Petri dishes containing gauze and a filter paper disc moistened with sterile saline in triplicate (Fig. 1a, b). Then, the plates were transferred to a refrigerator with temperature and humidity recorded by a thermohygrograph (Sigma II) and kept for the following time periods: 12, 24, 48 and 72 h (Fig. 1c) which corresponded to T1, T2, T3 and T4 treatments, respectively. The control consisted in the use of larvae without sterilization and without refrigeration, in triplicate, kept in a climatized chamber under controlled conditions an acclimatized chamber (Quimis) regulated at 30 ºC/day and 28 ºC/night, with 60±10% relative humidity and 12 h of photophase (beginning at 6:00 am). The choice of a dipteran species that has ideal biological characteristics for biotherapy is fundamental. Thus, the Chrysomya putoria (Weidmann 1819) species was selected for this study, which is a necrophagous dipteran found in abundance in Brazil, mainly in urban centers (Marinho et al. 2006). The mastery of breeding techniques for this species enables obtaining females with a high capacity for oviposition, generating a large amount of egg masses to be submitted to the sterilization process, which is an important prerequisite for its use in biotherapy. INTRODUCTION potential therapeutic use have underpinned the emergence of a series of companies created to explore insect-derived bioactives, the so-called “Drugs from bugs” (Zainzinger 2019). Nature’s pharmacy is vast and its products have been used throughout human history as raw material for use in traditional medicine. Plants were the first to be used by early humans in the search for cures of diseases, and after positive experiences with medicinal formulations obtained from vegetables, early humans sought to use products of animal origin. Today, we have several medicines that are derived from plants, microorganisms, algae or animals, such as aspirin, penicillin, cytarabine, heparin, and captopril, among others (Ratcliffe et al. 2011). Although insects are seen as pests in many crops, recent developments in the identification and bioengineering of natural products with The best known among the natural therapies that use insects is biotherapy, also known as larval therapy or larval debridement therapy. This consists in applying sterile live larvae of necrophagous flies (Diptera: Calliphoridae) in wounds with devitalized tissues in order to remove the necrotic material and promote local healing. Many health professionals have recognized the importance of this biotherapy, especially in cases of wounds that are difficult to heal or infected with antimicrobial-resistant bacteria. Clinical-laboratory studies point to C. putoria AFTER REFRIGERATION: USE IN BIOTHERAPY DANIELE L. DALLAVECCHIA et al. An Acad Bras Cienc (2023) 95(Suppl. 1) e20220578 2 \| 9 RESULTS The maximum temperature recorded in the refrigerator during the experiment was 9 ºC and the minimum was 3 ºC, while the maximum air humidity was 100% and the minimum was 19% (Fig. 2). The body mass of third instar larvae that abandoned the diet (treated and control) differed significantly between treatments (p < 0.05) (Table I), the same we can see duration of larval stages. The development time of T4 larvae was significantly longer than in the other treatments and in the control (p < 0.01). In pupal stage the treatments T3 and T4 was lower than T1, T2 and control treatments, and the difference was significantly. The complete developmental stage was significantly higher for T4 (p < 0.01). Figure 1. a) Petri dish with gauze and filter paper; b) Petri dish containing sterilized eggs of Chrysomya putoria in the hatching phase on filter paper and gauze moistened with sterile saline; c) Thermohygrograph; d) and e) 40 first instar larvae incubated in beakers containing chicken gizzard for all treatments; and f) Test tube with C. putoria larvae and wood shavings after diet abandonment. Figure 1. a) Petri dish with gauze and filter paper; b) Petri dish containing sterilized eggs of Chrysomya putoria in the hatching phase on filter paper and gauze moistened with sterile saline; c) Thermohygrograph; d) and e) 40 first instar larvae incubated in beakers containing chicken gizzard for all treatments; and f) Test tube with C. putoria larvae and wood shavings after diet abandonment. Figure 1. a) Petri dish with gauze and filter paper; b) Petri dish containing sterilized eggs of Chrysomya putoria in the hatching phase on filter paper and gauze moistened with sterile saline; c) Thermohygrograph; d) and e) 40 first instar larvae incubated in beakers containing chicken gizzard for all treatments; and f) Test tube with C. putoria larvae and wood shavings after diet abandonment. an acclimatized chamber regulated at 30 ºC/day and 28 ºC/night, with 60±10% relative humidity and 12 h of photophase (beginning at 6:00 am) to observe the viability of the immature stages and larval, pupal and total development. After abandoning the diet, samples with five larvae (3rd instar) had their body mass determined using then weighed on semi- analytical scales, then were transferred to test tubes with sterilized wood shavings until the insects emerged (Fig. 1f). All five treatments (Control, T1, T2, T3 and T4) were performed in triplicate. MATERIALS AND METHODS In addition, the possibility of storing the larvae obtained under refrigeration improves their distribution logistics for use (Dallavecchia et al. 2015, Ferraz et al. 2014). Thus, the objective of this work was to evaluate the larval, pupal and total viability, as well as the body mass of the larvae, the development of the insects after storage at low temperatures for different time periods (12, 24, 48 and 72 h) and the sex ratio of adult insects of the C. putoria species. The plates were removed from the refrigerator after 12, 24, 48 and 72h, then 40 first- instar larvae were transferred to 50 g of chicken gizzards placed in 100 mL beakers. These were inserted into beakers larger than 500mL with sterilized wood shavings as a substrate for pupariation, and then sealed with scaline fabric secured with an elastic band and labelled (Fig. 1d, e). The rearing containers were transferred to An Acad Bras Cienc (2023) 95(Suppl. 1) e20220578 2 \| 9 C. putoria AFTER REFRIGERATION: USE IN BIOTHERAPY DANIELE L. DALLAVECCHIA et al. Figure 1. a) Petri dish with gauze and filter paper; b) Petri dish containing sterilized eggs of Chrysomya putoria in the hatching phase on filter paper and gauze moistened with sterile saline; c) Thermohygrograph; d) and e) 40 first instar larvae incubated in beakers containing chicken gizzard for all treatments; and f) Test tube with C. putoria larvae and wood shavings after diet abandonment. The Microsoft Excel program was used to analyze the raw data and the other analyses were performed with the R program, version 3.4.4. Variations in mean larval body mass and duration of larval, pupal and total development stages (from first instar to adult) were analyzed by Kruskal-Wallis test followed by Pairwise comparisons using Wilcoxon rank sum test with adjust p-value by BH method (Benjamini & Hochberg 1995). The viability was analyzed by proportion test. In all tests a significance level of 0.05 was considered. The sex ratio (SR) was determined as follows: SR = F/M+F, where F is the number of females and M is the number of males. The expected frequency (%) is a 1:1 ratio (= 0.50). The normality of the insects, that is, the absence of morphological changes, were also observed in terms of their frequency. An Acad Bras Cienc (2023) 95(Suppl. 1) e20220578 3 \| 9 RESULTS The average time of development from neo-larvae to adult was: 8.113 days for control; 8.104 days for T1; 8.067 days for T2; 8.063 days for T3; and 9.138 days for T4 (Table I). The diet abandonment rate of C. putoria larvae reached its peak on the 4th day after the beginning of An Acad Bras Cienc (2023) 95(Suppl. 1) e20220578 3 \| 9 C. putoria AFTER REFRIGERATION: USE IN BIOTHERAPY DANIELE L. DALLAVECCHIA et al. Figure 2. a) Record of the variation of temperature and b) Record of the variation of relative humidity of the air during the days of experimentation. Figure 2. a) Record of the variation of temperature and b) Record of the variation of relative humidity of the air during the days of experimentation. the experiment in all treatments, indicating homogeneity in the development of these insects. Only 8 (n=120) larvae from the control and 17 (n=120) from the T1 abandoned on the 3rd day. Pupariation peaked on the 5th day after the start of the experiment in all treatments, except for T4, which peaked on the 6th day. The emergence peak in all treatments was on the 8th day; however, emergence at T4 started on the 9th day (Fig. 3). without the danger of these interfering with the therapeutic action of the larvae (Ferraz et al. 2014). It is indicated for different types of infected wounds and its effectiveness has already been widely proven (Sherman et al. 2013, Siribumrungwong et al. 2018, Borkataki et al. 2018). Another increasingly important use of larval therapy is in the context of wars and disasters where production of medicinal maggots needs to be located close to the point of care (Stadler et al. 2016). In these extreme situations, it is important to know the entire process of rearing, maintaining, sterilizing and storing the larvae (Stadler 2020). The larval viability differed significantly between the treatments tested (p < 0.05), being significantly lower for T4 (70%) when compared to the other treatments (p < 0.05). Could be observed in pupal viability significantly difference between control, T1 and T2 against T3 and T4 (p < 0.05). In T4 (72h of refrigeration) only 38% of the insects reached adulthood (p < 0.05), there was significantly difference between other treatments. The sex ratio of adult insects was below the expected value (SR= 0.5) for the control - SR= 0.38 and T3 - SR= 0.33. RESULTS Normality was 100% for all insects in all treatments (Table II). Mumcuoglu et al. (2001) reported the survival of larvae (1st intar) of Lucilia sericata for up to 5 days at a temperature between 5 and 8 ºC without interfering with their viability. The same could be observed in the present study after the storage of larvae from sterilized eggs under a minimum temperature that varied between 3 and 9 ºC for a period of up to 72 h. The storage of newly hatched larvae from sterilized eggs enables these sterile larvae to be sent to places far from their original place of production. The low temperature slows the metabolism of the larvae, delaying their development and allowing them to withstand starvation longer, DISCUSSION ** Control - without treatment, T1- sterile larvae submitted to 12 h of refrigeration, T2- sterile larvae to 24 h of refrigeration, T3- sterile larvae submitted to 48 h of refrigeration, T4- sterile larvae submitted to 72 h of refrigeration (experiments with 40 first instar larvae in triplicate). thereby reaching their final destination in viable condition. conditions. This fact shows that the egg sterilization process followed by refrigeration did not affect the duration of the post- embryonic development stages, nor the larval abandonment rate, which is the most important phase for biotherapy. Larvae stay in patient wounds for up to 72 h (Sherman 2014), which is the period of time they need to consume enough food for their full development. After this period, in nature or in the laboratory, the larvae abandon the diet to pupate. With this, it is necessary that the dressings of the patients are changed after 72 h, for the larvae to be removed from the wound bed. This periodicity is also convenient for the maintenance of colonies in the laboratory. Ferraz et al. (2016) obtained a similar mean body mass (control - 0.560 mg, T1 - 0.560 mg and T2 - 0.1520 mg) in testing different diets for C. putoria larvae to that found in the present study. This fact shows that, although there was a significant difference in the body mass of T1 and T3 compared to the control, the larvae remained healthy, even after the egg sterilization, starvation and refrigeration process, reaching equivalent body masses to those obtained under non-adverse conditions, reflecting an adequate post-embryonic growth. Regarding development, the larvae almost entirely abandoned the diet on the 4th day in all treatments. The same could be observed with pupariation, which mostly peaked on the 5th day, with the exception of T4, which peaked on the 6th day. The birth of adults occurred on the 8th day after beginning the experiment for the control and T1, T2 and T3 treatments. The exception was T4, which had its birth peak on the 9th day. Although T4 had pupated and emerged within a day of the other treatments, the development generally showed homogeneity in all evaluated In one study to evaluate the post-embryonic behavior of C. putoria larvae in different diets under an average temperature of 20.6 ºC and an average Relative Humidity of 67.7%, Ferraz et al. An Acad Bras Cienc (2023) 95(Suppl. 1) e20220578 5 \| 9 DISCUSSION Chronic wound debridement therapy with larvae is simple, and highly effective. It can even be used in patients undergoing antibiotic therapy An Acad Bras Cienc (2023) 95(Suppl. 1) e20220578 4 \| 9 C. putoria AFTER REFRIGERATION: USE IN BIOTHERAPY DANIELE L. DALLAVECCHIA et al. Table I. Mean (standard deviation) body mass (mg) and mean duration of post-embryonic development stages* of Chrysomya putoria from control and refrigeration treatments** reared in chicken gizzards. Body mass and duration of stages in days Treatment Mass (g) Larvae (days) Pupae (days) Adult (days)  ± p  ± p  ± p  ± p Control 0.053a ± 0.008 5.025a ± 0.406 3.138a ± 0.351 8.113a ± 0.166 T1 (12 h) 0.056b ± 0.004 4.904a ± 0.318 3.313a ± 0.469 8.104a ± 0.148 T2 (24h) 0.052a ± 0.001 5.000a ± 0 3.067a ± 0.105 8.067a ± 0.105 T3 (48h) 0.057b ± 0.003 5.000a ± 0 3.027b ± 0.128 8.063a ± 0.179 T4 (72h) 0.053a ± 0.002 6.160b ± 0.339 3.000b ± 0 9.138b ± 0.341 Means followed by the same letter in the same column do not differ significantly by Kruskal-Wallis followed by Pairwise comparisons. * Control - without treatment, T1- sterile larvae submitted to 12 h of refrigeration, T2- sterile larvae to 24 h of refrigeration, T3- sterile larvae submitted to 48 h of refrigeration, T4- sterile larvae submitted to 72 h of refrigeration (experiments with 40 first instar larvae in triplicate). Table I. Mean (standard deviation) body mass (mg) and mean duration of post-embryonic development stages* of Chrysomya putoria from control and refrigeration treatments** reared in chicken gizzards. Means followed by the same letter in the same column do not differ significantly by Kruskal-Wallis followed by Pairwise comparisons. * Control - without treatment, T1- sterile larvae submitted to 12 h of refrigeration, T2- sterile larvae to 24 h of refrigeration, T3- sterile larvae submitted to 48 h of refrigeration, T4- sterile larvae submitted to 72 h of refrigeration (experiments with 40 first instar larvae in triplicate). Means followed by the same letter in the same column do not differ significantly by Kruskal-Wallis followed by Pairwise comparisons. DISCUSSION (2012) observed that the larvae completed their biological cycle (larva to adult) in 8.88 days for insects raised on beef; 8.68 days in chicken gizzards; and 9.07 days in gizzard agar. The larvae in the present study completed their total cycle more quickly, completing it in 8.113 C. putoria AFTER REFRIGERATION: USE IN BIOTHERAPY DANIELE L. DALLAVECCHIA et al. Figure 3. Abandonment rate of mature larvae from the diet, pupariation and emergence of C. putoria after sterilization of eggs with glutaraldehyde and submitted to refrigeration (treatments: T1 - 12 h, T2 - 24 h, T3 - 48 h and T4 - 48 h). In the control, eggs without sterilization and submitted to refrigeration. Figure 3. Abandonment rate of mature larvae from the diet, pupariation and emergence of C. putoria after sterilization of eggs with glutaraldehyde and submitted to refrigeration (treatments: T1 - 12 h, T2 - 24 h, T3 - 48 h and T4 - 48 h). In the control, eggs without sterilization and submitted to refrigeration. days for the control, 8.104 days for T1, 8.067 days for T2, and 8.063 days for T3; however, the insects in T4 needed a longer time (9.138 days) to complete their development. This acceleration in development time is probably due to the higher temperature of the climatized chamber (30 ºC day/28 ºC night) where the insects were kept after the refrigeration process. However, the insects required a longer time period in T4, which can be explained by a longer refrigeration period (72 h), significantly interfering in this biological parameter. These results generally showed that C. putoria larvae have great resistance to the sterilization, starvation and refrigeration process without affecting their biological cycle, except when submitted to 72 h of refrigeration. low temperatures (between 3 and 9 ºC). Even so, 70% of the larvae were able to adapt to the adverse conditions (previous sterilization process, starvation, low temperature) to which they were subjected. The high viability of C. putoria larvae observed in the T1 to T3 treatments is of paramount importance for implementing larval therapy. This is because the larvae are subjected to processing before being applied to the wounds which could compromise their viability, such as egg separation (mechanical disturbance), chemical sterilization, rinsing and filtration at the end of sterilization, and storage at low temperature (physical stress) (Dallavecchia et al. 2019). An Acad Bras Cienc (2023) 95(Suppl. 1) e20220578 6 \| 9 DISCUSSION Laboratories which commercially produce and distribute larvae for medicinal use generally need to maintain larval viability for a considerable period of time, since the larvae are highly perishable and go through a long The significant difference observed in T4 larval viability (70%) was probably due to the fact that the larvae remained without any food for up to 72 h after hatching and were kept at C. putoria AFTER REFRIGERATION: USE IN BIOTHERAPY DANIELE L. DALLAVECCHIA et al. Table II. Viability of larval, pupal and total stages of Chrysomya putoria reared in chicken gizzards in treatments (T1-12 h, T2-24 h, T3-48 h, T4-72 h after sterilization and refrigeration, Control - larvae from eggs without treatment) Table II. Viability of larval, pupal and total stages of Chrysomya putoria reared in chicken gizzards in treatments (T1-12 h, T2-24 h, T3-48 h, T4-72 h after sterilization and refrigeration, Control - larvae from eggs without treatment). % Viability Treatment Cooling time(h) Larvae Pupae Adults  Female  Male Sex Ratio** C Control 95,00a 97,36a 92,50a 14 23 0,38 T1 12 95,00a 94,73a 90,00a 17 19 0,47 T2 24 95,00a 89,47a 87,50a 16 19 0,46 T3 48 95,00a 78,94b 75,00a 10 20 0,33 T4 72 70,00b 53,57b 37,50b 8,7 6 0,59 * Larval, pupal and adult viability were analyzed by the proportion test and a significance level of 0.05 was considered. *The Sex Ratio (SR) was obtained as follows: SR = F / M + F, where F is the number of females and M the number of males. Larval, pupal and adult viability were analyzed by the proportion test and a significance level of 0.05 was considered. *The Sex Ratio (SR) was obtained as follows: SR = F / M + F, where F is the number of females and M the number of males. Larval, pupal and adult viability were analyzed by the proportion test and a significance level of 0.05 was considered. **The Sex Ratio (SR) was obtained as follows: SR = F / M + F, where F is the number of females and M the number of males. logistical process until they reach their final destination, which can be in another city or even country, including dispatch by air. The recommended time period for the larvae to be distributed and applied to the wound is up to 48 h (Stadler 2020). DISCUSSION undergo chemical or refrigeration processes, which were the objectives of this study. The sex ratio in T1, T2 and T3 treatments was as expected (0.5), based on the formula presented in the methodology, indicating stability in the population. Fisher (1930) advocates that there will only be stability in the population when the sex ratio is 1:1; a deviated sex ratio is not evolutionarily stable, because there will be a disproportion in the sex of individuals in the population in future generations. The control and T3 had a lower sex ratio than expected. Although this fact is relevant for maintaining dipteran colonies in the laboratory, it does not affect the development of larvae for biotherapy which feed voraciously when inserted into the bed of infected wounds. The pupal viability ranged from 97,36% to 78,94% in all treatments, with the exception of T4, where only 53,57% of the insects pupated. This same behaviour could be observed in the work by Ferraz et al. (2011) when testing three diets for raising Diptera, where pupal viability ranged from 98% to 71%. Furthermore, approximately 90% of flies successfully completed development to adult stage in the control, T1, and T2 groups, but only 75% in the T3 and 37,50% in the T4 group. Total viability was lower for T4, which can be explained by the starvation time (72 h) to which the larvae were submitted. Feeding after hatching larvae is extremely important for the normal life cycle of dipterans. Although T4 showed a total viability below 50%, this fact is not important for larval therapy, since adult insects are not used in biotherapy, only larvae. We can conclude in this study that C. putoria larvae are very resistant to the various stressful logistical processes for their use in biotherapy. They remained healthy after feeding and development even in a state of starvation. The observed viability of almost 70% after 72 h at low temperatures allows enough time to transport the larvae to distant locations until their final destination: the patient. 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FERRAZ ACP, DALLAVECCHIA DL, SILVA DC, CARVALHO RP, SILVA FILHO RG & AGUIAR-COELHO VM. 2012. Alternative diets for Chrysomya putoria, an old world screwworm fly. J Insect Sci 12: 1-11. SHERMAN RA, MUMCUOGLU KY, GRASSBERGER M & TANTAWI TI. 2013. Maggot Therapy. Biotherapy - History, Principles and Practice. In: Grassberger M, Sherman R, Gileva O, Kim C & Mumcuoglu K (Eds), Springer, Dordrecht, p. 5-29. SIRIBUMRUNGWONG B, WILASRUSMEE C & RERKASEM K. 2018. Maggot Therapy in Angiopathic Leg Ulcers: A Systematic SHERMAN RA, MUMCUOGLU KY, GRASSBERGER M & TANTAWI TI. 2013. Maggot Therapy. Biotherapy - History, Principles and Practice. In: Grassberger M, Sherman R, Gileva O, Kim C & Mumcuoglu K (Eds), Springer, Dordrecht, p. 5-29. SIRIBUMRUNGWONG B, WILASRUSMEE C & RERKASEM K. 2018. Correspondence to: Daniele Lourinho Dallavecchia E-mail: danieledallavecchia@gmail.com Correspondence to: Daniele Lourinho Dallavecchia E-mail: danieledallavecchia@gmail.com ZAINZINGER V. 2019. Drugs from bugs Chemistry & Industry. Power Insect SCI Issue 10. Available AT: https://www.soci. org/chemistry-and-industry/cni-data/2019/10/drugs- from-bugs. Accessed: 02/04/2022. VALÉRIA M. AGUIAR2 https://orcid.org/0000-0003-3765-3630 VAN DER PLAS MJA, BALDRY M, VAN DISSEL JT, JUKEMA GN & NIBBERING PH. 2009. Maggot secretions suppress pro- inflammatory responses of human monocytes through elevation of cyclic AMP. Diabetologia 52: 1962-1970. 1Instituto de Investigação, Inovação e Desenvolvimento (FP- I3ID), Universidade Fernando Pessoa (UFP), Escola Superior de Saúde, Praça de 9 de Abril, 349, 4249-004 Porto, Portugal 2Universidade Federal do Estado do Rio de Janeiro (UNIRIO), Departamento de Microbiologia e Parasitologia, Rua Frei Caneca, 94, Centro, 20211-040 Rio de Janeiro, RJ, Brazil 3Universidade Federal do Estado do Rio de Janeiro (UNIRIO), Departamento de Métodos Quantitativos, Av. Pasteur, 458, Urca, 22290-255 Rio de Janeiro, RJ, Brazil 1Instituto de Investigação, Inovação e Desenvolvimento (FP- I3ID), Universidade Fernando Pessoa (UFP), Escola Superior de Saúde, Praça de 9 de Abril, 349, 4249-004 Porto, Portugal VAN DER PLAS MJA, JUKEMA GN, WAI SW, DOGTEROM- BALLERING HCM, LAGENDIJK EI, VAN GULPEN C, VAN DISSEL JT, BLOEMBERG GV & NIBBERING PH. 2008. Maggot excretions/ secretions are differentially effective against biofilms of Staphylococcus aureus and Pseudomonas aeruginosa. J Antimicrob Chemother 61: 117-122. VAN DER PLAS MJA, JUKEMA GN, WAI SW, DOGTEROM- BALLERING HCM, LAGENDIJK EI, VAN GULPEN C, VAN DISSEL JT, BLOEMBERG GV & NIBBERING PH. 2008. Maggot excretions/ secretions are differentially effective against biofilms of Staphylococcus aureus and Pseudomonas aeruginosa. J Antimicrob Chemother 61: 117-122. 2Universidade Federal do Estado do Rio de Janeiro (UNIRIO), Departamento de Microbiologia e Parasitologia, Rua Frei Caneca, 94, Centro, 20211-040 Rio de Janeiro, RJ, Brazil 3Universidade Federal do Estado do Rio de Janeiro (UNIRIO), Departamento de Métodos Quantitativos, Av. Pasteur, 458, Urca, 22290-255 Rio de Janeiro, RJ, Brazil WANG TY ET AL. 2020. Maggot excretions/secretions promote diabetic wound angiogenesis via miR18a/19a – TSP-1 axis. Diabetes Res Clin Pract 165: 108-140. Author contributions Daniele Lourinho Dallavecchia - Theoretical research, research execution, data analysis and article writing. Renato Geraldo da Silva Filho - Data analysis and article writing. Alexandre Sousa da Silva - Data analysis, mainly the statistical part and writing of the article. Valéria Magalhães Aguiar - Theoretical research, data analysis and article writing. REFERENCES Maggot Therapy in Angiopathic Leg Ulcers: A Systematic SHERMAN RA, MUMCUOGLU KY, GRASSBERGER M & TANTAWI TI. 2013. Maggot Therapy. Biotherapy - History, Principles and Practice. In: Grassberger M, Sherman R, Gileva O, Kim C & Mumcuoglu K (Eds), Springer, Dordrecht, p. 5-29. FERRAZ ACP, DALLAVECCHIA DL, SILVA DC, FIGUEIREDO AL, PROENÇA B, SILVA-FILHO RG, AGUIAR VM & EWER J. 2014. Effects of the antibiotics gentamicin on the postembryonic SIRIBUMRUNGWONG B, WILASRUSMEE C & RERKASEM K. 2018. Maggot Therapy in Angiopathic Leg Ulcers: A Systematic An Acad Bras Cienc (2023) 95(Suppl. 1) e20220578 8 \| 9 C. putoria AFTER REFRIGERATION: USE IN BIOTHERAPY DANIELE L. DALLAVECCHIA et al. C. putoria AFTER REFRIGERATION: USE IN BIOTHERAPY Review and Meta-Analysis. Int J Low Extrem Wounds 17: 227-235. Review and Meta-Analysis. Int J Low Extrem Wounds 17: 227-235. DANIELE L. DALLAVECCHIA1,2 https://orcid.org/0000-0002-8982-3315 DANIELE L. DALLAVECCHIA1,2 https://orcid.org/0000-0002-8982-3315 STADLER F. 2020. The maggot therapy supply chain: a review of the literature and practice. Med Vet Entomol 34: 1-9. RENATO G. DA SILVA FILHO2 https://orcid.org/0000-0001-6960-9314 ALEXANDRE S. SILVA3 https://orcid.org/0000-0002-5573-4111 STADLER F, SHABAN R & TATHAM P. 2016. Maggot Debridement Therapy in Disaster Medicine. Prehosp Disaster Med 31: 79-84. Manuscript received on July 26, 2022; accepted for publication on November 11, 2022 An Acad Bras Cienc (2023) 95(Suppl. 1) e20220578 9 \| 9 How to cite DALLAVECCHIA DL, DA SILVA FILHO RG, SILVA AS & AGUIAR VM. 2023. Biological behavior of Chrysomya putoria (Wiedemann, 1819) (Diptera: Calliphoridae) after refrigeration: Logistics for use in Biotherapy. An Acad Bras Cienc 95: e20220578. DOI 10.1590/0001-3765202320220578. Manuscript received on July 26, 2022; accepted for publication on November 11, 2022 An Acad Bras Cienc (2023) 95(Suppl. 1) e20220578 9 \| 9 An Acad Bras Cienc (2023) 95(Suppl. 1) e20220578 9 \| 9
https://openalex.org/W2345628103	http://ojs.aas.bf.uni-lj.si/index.php/AAS/article/download/198/127	Slovene	null	Camelina (Camelina sativa (L.) Crantz) oilcake – untapped resource of phenolic compounds	Acta agriculturae Slovenica	2,016	cc-by	5,005	Petra TERPINC1, Helena ABRAMOVIČ2 Petra TERPINC1, Helena ABRAMOVIČ2 Received December 23, 2015; accepted January 04, 2016. Delo je prispelo 23. decembra 2015, sprejeto 04. januarja 2016. IZVLEČEK Delo zajema celovito študijo fenolnih spojin, njihovo zastopanost in identifikacijo v preostankih po stiskanju olja iz semen navadnega rička slovenskega porekla, to je v oljni pogači. Poleg tega poda rezultate določitve njihove učinkovitosti z metodami, ki vključujejo različne mehanizme antioksidativnega delovanja. Izkazalo se je, da se po stiskanju olja večina fenolnih spojin akumulira v pogači. Potrjena je bila prisotnost številnih antioksidantov: sinapina, 4-vinilgvajakola, 4-vinilkatehola, 4-vinilfenola, 4-vinilsiringola, elagne kisline, protokatehulne kisline, p-hidroksibenzojske kisline, sinapinske kisline, salicilne kisline, katehina, kvercetina in kvercetin glukozida. Izvlečki pogače so pokazali dobro redukcijsko moč in sposobnost lovljenja radikalov. Toplotna obdelava semen vpliva na količino prostih fenolnih spojin, topnih konjugatov in netopno vezanih fenolov, kot tudi na antioksidativno učinkovitost posameznih frakcij. Tudi primerjava z ostalimi oljnicami in sintetičnim antioksidantom opravičuje smotrnost uporabe ričkove pogače v živilski industriji. The work includes a comprehensive study of phenolic compounds, their occurrence and identification in the residues after pressing of the oil from camelina seeds of Slovenian origin, i.e. oilcake. In addition, the efficiencies of antioxidant determinations using different methods according to different mechanisms are presented. These data demonstrate that almost all of the phenolic compounds in these seeds remain in the seed oilcake. The following antioxidants were confirmed: sinapine, 4-vinylphenol, 4-vinylguaiacol, 4-vinylsyringol, 4- vinylcatechol, ellagic acid, protocatechuic acid, p- hydroxybenzoic acid, sinapic acid, salicylic acid, catechin, quercetin and quercetin glucoside. The oilcake has high reducing power and radical scavenging activity. Heat treatment of seeds affects the amount of free, soluble and insoluble bound phenolic compounds as well as antioxidant capacity of individual fractions. Potential applications of camelina oilcake in the food industry are further justified by comparisons with other oilcakes and synthetic antioxidant. Ključne besede: navadni riček (Camelina sativa (L.) Crantz), oljna pogača, antioksidativno delovanje, fenolne spojine Ključne besede: navadni riček (Camelina sativa (L.) Crantz), oljna pogača, antioksidativno delovanje, fenolne spojine Key words: Camelina (Camelina sativa (L.) Crantz), oilcake, antioxidant activity, phenolic compound Key words: Camelina (Camelina sativa (L.) Crantz), oilcake, antioxidant activity, phenolic compound DOI: 10.14720/aas.2016.107.1.22 DOI: 10.14720/aas.2016.107.1.22 Agrovoc descriptors: camelina; camelina sativa; oilseed cakes; phenolic compounds; phenols; antioxidants Agrovoc descriptors: camelina; camelina sativa; oilseed cakes; phenolic compounds; phenols; antioxida Agris category code: Q04 1 asist. dr., Jamnikarjeva 101, SI-1000 Ljubljana, e-mail: petra.terpinc@bf.uni-lj.si 2 izr. prof. dr., Jamnikarjeva 101, SI-1000 Ljubljana, e-mail: helena.abramovic@bf.uni-lj.si 2 izr. prof. dr., Jamnikarjeva 101, SI-1000 Ljubljana, e-mail: helena.abramovic@bf.uni-lj.si Ključne besede: navadni riček (Camelina sativa (L.) Crantz), oljna pogača, antioksidativno delovanje, fenolne spojine Acta agriculturae Slovenica, 107 - 1, marec 2016 str. 243 - 250 Key words: Camelina (Camelina sativa (L.) Crantz), oilcake, antioxidant activity, phenolic compound 1 asist. dr., Jamnikarjeva 101, SI-1000 Ljubljana, e-mail: petra.terpinc@bf.uni-lj.si 1 UVOD Navadni riček (Camelina sativa (L.) Crantz), je oljnica, ki je na Koroškem znana z imenom toter (beseda izhaja iz nemškega izraza Dotter). Arheološke raziskave so potrdile, da so ga že v bronasti in železni dobi na območjih ob Severnem morju in ob Renu uporabljali za pripravo kašnatih jedi in kruha. Po sporadični gojitvi značilni za srednji vek, se je navadni riček v 20. stoletju razširil po deželah Evrope, kjer so iz semen pridobivali olje, proteinsko bogate oljne pogače pa so uporabljali za krmo živali (Zubr, 1997; Zubr, 2003a). Slovenija je ena redkih držav, kjer se je tradicionalna pridelava rička ohranila do danes, sejejo ga na Koroškem, medtem ko je drugod po Acta agriculturae Slovenica, 107 - 1, marec 2016 str. 243 - 250 Petra TERPINC, Helena ABRAMOVIČ Sloveniji skoraj neznan. Zaradi specifične prehranske vrednosti (velika vsebnost večkrat nenasičenih maščobnih kislin, prisotnost esencialnih in n-3 maščobnih kislin) se zanimanje za to alternativno oljnico v deželah Severne Evrope in Severne Amerike zadnja leta povečuje. Izsledki raziskav, ki so bile na olju in oljnih pogačah rička opravljene v Sloveniji (Abramovič in Abram, 2005; Abramovič in sod., 2007; Hrastar in sod., 2009; Hrastar in sod., 2011a; Hrastar in sod., 2011b; Hrastar in sod., 2013, Terpinc in sod., 2011a; Terpinc in sod., 2012a; Terpinc in sod., 2012b), opravičujejo potrebo po njegovi večji prepoznavnosti tudi na domačih tleh. Fenolne spojine, ki jih pridobimo iz semen, pogače in olja navadnega rička, bi se namreč lahko uporabile v živilski industriji za razvoj novih ter izboljšanje varnosti in kakovosti že obstoječih izdelkov. Posebno pozornost zasluži pogača, ki kot preostanek pri stiskanju olja predstavlja zanimiv in poceni naravni vir antioksidantov. Med nasičenimi maščobnimi kislinami prevladujeta palmitinska in stearinska kislina (Zubr, 1997). Uživanje ričkovega olja, kjer linolna kislina (LA; 18:2n – 6) predstavlja približno 15 %, α-linolenska kislina (ALA; 18:3n – 3) pa do 40 % vseh maščobnih kislin, pomembno prispeva k manjšemu razmerju med n-6 in n-3 maščobami v prehrani. Naslednja posebnost tega olja je relativno velika vsebnost gondojske kisline (20:1n-9) (Zubr in Matthäus, 2002). Olje navadnega rička je torej pomemben vir tako esencialnih nenasičenih (LA, ALA) kot n-3 maščobnih kislin (ALA) (Zubr in Matthäus, 2002). Med nasičenimi Olje navadnega rička se je tradicionalno uporabljalo kot domače zdravilo. Kot splošno krepilno sredstvo so ga predpisovali pri težavah zaradi rane na želodcu in dvanajsterniku, prav tako pa tudi pri raznih vnetjih ter za oskrbo opeklin in ran (Rode, 2001). 1 UVOD Svoje mesto je navadni riček našel celo v proizvodnji biogoriv, mil, barv in lakov (Zubr, 1997; Moser, 2010). Specifični dermatološki učinki večkrat nenasičenih maščobnih kislin pa omogočajo uporabo tega olja tudi v farmacevtski in kozmetični industriji kot sestavni del raznih olj, krem in losjonov (Schuster in Friedt, 1998; Zubr, 1997). Ričkovo olje se je ob uporabi zmerne temperature izkazalo kot primerno za kuhanje, pečenje, cvrenje, poskusno so ga uporabili tudi za izdelavo raznih namazov, solatnih prelivov, majonez, sladoleda (Zubr, 1997). Pri pripravi hrane, predvsem pa kot naravno ljudsko zdravilo ga nekateri domačini na Koroškem uporabljajo še danes. Navadni riček spada v družino križnic (Brassicaceae); ločimo ozimne in jare sorte (Zubr, 1997). Značilni so majhni rumeni cvetovi (približno 5 mm v premeru), plodovi hruškaste oblike vsebujejo več drobnih, rumeno rdečkastih semen, prevladuje samooprašitev (Schuster in Friedt, 1998). Semena vsebujejo med 35 in 45 ut. % olja. Viri navajajo, da je pridelek ričkovega olja na hektar do 907 litrov, kar je več kot pri sončnici in soji (Moser, 2010). Delež surovih proteinov v semenih navadnega rička je od 25 do 45 %, delež surovih vlaknin pa okoli 10 %. Kakovost semen je odvisna tako od sorte, kot tudi od rastnih razmer (Zubr, 2003b). Oljnica najbolje uspeva v zmernem podnebju, a dobro prenaša tudi sušo in nizke temperature. Ker je rastlina nezahtevna glede tal ter odporna na številne insekte, njeno gojenje ne zahteva visokih stroškov za gnojila in pesticide (Zubr, 2003b). Raziskave so potrdile, da je navadni riček primeren tudi za ekološko pridelavo (Kirkhus in sod, 2013). Abramovič in Abram (2005) sta ugotavljali vpliv razmer skladiščenja na potek oksidacije ričkovega olja slovenskega izvora. Stopnjo primarnih oksidacijskih produktov sta spremljali s peroksidnim, stopnjo sekundarnih produktov oksidacije pa z anizidinskim številom. Izkazalo se je, da je ričkovo olje dokaj stabilno. Naslednji poskus (Hrastar in sod., 2011a), ki je vključeval vzorce navadnega rička treh zaporednih sezon, je pokazal, da semena navadnega rička iz različnih lokacij Koroške vsebujejo 29 – 40 ut. % olja, medtem ko so avtorji v semenih potrdili tudi veliko vsebnost glukozinolatov, ki neugodno vplivajo na razvoj rakastih celic (Hayes in sod., 2008). Na podlagi dobljenih vrednosti za jodno in peroksidno število ter vsebnost prostih maščobnih Ričkovo ali totrovo olje je rumeno oranžne barve, značilnega vonja in okusa. Tradicionalno ga pridobivajo iz zmletih semen, ki jih pred stiskanjem zmešajo z vodo in toplotno obdelajo pri temperaturi do 90 °C. Acta agriculturae Slovenica, 107 - 1, marec 2016 44 2 OLJNA POGAČA NAVADNEGA RIČKA – MOŽNOSTI APLIKACIJE NA PODLAGI DOSEDANJIH RAZISKAV V okviru svoje raziskave so Terpinc in sod. (2012b) želeli ovrednotiti antioksidativni potencial pogače navadnega rička in pokazati, da je le-ta pomemben vir spojin z antioksidativno učinkovitostjo. Fenolne spojine so pridobili iz izbranih vzorcev s pomočjo ekstrakcije ob uporabi 80 % (v/v) metanola. Vsebnost skupnih fenolnih spojin so izrazili kot ekvivalent klorogenske kisline (KK) in ugotovili, da med stiskanjem olja večina fenolnih spojin preostane v pogači (1666 mg KK/100 g). Nekoliko manjše vrednosti, ki so jih določili v semenih, potrjujejo, da prisotnost vode in visoka temperatura med praženjem semen pospešita sprostitev fenolnih spojin iz vezanih oblik. S kvalitativno LC-MS analizo fenolnih spojin so določili zastopanosti le-teh v posameznih metanolnih izvlečkih pogače navadnega rička. Potrjena je bila prisotnost različnih pomembnih spojin. da je med praženjem prišlo do njune sprostitve iz konjugiranih in netopno vezanih oblik. Med fenolnimi kislinami so avtorji (Terpinc in sod., 2012b) v izvlečkih pogače navadnega rička uspešno identificirali tri hidroksibenzojske (protokatehulna, p-hidroksibenzojska in salicilna kislina) in eno hidroksicimetno kislino (sinapinska kislina). Slednja je bila že pred leti razglašena za enega najpomembnejših antioksidantov pogače navadnega rička (Salminen in sod., 2006). Med fenolnimi kislinami se je v olje prenesla v manjši meri le sinapinska kislina, medtem ko je bila prisotnost salicilne in p-hidroksibenzojske kisline skorajda zanemarljiva, protokatehulna pa celo pod pragom detekcije. Količina slednje je v pogači glede na semena precej večja, tudi zastopanost p- hidroksibenzojske in salicilne kisline je bila večja v pogači kot v semenih, kar nakazuje, da so visoke temperature omogočile omenjenim spojinam sprostitev iz vezanih oblik. Naslednji pomembni antioksidant, ki so ga uspešno identificirali v izvlečkih omenjene oljnice slovenskega porekla, je elagna kislina, ki ima v svoji strukturi štiri hidroksilne skupine, kar ji vsekakor omogoča velik potencial za lovljenje prostih radikalov (Hayes in sod., 2009). Med stiskanjem se le v manjši meri prenese v olje, v večji meri pa ostane v pogači. Sinapin je ester sinapinske kisline in holina, njegova prisotnost v pogači navadnega rička pa je bila potrjena že pred leti (Matthäus in Zubr, 2000; Salminen in sod., 2006). V raziskavi slovenskih vzorcev so ugotovili, da je vsebnost sinapina v pogači in semenih praktično enaka, medtem ko v olje ne prehaja, kar je v skladu tudi z drugimi publikacijami (Matthäus in Zubr, 2000). Zadnjo Katehin in kvercetin sta v naravi precej razširjena flavonoida, ki se ponašata z antimikrobno, antikarcenogeno in antimutageno aktivnostjo (Rice-Evans in sod., 1996). 1 UVOD Zanimanje za ričkovo olje je posledica njegove specifične sestave. Vsebuje 7- 14 % nasičenih, 26-41 % enkrat nenasičenih ter kar 46-64 % večkrat nenasičenih maščobnih kislin. 244 Acta agriculturae Slovenica, 107 - 1, marec 2016 244 Oljna pogača navadnega rička (Camelina sativa (L.) Crantz) – neizkoriščeni vir fenolnih spojin prašičev in prežvekovalcev; omejujoč dejavnik so prisotni glukozinolati (15-20 µmol/g pogače) (Schuster in Friedt, 1998; Zubr, 1997). Oljna pogača ima ugodno sestavo maščobnih kislin; izkazala se je kot pomemben vir vitaminov, mineralov in antioksidantov (Zubr, 2010; Matthäus, 2002). kislin, so avtorji zaključili, da se ričkovo olje lahko uporablja za najrazličnejše prehranske namene. Analizirano ričkovo olje je bilo nadalje izredno bogato z esencialno n-3 α-linolensko kislino (33,3 - 37,7 %) in γ –tokoferolom (532-798 mg/kg). Glavni namen gojenja navadnega rička je olje. Pogača je preostanek semen po stiskanju olja. Ker je bogata s proteini, se uporablja za krmo domačih živali, predvsem perutnine (Zubr, 1997; Zubr, 2003b). V omejenih količinah se lahko dodaja krmi Prispevek povzema rezultate raziskav, ki so bile opravljene na oljnih pogačah navadnega rička slovenskega porekla. 2 OLJNA POGAČA NAVADNEGA RIČKA – MOŽNOSTI APLIKACIJE NA PODLAGI DOSEDANJIH RAZISKAV Pri tem je potrebno upoštevati, da je bila koncentracija dodanih izvlečkov znatno pod dovoljeno za sintetične antioksidante v živilski industriji. Polifenoli so resda glavna komponenta rastlin z antioksidativnimi lastnostmi, a vsekakor ne edina (vitamin C, vitamin E, karotenoidi, itd.) (Moure in sod., 2001). Še več, celo vzorci s podobno koncentracijo skupnih fenolnih spojin neredko vsebuje pogača navadnega rička več fenolov kot pogača oljne ogrščice (Brassica napus L. var. napus) in lanu (Linum usitatissimum L.), vendar manj kot bela gorjušica (Sinapis alba L.), katero je kot najbogatejši vir med naštetimi razglasil že Matthäus (2002). Nadalje so avtorji z metodo določitve redukcijske sposobnosti ugotovili, da so spojine, ki se nahajajo v pogači navadnega rička, boljši reducenti od tistih v beli gorjušici, medtem ko sta se oljna ogrščica in lan izkazala bolje. DPPH· test je pokazal, da je izvleček navadnega rička primerljiv z izvlečki nekaterih drugih oljnic in učinkovitejši od BHT. Rezultati določitve sposobnosti keliranja so pokazali, da imajo fenolne spojine ekstrahirane iz navadnega rička boljšo sposobnost vezave kovinskih ionov od izvlečkov oljne ogrščice in bele gorjušice, medtem ko se je kot najučinkovitejši izkazal izvleček iz pogače lanu. V sistemu emulgirane linolne kisline v vodi in β-karotena so se antioksidanti prisotni v izvlečku pogače navadnega rička med vsemi testiranimi oljnicami izkazali kot najučinkovitejši, boljši od navadnega rička je bil le nepolarni BHT. Med začetno fazo oksidacije nenasičenih maščobnih kislin pride v maščobnokislinski verigi do premestitve dvojne vezi in nastanka konjugiranega sistema, ki absorbira svetlobo pri 234 nm. Vpliv dodanih metanolnih izvlečkov iz pogač testiranih oljnic oz. BHT na tvorbo konjugiranih dienov (primarnih produktov oksidacije) in trienov (sekundarnih produktov oksidacije) so avtorji omenjene raziskave (Terpinc in sod., 2012a) spremljali v olju barvilnega rumenika, žafranike (Carthamus tinctorius L.), ki so ga inkubirali pri temperaturi 50 °C. Salminen in sod. (2006) so ugotovili, da je izvleček iz pogače navadnega rička učinkovit antioksidant tako v preprečevanju proteinske kot lipidne oksidacije. Rezultati raziskave slovenskih vzorcev so potrdili, da je nastanek konjugiranih dienov in trienov v olju ob prisotnosti izvlečka pogače navadnega rička omiljen v primerjavi s kontrolo (olje brez dodatka antioksidantov), primerljiv s preostalimi izvlečki, vendar obsežnejši v primerjavi z BHT. Pri tem je potrebno upoštevati, da je bila koncentracija dodanih izvlečkov znatno pod dovoljeno za sintetične antioksidante v živilski industriji. Polifenoli so resda glavna komponenta rastlin z antioksidativnimi lastnostmi, a vsekakor ne edina (vitamin C, vitamin E, karotenoidi, itd.) (Moure in sod., 2001). Acta agriculturae Slovenica, 107 - 1, marec 2016 246 2 OLJNA POGAČA NAVADNEGA RIČKA – MOŽNOSTI APLIKACIJE NA PODLAGI DOSEDANJIH RAZISKAV O prisotnosti kvercetina v listih navadnega rička so pisali že Onyilagha in sod. (2003), medtem ko so o pomembnem antioksidativnem doprinosu flavonolov v pogači, zlasti kvercetin glikozidov, nekaj let kasneje poročali tudi Salminen in sod. (2006). Kot je pokazala analiza slovenskih vzorcev, se tako katehin, kot tudi kvercetin in kvercetin glukozid nahajajo v pogači, medtem ko je bilo v olju moč zaznati le manjše deleže katehina in kvercetina. Vsebnost slednjih dveh je bila v pogači večja kot v semenih, kar kaže na to, 245 Acta agriculturae Slovenica, 107 - 1, marec 2016 Petra TERPINC, Helena ABRAMOVIČ vsebuje pogača navadnega rička več fenolov kot pogača oljne ogrščice (Brassica napus L. var. napus) in lanu (Linum usitatissimum L.), vendar manj kot bela gorjušica (Sinapis alba L.), katero je kot najbogatejši vir med naštetimi razglasil že Matthäus (2002). Nadalje so avtorji z metodo določitve redukcijske sposobnosti ugotovili, da so spojine, ki se nahajajo v pogači navadnega rička, boljši reducenti od tistih v beli gorjušici, medtem ko sta se oljna ogrščica in lan izkazala bolje. DPPH· test je pokazal, da je izvleček navadnega rička primerljiv z izvlečki nekaterih drugih oljnic in učinkovitejši od BHT. Rezultati določitve sposobnosti keliranja so pokazali, da imajo fenolne spojine ekstrahirane iz navadnega rička boljšo sposobnost vezave kovinskih ionov od izvlečkov oljne ogrščice in bele gorjušice, medtem ko se je kot najučinkovitejši izkazal izvleček iz pogače lanu. V sistemu emulgirane linolne kisline v vodi in β-karotena so se antioksidanti prisotni v izvlečku pogače navadnega rička med vsemi testiranimi oljnicami izkazali kot najučinkovitejši, boljši od navadnega rička je bil le nepolarni BHT. Med začetno fazo oksidacije nenasičenih maščobnih kislin pride v maščobnokislinski verigi do premestitve dvojne vezi in nastanka konjugiranega sistema, ki absorbira svetlobo pri 234 nm. Vpliv dodanih metanolnih izvlečkov iz pogač testiranih oljnic oz. BHT na tvorbo konjugiranih dienov (primarnih produktov oksidacije) in trienov (sekundarnih produktov oksidacije) so avtorji omenjene raziskave (Terpinc in sod., 2012a) spremljali v olju barvilnega rumenika, žafranike (Carthamus tinctorius L.), ki so ga inkubirali pri temperaturi 50 °C. Salminen in sod. (2006) so ugotovili, da je izvleček iz pogače navadnega rička učinkovit antioksidant tako v preprečevanju proteinske kot lipidne oksidacije. Rezultati raziskave slovenskih vzorcev so potrdili, da je nastanek konjugiranih dienov in trienov v olju ob prisotnosti izvlečka pogače navadnega rička omiljen v primerjavi s kontrolo (olje brez dodatka antioksidantov), primerljiv s preostalimi izvlečki, vendar obsežnejši v primerjavi z BHT. 2 OLJNA POGAČA NAVADNEGA RIČKA – MOŽNOSTI APLIKACIJE NA PODLAGI DOSEDANJIH RAZISKAV Izsledki proučevanja vpliva razmer predpriprave semen na vsebnost najbolj zastopanih fenolnih spojin v posameznih frakcijah so pokazali, da se skoraj ves sinapin nahaja v prosti obliki in sicer največ v pogači po praženju semen pri 160 °C. Večina protokatehulne kisline je vezana kot sestavni del celične stene, njena vsebnost v izvlečkih je naraščala s temperaturo toplotne obdelave. 4-vinilsiringol je bil med vsemi dekarboksilacijskimi produkti hidroksicimetnih kislin daleč najbolj zastopan, kot prost je dosegel maksimalno vrednost v izvlečku pogače po obdelavi pri 160°C. Prosta frakcija 4-vinilkatehola je s praženjem naraščala in v nasprotju s stanjem v toplotno neobdelanih semenih prevladala nad vezano obliko. Fenolne spojine v rastlinski celici ne obstajajo le v prosti obliki (Huang in sod., 2009). T.i. netopno vezane fenolne spojine so sestavni del celične stene, kjer so običajno vezane na kompleksne ogljikove spojine kot sta lignin in arabinoksilan (Adom in Liu, 2002). Druge se nahajajo v vakuolah, kjer so povezane z različnimi sladkorji, alkoholi, organskimi kislinami in drugimi fenolnimi spojinami. To skupino pojmujemo topni konjugati (Liyana-Pathirana in Shahidi, 2006). Z ustrezno predpripravo semen (toplotna obdelava, mletje in razmastitev) in izbranim ekstrakcijskim postopkom (80 % metanol (v/v) ter alkalna hidroliza) lahko pridobimo posamezne frakcije fenolnih spojin. Izkazalo se je, da so fenolne spojine v razmaščenih zmletih semenih (v pogači) navadnega rička prisotne kot proste, kot topni konjugati in kot netopno vezane (Terpinc in sod., 2011a). Najmanjši delež so predstavljale ravno proste fenolne spojine. To pomeni, da se večina fenolnih spojin ne ekstrahira že s samim metanolom, ampak je za to potrebna hidroliza, ki omogoči ustrezno cepitev vezi. Raziskovalci so izvedli alkalno hidrolizo, ki je v primerjavi s kislinsko ali encimsko hidrolizo pogosteje uporabljena. Praženje pri višjih temperaturah je znatno povečalo vsebnost prostih fenolov. Temperatura je vplivala tudi na dobit topnih konjugatov, ki so bili v pogači toplotno netretiranih semen navadnega rička slabše zastopani od fenolov vezanih na celično steno. Tako je bilo mogoče ugotoviti, da je bilo za sprostitev slednjih in cepitve vezi v topnih konjugatih potrebno praženje pri 160 °C, medtem ko je prišlo do sprostitve topnih konjugatov kot takih že pri nižjih temperaturah. Hkrati je potekala tudi študija vpliva različnih načinov predpriprave semen na vsebnost skupnih flavonoidov, izraženih kot ekvivalent rutina. Dvig temperature praženja je pozitivno vplival na vsebnost prostih flavonoidov v pogači; optimalna temperatura je tako znašala 160 °C, medtem ko so bile za sprostitev netopno vezane frakcije primernejše nižje temperature (80 °C). 2 OLJNA POGAČA NAVADNEGA RIČKA – MOŽNOSTI APLIKACIJE NA PODLAGI DOSEDANJIH RAZISKAV Še več, celo vzorci s podobno koncentracijo skupnih fenolnih spojin neredko skupino uspešno identificiranih fenolnih spojin predstavljajo 4-vinil derivati hidroksicimetnih kislin (4-vinilgvajakol, 4-vinilkatehol, 4-vinilfenol, 4-vinilsiringol), ki se prav vsi ponašajo s pomembnimi antioksidativnimi lastnostmi (Terpinc in sod., 2011b). Antioksidanti se razlikujejo tako po kemijskih kot fizikalnih lastnostih in delujejo na različne načine - kot reducirajoči dejavniki, lovilci prostih radikalov in/ali kelatorji kovin. V opravljeni raziskavi (Terpinc in sod., 2012b) se je izkazalo, da so fenolne spojine prisotne v izvlečku iz pogače slabši reducenti kot tiste v izvlečkih semen in olja, medtem ko so najuspešnejši lovilci radikalov prisotni v pogači. Metoda beljenja β-karotena se od ostalih analiznih metod razlikuje po tem, da reakcija med antioksidantom in radikalom poteka v sistemu emulzije linolne kisline v vodi. Ta metoda predstavlja enega bolj realnih medijev, saj so živila pogosto heterogeni koloidni sistemi. Kot najučinkovitejši se je izkazal izvleček iz olja. Glede na majhno število identificiranih spojin v olju ter na njihov majhen delež, Terpinc in sod. (2012b) predvidevajo, da so sodelovale zlasti neidentificirane in nekvantificirane fenolne spojine. Olje je namreč nepolaren medij in kot tak se vanj med stiskanjem semen prenesejo zlasti manj polarne spojine. Kot je splošno znano, se polarni antioksidanti zadržujejo v vodni fazi emulzije, zaradi česar so v oljni fazi slabše zastopani in tako posledično manj učinkoviti v zaščiti linolne kisline. Pravilnik o aditivih za živila (2010) omejuje uporabo sintetičnih antioksidantov, kot je butilirani hidroksitoluen (BHT), na 100 ppm. Kot se je izkazalo, so fenolne spojine v izvlečku iz olja izjemno učinkovite že pri precej manjši koncentraciji (30 ppm). Sposobnost vezave kovinskih ionov je naslednji način delovanja antioksidantov. Kot je znano, kovinski ioni katalizirajo verižno radikalsko reakcijo lipidne oksidacije, ki vodi v kvar živil (Andjelković in sod., 2006). Rezultati opravljene analize (Terpinc in sod., 2012b) so pokazali, da je za fenole iz pogače in semen značilna znatno manjša aktivnost kot jo ima izvleček iz olja s skoraj absolutno sposobnostjo keliranja kovinskih ionov. Opravljena je bila tudi študija primerjave pogače navadnega rička z izvlečki pogač nekaterih drugih oljnic in sintetičnim antioksidantom BHT (Terpinc in sod., 2012a). Izsledki raziskave so pokazali, da Acta agriculturae Slovenica, 107 - 1, marec 2016 246 Oljna pogača navadnega rička (Camelina sativa (L.) Crantz) – neizkoriščeni vir fenolnih spojin izražajo povsem različen antioksidativni potencial, ki ga med drugim lahko pripišemo tudi medsebojnemu delovanju antioksidantov ter vplivu medija na reaktivnost antioksidanta. 2 OLJNA POGAČA NAVADNEGA RIČKA – MOŽNOSTI APLIKACIJE NA PODLAGI DOSEDANJIH RAZISKAV Za cepitev vezi v tej frakciji pa temperatura nad 120 °C. V okviru iste raziskave (Terpinc in sod., 2011a) so proučili tudi antioksidativne lastnosti fenolnih spojin, ki so prisotne kot proste, zaestrene ali netopno vezane. Rezultati so pokazali, da, neodvisno od toplotne obdelave, posedujejo največji redukcijski potencial tisti fenoli, ki so sestavni del celične stene, medtem ko so najmanj učinkoviti tisti, ki tvorijo topne konjugate. Ugotovili so, da dosežejo fenolne spojine, ki so prisotne kot proste oz. netopno vezane, maksimalno redukcijsko moč, če semena predhodno obdelamo pri 120 °C, medtem, ko se je za topne konjugate izkazalo, da so kot reducenti najučinkovitejši, če semen ne pražimo. Nadalje so ugotovili, da temperatura toplotne obdelave močno zaznamuje tudi sposobnost lovljenja DPPH· radikalov, pri čemer je najboljšo učinkovitost pokazala frakcija prostih fenolov, kot optimalna pa se je izkazala temperatura praženja 120 °C. Kot so zapisali že Daglia in sod. (2000), je antioksidativna aktivnost odvisna od razgradnje obstoječih fenolnih spojin ter na drugi strani od uspešne tvorbe novih spojin z antioksidativno učinkovitostjo, predvsem produktov Maillardove reakcije, kadar so semena podvržena intenzivnejši toplotni obdelavi. V emulziji so se kot najslabši antioksidanti izkazale fenolne spojine, ki so prisotne kot topni konjugati, medtem ko so bili najučinkovitejši zoper alkilperoksilne radikale tisti fenoli, ki se v preiskovanem rastlinskem matriksu nahajajo v netopno vezani obliki. Nasprotno pa so se prosti fenoli izkazali za najučinkovitejše kelatorje. Frakcija topnih konjugatov v izvlečku iz pogače tako praženih kot nepraženih semen ni izkazala aktivnosti. Ker pa je praženje semen 247 Acta agriculturae Slovenica, 107 - 1, marec 2016 Petra TERPINC, Helena ABRAMOVIČ priporočili toplotno obdelavo pri 80 °C, saj je pri omenjenih pogojih obseg razpada kelatorjev najmanjši. pogosto neizogibno tako zaradi nastanka želene arome, barve, teksture, kot tudi povečane hranilne vrednosti, so avtorji kot sprejemljivo izbiro Slika 1: Oljna pogača navadnega rička (Camelina sativa (L.) Crantz) Slika 1: Oljna pogača navadnega rička (Camelina sativa (L.) Crantz) 4 VIRI Abramovič H., Abram V. 2005. Physico-chemical properties, composition and oxidative stability of Camelina sativa oil. Food technology and biotechnology, 43: 63-70 Hrastar R., Terpinc P., Košir I. J., Abramovič H. 2013. Effect of deodorization of camelina (Camelina sativa) oil on its phenolic content and the radical scavenging effectiveness of its extracts. Journal of agricultural and food chemistry, 61: 8098-8103. DOI: 10.1021/jf400309j Abramovič H., Butinar B., Nikolič V. 2007. Changes occurring in phenolic content, tocopherol composition and oxidative stability of Camelina sativa oil during storage. Food chemistry, 104: 903- 909. DOI: 10.1016/j.foodchem.2006.12.044 Huang P.V., Maeda T., Miyatake K., Morita N. 2009. Total phenolic compounds and antioxidant capacity of wheat graded flours by polishing method. Food Research International, 42: 185-190. DOI: 10.1016/j.foodres.2008.10.005 Adom K.K., Liu R.H. 2002. Antioxidant activity of grains. Journal of Agricultural and Food Chemistry, 50: 6182-6187. DOI: 10.1021/jf0205099 Kirkhus B., Lundon A.R., Haugen J.E., Vogt G., Borge G.I., Henriksen B.I. 2013. Effects of environmental factors on edible oil quality of organically grown Camelina sativa. Journal of Agricultural and Food Chemistry, 61: 3179-3185. DOI: 10.1021/jf304532u Andjelković M., Van Camp J., De Meulenaer B., Depaemelaere G., Socaciu C., Verloo M., Verhe R. 2006. Iron-chelation properties of phenolic acids bearing catechol and galloyl groups. Food Chemistry, 98: 23-31. DOI: 10.1016/j.foodchem.2005.05.044 Liyana-Pathirana C.M., Shahidi F. 2006. Importance of insoluble-bound phenolics to antioxidant properties of wheat. Journal of Agricultural and Food Chemistry, 54: 1256-1264. DOI: 10.1021/jf052556h Daglia M., Papetti A., Gregotti C., Bertè F., Gazzani G. 2000. In vitro antioxidant and ex vivo protective activities of green and roasted coffee. Journal of Agricultural and Food Chemistry, 48: 1449-1454. DOI: 10.1021/jf990510g Matthäus B. 2002. Antioxidant activity of extracts obtained from residues of different oilseeds. Journal of Agricultural and Food Chemistry, 50: 3444- 3452. DOI: 10.1021/jf011440s Hayes J., Kelleher M., Eggleston I. 2008. The cancer chemopreventive actions of phytochemicals derived from glucosinolates. European Journal of Nutrition, 47: 73-88. DOI: 10.1007/s00394-008-2009-8 Matthäus B., Zubr J. 2000. Variability of specific components in Camelina sativa oilseed cakes. Industrial Crops and Products, 12: 9-18. DOI: 10.1016/S0926-6690(99)00040-0 Hayes J.E., Stepanyan V., Allen P., O’Grady M.N., O’Brien N.M., Kerry J.P. 2009. The effect of lutein, sesamol, ellagic acid and olive leaf extract on lipid oxidation and oxymyoglobin oxidation in bovine and porcine muscle model systems. Meat Science, 83: 201-208. DOI: 10.1016/j.meatsci.2009.04.019 Moser B. R. 2010. Camelina (Camelina sativa L.) oil as a biofuels feedstock: Golden opportunity or false hope? 3 ZAKLJUČKI Tako določitev vsebnosti skupnih fenolov, kot tudi antioksidativni testi ter analize fenolnega profila potrjujejo, da se med stiskanjem olja iz semen večina fenolnih spojin ne prenese v olje, temveč preostane v pogači. V slednji je poleg 4-vinil derivatov in sinapina, potrjena prisotnost še drugih antioksidantov: elagne kisline, protokatehulne kisline, p-hidroksibenzojske kisline, sinapinske kisline, salicilne kisline, katehina, kvercetina in kvercetin glukozida. Slednje je dobrodošel podatek za živilsko industrijo, saj predstavlja pogača navadnega rička, ki je sicer stranski proizvod pri pridelavi omenjenega olja, izredno pomemben in poceni, a do sedaj neizkoriščen naravni vir antioksidantov. Še zlasti kot nadomestek za potencialno škodljive sintetične antioksidante. Različne razmere toplotne obdelave semen so močno vplivale tako na vsebnost posameznih fenolnih spojin v posameznih frakcijah kot tudi na antioksidativne značilnosti izvlečkov iz pogače. Toplotno labilnejše spojine so med praženjem razpadle, hkrati pa se je povečala vsebnost drugih; slednje predvsem kot posledica sprostitve flavonoidov in ostalih fenolnih spojin iz strukturnih materialov celice. Raziskave opravljene na slovenskih vzorcih navadnega rička so nadalje pokazale, da je izbira temperaturnega režima pri toplotni obdelavi semen odvisna od želenih antioksidativnih lastnosti izvlečkov iz pogače: 80°C za boljšo sposobnost keliranja kovinskih ionov in lovljenje prostih radikalov v emulgiranih sistemih, 120 °C pa v primeru potrebe po optimalni redukcijski sposobnosti in anti-radikalski aktivnosti v homogenem mediju. Acta agriculturae Slovenica, 107 - 1, marec 2016 248 Oljna pogača navadnega rička (Camelina sativa (L.) Crantz) – neizkoriščeni vir fenolnih spojin Acta agriculturae Slovenica, 107 - 1, marec 2016 4 VIRI Lipid Technology, 22: 270-273. DOI: 10.1002/lite.201000068 Hrastar R., Gams Petrišič M., Ogrinc N., Košir, I.J. 2009. Fatty acid and stable carbon isotope characterization of Camelina sativa oil: implications for authentication. Journal of agricultural and food chemistry, 57: 579-585. 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DOI: 10.1002/ejlt.201000438 Pravilnik o aditivih za živila. 2010. Uradni list Republike Slovenije, 100: 15516-15612 Rice-Evans C.A., Miller N.J., Paganga G. 1996. Structure-antioxidant activity relationships of 249 Acta agriculturae Slovenica, 107 - 1, marec 2016 Petra TERPINC, Helena ABRAMOVIČ flavonoids and phenolic acids. Free Radical Biology and Medicine, 20: 933-956. DOI: 10.1016/0891-5849(95)02227-9 (Camelina sativa) seeds on the antioxidant potential of their extracts. Journal of Agricultural and Food Chemistry, 59: 8639-8645. DOI: 10.1021/jf2016072 Rode J. 2001. Tradicionalno domace zdravilo: navadni riček - Camelina sativa (L.) Crantz. Herbika, 40-42 Terpinc P., Polak T., Šegatin N., Hanzlowsky A., Poklar Ulrih N., Abramovič H. 2011b. Antioxidant properties of 4-vinyl derivatives of hydroxycinnamic acids. Food Chemistry, 128: 62- 69. DOI: 10.1016/j.foodchem.2011.02.077 Salminen H., Estévez M., Kivikari R., Heinonen M. 2006. Inhibition of protein and lipid oxidation by rapeseed, camelina and soy meal in cooked pork meat patties. European Food Research and Technology, 223: 461-468. DOI: 10.1007/s00217- 005-0225-5 Zubr J. 1997. Oil-seed crop: Camelina sativa. Industrial Crops and Products, 6: 113-128. DOI: 10.1016/S0926-6690(96)00203-8 Schuster A., Friedt W. 1998. Glucosinolate content and composition as parameters of quality of Camelina seed. Industrial Crops and Products, 7: 297-302. DOI: 10.1016/S0926-6690(97)00061-7 Zubr J. 2003a. Dietary fatty acids and amino acids of Camelina sativa seed. Journal of Food Quality, 26: 451-462. DOI: 10.1111/j.1745- 4557.2003.tb00260.x Terpinc P., Čeh B., Poklar Ulrih N., Abramovič H. 2012a. Acta agriculturae Slovenica, 107 - 1, marec 2016 250 4 VIRI Studies of the correlation between antioxidant properties and the total phenolic content of different oil cake extracts. Industrial Crops and Products, 39: 210-217. DOI: 10.1016/j.indcrop.2012.02.023 Zubr J. 2003b. 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https://openalex.org/W3028772196	https://vtechworks.lib.vt.edu/bitstream/10919/99179/1/sensors-20-03111.pdf	English	null	Acoustic Energy Harvesting and Sensing via Electrospun PVDF Nanofiber Membrane	Sensors	2,020	cc-by	8,076	Received: 17 April 2020; Accepted: 21 May 2020; Published: 31 May 2020 Abstract: This paper introduces a new usage of piezoelectric poly (vinylidene ﬂuoride) (PVDF) electrospun nanoﬁber (NF) membrane as a sensing unit for acoustic signals. In this work, an NF mat has been used as a transducer to convert acoustic signals into electric voltage outcomes. The detected voltage has been analyzed as a function of both frequency and amplitude of the excitation acoustic signal. Additionally, the detected AC signal can be retraced as a function of both frequency and amplitude with some wave distortion at relatively higher amplitudes and within a certain acoustic spectrum region. Meanwhile, the NFs have been characterized through piezoelectric responses, beta sheet calculations and surface morphology. This work is promising as a low-cost and innovative solution to harvest acoustic signals coming from wide resources of sound and noise. Keywords: PVDF; nanoﬁbers; piezoelectricity; acoustic sensors; energy harvesting Acoustic Energy Harvesting and Sensing via Electrospun PVDF Nanoﬁber Membrane Nader Shehata 1,2,3,* , Ahmed H. Hassanin 4,5 , Eman Elnabawy 4 , Remya Nair 1, Sameer A. Bhat 1 and Ishac Kandas 1,4 1 Department of Engineering Mathematics and Physics, Faculty of Engineering, Alexandria University, Alexandria 21544, Egypt; r.nair@kcst.edu.kw (R.N.); s.bhat@kcst.edu.kw (S.A.B.); ishac@vt.edu (I.K.) 2 1 Department of Engineering Mathematics and Physics, Faculty of Engineering, Alexandria University, Alexandria 21544, Egypt; r.nair@kcst.edu.kw (R.N.); s.bhat@kcst.edu.kw (S.A.B.); ishac@vt.edu (I.K.) 2 Faculty of Science, Utah State University, Logan, UT 84341, USA 3 The Bradley Department of Electrical and Computer Engineering, Virginia Tech, Blacksburg, VA 24061, USA 4 Center of Smart Nanotechnology and Photonics (CSNP), SmartCI Research Center of Excellence, Alexandria University, Alexandria 21544, Egypt; ahassan@ncsu.edu (A.H.H.); eman.elnabawy@smartci.alexu.edu.eg (E.E.) 3 The Bradley Department of Electrical and Computer Engineering, Virginia Tech, Blacksburg, VA 24061, USA 4 Center of Smart Nanotechnology and Photonics (CSNP), SmartCI Research Center of Excellence, Alexandria University, Alexandria 21544, Egypt; ahassan@ncsu.edu (A.H.H.); eman.elnabawy@smartci.alexu.edu.eg (E.E.) y g 5 Department of Textile Engineering, Faculty of Engineering, Alexandria University, Alexandria 21544, Egypt * Correspondence: nader83@vt.edu; Tel.: +20-1091-165-300 5 Department of Textile Engineering, Faculty of Engineering, Alexandria University, Alexandria 21544, Egypt * Correspondence: nader83@vt.edu; Tel.: +20-1091-165-300 sensors sensors sensors Sensors 2020, 20, 3111; doi:10.3390/s20113111 1. Introduction Multi-walled carbon nanotubes (MWCNTs) were added by diﬀerent concentrations starting from 5 to 1 wt. % with steps of 0.05 wt.%. At the beginning, the β-phase increased at a high rate. Then, by increasing the concentration of CNTs, the β-phase increased at a slow rate until it reached saturation [17,18]. Another study added MWCNTs with concentrations of 0.6, 1 and 2 wt.%. FTIR spectra showed that the infrared band at 837 and 1273 cm−1 became more prominent. That means an enhancement of β-phase with increasing concentrations of MWCNTs. On the other hand, XRD analysis showed a notable diﬀraction peak at 2 theta equals 19.9◦[19]. This is further evidence of the formation of the β-phase and the positive eﬀect of adding MWCNTs to PVDF solution for enhancing the piezoelectric eﬀect. Poly(vinylidene ﬂuoride-co-hexaﬂuoropropene) (PVDF-HFP)/poly (3,4-ethylenedioxythio-phene) (PEDOT) composite membranes with increased pore volume and high mechanical and electrical properties were introduced as piezoresistive pressure sensors [20]. The synthesized NF mat provided high sensitivity (up to 13.5 kPa−1) with a minimum pressure detection of ~1 Pa and stable operation over 10,000 strain cycles. Electrospun PVDF nanoﬁbers have been reported as ultrasensitive acoustic nanogenerators. The composite membranes exhibited a high-generating output voltage of 11 V with a current density of 6 nA/cm2 upon repetitive mechanical stress that could light up 10 blue light-emitting diodes (LEDs) instantaneously. The development of a ﬂexible pressure sensor based on a PVDF-TrFE nanoﬁber mat was also reported [21]. The developed sensor demonstrated a maximum sensitivity of 60.5 mV. N−1 and reliability up to a frequency of 20 Hz. A poly(vinylidene ﬂuoride) sensor device was prepared and showed signiﬁcant detection of low-frequency acoustic waves with a sensitivity as high as 266 mV Pa−1 [22], which can be used as an acoustic sensor that can eﬃciently distinguish sound waves along with noise detection. Regarding acoustic energy harvesting, PVDF and lead zirconate titanate (PZT) piezoelectric cantilever beams were placed inside a quarter-wavelength straight-tube resonator to harvest acoustic energy at 146 and 199 Hz, respectively. A single PVDF beam produced a voltage of 0.105 V at a frequency of 146 Hz when the incident beam level was 100 dB. For the PZT beam, the maximum voltage was 1.433 V at a frequency of 199 Hz [23]. A noise from a Disk Jockey (DJ) was converted to electrical energy using a piezoelectric transducer (model 7BB-41-2). The maximum generated power was 4.96 mW [24]. 1. Introduction Alternative energy sources, such as mechanical, thermal, wind and water energies, have been extensively studied over recent decades [1,2]. These environmental resources are promising selections to be transduced into electrical engineering for both clean environment considerations and cheaper economic costs [3]. Among the diﬀerent transducing mechanisms, one of the most exciting mechanisms is the piezoelectric one where mechanical energy, including vibrations, pressures, bending and tension stresses, can be converted into electrical energy [4]. This oﬀers a promising property for a wide variety of energy harvesting applications, such as biomedical sensors [5], wearable electronics [6] and smart textiles [7]. Acoustic sensors have been widely introduced in several applications, including medical, environmental, industrial and scientiﬁc research [8,9], based on various transducing mechanisms, such as piezoelectricity, piezocapacitance, piezoresistance and piezo-optics [10–13]. Polymeric piezoelectric acoustic sensors are considered among the most distinct types of materials due to their higher sensitivity and ﬂexibility [14]. However, most of the piezoelectric polymer processes have some drawbacks due to the need for high-voltage poling or mechanical stretching, which entail more energy consumption and higher costs. Recently, piezoelectric nanoﬁbers (NFs) prepared through electrospinning have been strongly introduced for various applications. Many studies have indicated that electrospun NFs from piezoelectric polymers, such as poly(vinylidene Sensors 2020, 20, 3111; doi:10.3390/s20113111 www.mdpi.com/journal/sensors www.mdpi.com/journal/sensors 2 of 12 Sensors 2020, 20, 3111 ﬂuoride) (PVDF), poly(vinylidene ﬂuoride-co-hexaﬂuoropropene) (PVDF-HFP) and poly(vinylidene ﬂuoride-co-triﬂuoroethylene) (PVDF-TrFE), have unique piezoelectric properties without the need for stretching or poling treatments. These piezoelectric nanoﬁbers are considered to be some of the best candidates for mechanical sensors and energy generator applications. PVDF nanoﬁbers were fabricated using an electrospinning technique with an applied voltage of 5 kV and a distance between the tip and the collector of 0.1 m. The solution concentration of the PVDF was 16 wt.%. The rotary speed of the collector was varied between 500, 1000, 1500 and 2000 rpm. FTIR spectra showed some peaks at 475, 510, 1275 and 1431 cm−1, respectively [15]. These can prove the alignment of the generated nanoﬁbers and the formation of the polar beta phase (β-phase) of PVDF. Some additives to the PVDF solution, such as hydrated salt and nickel chloride hexahydrate (NiCl2·6H2O) with a concentration less than 0.5 wt.%, could increase the polar beta phase by 30% due to the ionic interaction between salt and polymer molecules [16]. Moreover, addition of carbon nanotubes (CNTs) to PVDF can improve its piezoelectric performance. 2.3.2. Physical Characterization X-Ray diﬀraction (XRD) was measured on a diﬀractometer (Shimadzu Xlab 6100, Kyoto, Japan) with Cu radiation. The samples were scanned in the 2θ range of 5 to 80◦and with a scanning speed of 12 (deg/min). The Fourier-transform infrared spectroscopy (FTIR) spectra were measured on a FTIR spectrophotometer (Vertex 70 FTIR, Bruker, Billerica, MA, USA) in ATR mode. Samples were scanned 120 times at a resolution of 5 cm−1 over a range of 4000–400 cm−1. 2.1. Materials PVDF (Kynar, melt viscosity: 23.0–29.0) was supplied by ARKEMA (King of Prussia, PA, USA). N, N-Dimethyl Formamide (anhydrous, 98%) was purchased from LobaChemie (India). Chemicals were used without further dilution or puriﬁcation. 2.2. Membrane Fabrication The homogeneous PVDF (15 wt.%) solution was prepared by dissolving 3 g of PVDF powder into 20 mL of DMF and stirred overnight. The PVDF solution was then inserted into a plastic syringe tipped with a metal needle (21 gauge). The ﬂow rate was controlled at 1 mL. h−1 through a NE1000 syringe pump (New Era Pump Systems, Suﬀolk County, NY, USA), while the high voltage of 25 kV was applied to a syringe needle using a high-voltage power supply CZE1000R (Spellman, Hauppauge, NY, USA) and collected on a rotating drum collector at 1500 rpm, which was placed 10 cm away from the needle tip. The aforementioned parameters have been proved by earlier research work done by the authors to generate the best membrane morphology with minimum beads and defects [30,31]. 2.3. Material Characterizations 2.3. Material Characterizations 2.3.1. Morphological Characterization The morphological structure of nanoﬁber webs was observed using a scanning electron microscope (JEOL JSM-6010LV-SEM) with an accelerating voltage of 15 kV. The nanoﬁber mat was ﬁxed onto a carbon tape and sputtered with platinum before measuring. The ﬁber diameter was obtained using image processing software (ImageJ, Madison, WI, USA). 2.3.3. Piezoelectric Characterization Piezoelectric analysis of PVDF NFs was performed using a tool designed to control the applied force on PVDF nanoﬁbers through a motorized spring. Changing the length of the spring can control the applied force on piezonanoﬁbers. The nanoﬁber mat was sandwiched between two foil sheets and connected through shielded wires to a high-impedance oscilloscope (Tektronix MDO 3014). Then, peak-to-peak voltage was analyzed according to the changed applied force. 1. Introduction A PVDF cantilever beam was fabricated and placed inside a cavity of a Helmholtz resonator. The produced power was 0.10 µW [25]. Diﬀerent conﬁgurations of the Helmholz resonator were proposed to improve the acoustic energy harvesting (AEH) [26–28]. Many approaches to introduce AEH were illustrated, such as Helmholz resonator-based approaches, quarter-wavelength resonator-based approaches and acoustic metamaterial-based approaches [29]. However, the acoustic-to-electric energy conversion of polymeric nanoﬁbers still needs more research investigations to be applicable. In our work, we have prepared a ﬂexible PVDF nanoﬁber mat through an electrospinning method to be used for acoustic sensing applications. Detailed characterization for physical and structural properties of the pristine PVDF membrane has been investigated, such as FTIR, XRD and SEM, along with piezoelectric analysis under applied mechanical 3 of 12 Sensors 2020, 20, 3111 forces. In addition, the acoustic signals within variant amplitude and frequency have been converted to electrical energy using a simple hand-made setup, where the synthesized nanoﬁber mat is the main sensing unit. Additionally, the acoustic signals have been traced from the generated electric signal to check selective acoustic frequencies, where the nanoﬁber mat is eﬀective to detect the acoustic signals with minimum distortion. 2.3.4. Acoustic Sensing Analysis Figure 1 shows a photo of the acoustic sensing setup. In this setup, a source of acoustic signals, function generator Tektronix of Model AFG1062, 60 MHz, 300 MS/s, was connected through an acoustic ampliﬁer to a speaker. The generator controls both frequency and initial amplitude of the applied acoustic signal before being ampliﬁed. Then, the PVDF NFs mat is sandwiched between two aluminum 4 of 12 Sensors 2020, 20, 3111 2.3.4. Acoust foil parallel sheets and stacked to the speaker. The two foil sheets are connected through two shielded wires to a mixed domain oscilloscope (MDO) Tektronix 3014 with high impedance. The input voltage applied by the function generator to provide the acoustic signal was measured using a voltmeter connected to the power ampliﬁer. The oscilloscope was used to show the retraced acoustic signal detected by the mat. Using this signal, the peak-to-peak voltage was measured, and also the frequency of the distorted received signal was recorded, to study the impact of sound waves on PVDF NFs; we used a wide range of audio signal frequencies from 100 Hz to 20 KHz. The sound waves of diﬀerent frequencies at diﬀerent amplitudes were applied to the NF mat. In the beginning, for each frequency, the audio signal of a relatively low amplitude was applied and the output voltage generation from the NF mat was measured using the oscilloscope connected to the mat terminals. The input sound waves were modulated by ampliﬁcation using a power ampliﬁer connected to the source (speaker) where the samples of NFs were attached. These input-applied voltages were increased in steps of 0.6 V ten times until reaching 6 V, which resulted in an unbearable sound. Each time, the output voltage generated from the NFs was measured using the oscilloscope. function generator Tektronix of Model AFG1062, 60 MHz, 300 MS/s, was connected through an acoustic amplifier to a speaker. The generator controls both frequency and initial amplitude of the applied acoustic signal before being amplified. Then, the PVDF NFs mat is sandwiched between two aluminum foil parallel sheets and stacked to the speaker. The two foil sheets are connected through two shielded wires to a mixed domain oscilloscope (MDO) Tektronix 3014 with high impedance. The input voltage applied by the function generator to provide the acoustic signal was measured using a voltmeter connected to the power amplifier. 2.3.4. Acoustic Sensing Analysis The oscilloscope was used to show the retraced acoustic signal detected by the mat. Using this signal, the peak-to-peak voltage was measured, and also the frequency of the distorted received signal was recorded, to study the impact of sound waves on PVDF NFs; we used a wide range of audio signal frequencies from 100 Hz to 20 KHz. The sound waves of different frequencies at different amplitudes were applied to the NF mat. In the beginning, for each frequency, the audio signal of a relatively low amplitude was applied and the output voltage generation from the NF mat was measured using the oscilloscope connected to the mat terminals. The input sound waves were modulated by amplification using a power amplifier connected to the source (speaker) where the samples of NFs were attached. These input-applied voltages were increased in steps of 0.6 V ten times until reaching 6 V, which resulted in an unbearable sound. Each time, the output voltage generated from the NFs was measured using the oscilloscope. Figure 1. (a) Schematic diagram of the acoustic energy harvesting setup. (b) A photo of the detailed acoustic sensing setup. Figure 1. (a) Schematic diagram of the acoustic energy harvesting setup. (b) A photo of the detailed acoustic sensing setup. Figure 1. (a) Schematic diagram of the acoustic energy harvesting setup. (b) A photo of the detailed acoustic sensing setup. Figure 1. (a) Schematic diagram of the acoustic energy harvesting setup. (b) A photo of the detailed acoustic sensing setup. Figure 1. (a) Schematic diagram of the acoustic energy harvesting setup. (b) A photo of the detailed acoustic sensing setup. Figure 1. (a) Schematic diagram of the acoustic energy harvesting setup. (b) A photo of the detailed acoustic sensing setup. 3.1. Physical Characterizations Sensors 2020, 20, x; doi: FOR PEER REVIEW www.mdpi.com/journal/sensors PVDF NF morphology was examined by the FE-SEM, as shown in Figure 2. The morphological structure of the NF scaﬀold showed randomly distributed ﬁbers with an average diameter of 110 ± 63 nm. FTIR spectroscopy and XRD were performed to analyze the degree of crystallinity and β-phase content for the produced PVDF NFs. Figure 3a shows the infrared spectra of the electrospun PVDF nanoﬁbers. The graph shows characteristic bands for the C–F vibration of PVDF at 1400.2, 1191.9 and 881.4 cm−1 [32]. Furthermore, the β-absorption peak of PVDF appeared at 837.2 cm−1, which conﬁrms the eﬀect of electrospinning on enhancing the polarizability of PVDF. The amount of β-phase was 5 of 12 Sensors 2020, 20, 3111 quantitatively calculated by considering the relative absorption intensity of the β-phase (840 cm−1) and the α phase (760 cm−1), according to the proposed Gregorio and Cestari equation [33]: F (β) = Aβ / (1.3Aα + Aβ) (1) (1) where F (β) is the β-phase content, and Aα and Aβ are the absorbance at 766 and 840 cm−1, respectively. By calculating the previous equation according to the obtained IR result, the β-phase content for the PVDF nanoﬁber mat was 0.75, which conﬁrmed the high-piezoelectric response of our obtained nanoﬁber. Figure 3b represents the XRD pattern of the PVDF mat, the high intensity for the main characteristic peak corresponding to the PVDF β-phase (2θ = 20.3◦), which was attributed to the crystallographic (110) plane that resulted. The β-phase is known to be the most polar phase of PVDF, which shows spontaneous polarization by converting the mechanical energy into electrical energy and vice versa (piezoelectric and pyroelectric activity) [34]. Thus, the formation of nanoﬁbers by electrospinning promotes the formation of the β-phase, which in return enhances the piezoelectric properties. Sensors 2020, 20, x FOR PEER REVIEW 5 of 13 3. Results and Discussion 3.1. Physical Characterizations PVDF NF morphology was examined by the FE-SEM, as shown in Figure 2. The morphological structure of the NF scaffold showed randomly distributed fibers with an average diameter of 110 ± 63 nm. (a) (b) Figure 2. (a) SEM image with fiber diameter distribution for the electrospun poly (vinylidene fluoride) nanofiber (PVDF NF) mats, and (b) cross-section of the generated membrane. Figure 2. 3.1. Physical Characterizations (a) SEM image with ﬁber diameter distribution for the electrospun poly (vinylidene ﬂuoride) nanoﬁber (PVDF NF) mats, and (b) cross-section of the generated membrane. (a) (a) (a) (b) (b) Figure 2. (a) SEM image with fiber diameter distribution for the electrospun poly (vinylidene fluoride) nanofiber (PVDF NF) mats, and (b) cross-section of the generated membrane. Figure 2. (a) SEM image with ﬁber diameter distribution for the electrospun poly (vinylidene ﬂuoride) nanoﬁber (PVDF NF) mats, and (b) cross-section of the generated membrane. FTIR spectroscopy and XR content for the produced PVD 3.2. Piezoelectric Characterization FTIR spectroscopy and XR content for the produced PVD 3.2. Piezoelectric Characterization forces were exposed to the synthesized ectric voltage by applying cyclic forces output peak-to-peak voltage at different a fibers can be sensitive to a load of a few ne 10−5 V.m/N or 0.23 mV/Pa due to formed e Figure 3. (a) FTIR and (b) XRD analyses of PVDF nanofibers. Figure 3. (a) FTIR and (b) XRD analyses of PVDF nanoﬁbers. ctrospun nanofibers. 3.2. Piezoelectric Characterization For piezoelectric analysis, different applied forces were exposed to the synthesized PVDF nanofiber mat. Figure 4 shows the generated electric voltage by applying cyclic forces on the electrospun nanofiber mat. Figure 5 shows the output peak-to-peak voltage at different applied forces. It can be noticed that our synthesized nanofibers can be sensitive to a load of a few newtons with a piezoelectric response sensitivity of 1.05 × 10−5 V.m/N or 0.23 mV/Pa due to formed electric dipoles inside the electrospun nanofibers. Figure 4. Piezoelectric analysis of the PVDF nanofiber membrane at a cyclic applied force of 5 N and at a rate of 5 Hz. The shown y-axis presents electric voltage (in volts) and the x-axis is the time (in seconds). Figure 4. Piezoelectric analysis of the PVDF nanoﬁber membrane at a cyclic applied force of 5 N and at a rate of 5 Hz. The shown y-axis presents electric voltage (in volts) and the x-axis is the time (in seconds). 3.2. Piezoelectric Characterization For piezoelectric analysis, different applied forces were exposed to the synthesized PVDF nanofiber mat. Figure 4 shows the generated electric voltage by applying cyclic forces on the electrospun nanofiber mat. Figure 5 shows the output peak-to-peak voltage at different applied forces. It can be noticed that our synthesized nanofibers can be sensitive to a load of a few newtons with a piezoelectric response sensitivity of 1.05 × 10−5 V.m/N or 0.23 mV/Pa due to formed electric dipoles inside the electrospun nanofibers. Figure 4. Piezoelectric analysis of the PVDF nanofiber membrane at a cyclic applied force of 5 N and at a rate of 5 Hz. The shown y-axis presents electric voltage (in volts) and the x-axis is the time (in seconds). Figure 4. Piezoelectric analysis of the PVDF nanoﬁber membrane at a cyclic applied force of 5 N and at a rate of 5 Hz. The shown y-axis presents electric voltage (in volts) and the x-axis is the time (in seconds). Figure 4. FTIR spectroscopy and XR content for the produced PVD 3.2. Piezoelectric Characterization Figure 5 shows the output peak-to-peak voltage at different ap ces. It can be noticed that our synthesized nanofibers can be sensitive to a load of a few new th a piezoelectric response sensitivity of 1.05 × 10−5 V.m/N or 0.23 mV/Pa due to formed ele poles inside the electrospun nanofibers. Figure 3. (a) FTIR and (b) XRD analyses of PVDF nanofibers. 3.2. Piezoelectric Characterization For piezoelectric analysis, different applied forces were exposed to the synthesized PVDF nanofiber mat. Figure 4 shows the generated electric voltage by applying cyclic forces on the electrospun nanofiber mat. Figure 5 shows the output peak-to-peak voltage at different applied forces. It can be noticed that our synthesized nanofibers can be sensitive to a load of a few newtons with a piezoelectric response sensitivity of 1.05 × 10−5 V.m/N or 0.23 mV/Pa due to formed electric dipoles inside the electrospun nanofibers. Figure 3. (a) FTIR and (b) XRD analyses of PVDF nanoﬁbers. Figure 3. (a) FTIR and (b) XRD analyses of PVDF nanofibers. . Piezoelectric Characterization For piezoelectric analysis, different applied forces were exposed to the synthesized PV nofiber mat. Figure 4 shows the generated electric voltage by applying cyclic forces on ctrospun nanofiber mat. Figure 5 shows the output peak-to-peak voltage at different appl ces. It can be noticed that our synthesized nanofibers can be sensitive to a load of a few newto th a piezoelectric response sensitivity of 1.05 × 10−5 V.m/N or 0.23 mV/Pa due to formed elec poles inside the electrospun nanofibers. Figure 4. Piezoelectric analysis of the PVDF nanofiber membrane at a cyclic applied force of 5 N and at a rate of 5 Hz. The shown y-axis presents electric voltage (in volts) and the x-axis is the time (in seconds). Figure 4. Piezoelectric analysis of the PVDF nanoﬁber membrane at a cyclic applied force of 5 N and at a rate of 5 Hz. The shown y-axis presents electric voltage (in volts) and the x-axis is the time (in seconds). Figure 3. (a) FTIR and (b) XRD iezoelectric Characterization For piezoelectric analysis, different applied fiber mat. Figure 4 shows the generated el ospun nanofiber mat. Figure 5 shows the o s. It can be noticed that our synthesized nano a piezoelectric response sensitivity of 1.05 × analyses of PVDF nanofibers. FTIR spectroscopy and XR content for the produced PVD 3.2. Piezoelectric Characterization content for the produced PVDF NFs. Figure 3a shows the infrared spectra of the electrospun PVDF nanofibers. The graph shows characteristic bands for the C–F vibration of PVDF at 1400.2, 1191.9 and 881.4 cm−1 [32]. Furthermore, the β-absorption peak of PVDF appeared at 837.2 cm−1, which confirms the effect of electrospinning on enhancing the polarizability of PVDF. The amount of β-phase was quantitatively calculated by considering the relative absorption intensity of the β-phase (840 cm−1) d th h (760 1) di t th d G i d C t i ti [33] For piezoelectric analysis, diﬀerent applied forces were exposed to the synthesized PVDF nanoﬁber mat. Figure 4 shows the generated electric voltage by applying cyclic forces on the electrospun nanoﬁber mat. Figure 5 shows the output peak-to-peak voltage at diﬀerent applied forces. It can be noticed that our synthesized nanoﬁbers can be sensitive to a load of a few newtons with a piezoelectric 6 of 12 Sensors 2020, 20, 3111 characteristic ll response sensitivity of 1.05 × 10−5 V.m/N or 0.23 mV/Pa due to formed electric dipoles inside the electrospun nanoﬁbers. p p y g gy gy and vice versa (piezoelectric and pyroelectric activity) [34]. Thus, the formation of nanofibers by electrospinning promotes the formation of the β-phase, which in return enhances the piezoelectric properties. Figure 3. (a) FTIR and (b) XRD analyses of PVDF nanofibers. Figure 3. (a) FTIR and (b) XRD analyses of PVDF nanoﬁbers. Figure 3. (a) FTIR and (b) XRD analyses of PVDF nanofibers. .2. Piezoelectric Characterization For piezoelectric analysis, different applied forces were exposed to the synthesized PV anofiber mat. Figure 4 shows the generated electric voltage by applying cyclic forces on lectrospun nanofiber mat. Figure 5 shows the output peak-to-peak voltage at different app orces. It can be noticed that our synthesized nanofibers can be sensitive to a load of a few new with a piezoelectric response sensitivity of 1.05 × 10−5 V.m/N or 0.23 mV/Pa due to formed elec ipoles inside the electrospun nanofibers. Figure 3. (a) FTIR and (b) XRD analyses of PVDF nanofibers. Figure 3. (a) FTIR and (b) XRD analyses of PVDF nanoﬁbers. Figure 3. (a) FTIR and (b) XRD analyses of PVDF nanofibers. . Piezoelectric Characterization For piezoelectric analysis, different applied forces were exposed to the synthesized P nofiber mat. Figure 4 shows the generated electric voltage by applying cyclic forces on ctrospun nanofiber mat. FTIR spectroscopy and XR content for the produced PVD 3.2. Piezoelectric Characterization Piezoelectric analysis of the PVDF nanofiber membrane at a cyclic applied force of 5 N and at a rate of 5 Hz. The shown y-axis presents electric voltage (in volts) and the x-axis is the time (in seconds). Figure 4. Piezoelectric analysis of the PVDF nanoﬁber membrane at a cyclic applied force of 5 N and at a rate of 5 Hz. The shown y-axis presents electric voltage (in volts) and the x-axis is the time (in seconds). Figure 4. Piezoelectric analysis of 3.3. Acoustic Sensing Measurements Figure 4. Piezoelectric analysis of 3.3. Acoustic Sensing Measurements at a rate of 5 Hz. The shown y-axis presents electric voltage (in vo seconds). 3.3.1. Impact of Acoustic Signal Frequency and Amplitude Sensors 2020, 20, x; doi: FOR PEER REVIEW www.mdpi.com/journal/sensors Sensors 2020, 20, x; doi: FOR PEER REVIEW www.mdpi.com/journal/sensors As the frequency increased from 100 Hz to 10 kHz, the sound waves with increasing amplitude resulted in unendurable sound, but the sound waves were not audible above 10 kHz with amplitudes up to 6 V. All these cases resulted in an output voltage with some sort of distortion to the waves at certain amplitudes depending on the frequency level. Thus, the eﬀect of audio signals of diﬀerent frequencies at diﬀerent amplitudes on the NF mat was studied. The output voltage versus frequency at diﬀerent amplitudes obtained from the NF mat is shown in Figure 6a. For low-amplitude sound waves at input voltages from 0.6 to 1.2 V, the output voltage generated from the NF mat was found to be increasing almost linearly for low-applied voltages with increases in frequency up to 2000 Hz, as shown in Figure 6b, and then from 1 to 20 kHz, the generated output voltage was found to be almost constant. However, this behavior is found only at low-amplitude sound waves. As the input voltage increased from 1.8 to 6 V, the audio signal amplitude increased and the generated output voltage increased linearly as the frequency increased up to 4000 Hz; then, the output voltage started decreasing as the frequency increased to 20 kHz. The maximum generated voltage obtained from the NF mat was around 330 mV and was found at 4000 Hz, which may be correlated to a natural resonance response of our samples to the surface deformation at this frequency value. At this frequency, we plotted the 7 of 12 Sensors 2020, 20, 3111 generated output voltage as a function of the input voltage in Figure 6c, where the output voltage was linearly changing with the input voltage. Sensors 2020, 20, x FOR PEER REVIEW 7 of 13 Figure 5. Mean peak-to-peak converted voltage through the electrospun nanofibers under the impact of applied cyclic force. Figure 5. Mean peak-to-peak converted voltage through the electrospun nanoﬁbers under the impact of applied cyclic force. Sensors 2020, 20, x FOR PEER REVIEW 8 of 13 Figure 5. Mean peak-to-peak converted voltage through the electrospun nanofibers under the impact of applied cyclic force. Figure 5. Mean peak-to-peak converted voltage through the electrospun nanoﬁbers under the impact of applied cyclic force. at a rate of 5 Hz. The shown y-axis presents electric voltage (in vo seconds). 3.3.1. Impact of Acoustic Signal Frequency and Amplitude Sensors 2020, 20, x FOR PEER REVIEW 8 of 13 Sensors 2020, 20, x; doi: FOR PEER REVIEW www.mdpi.com/journal/sensors 3.3. Acoustic Sensing Measurements 3.3.1. Impact of Acoustic Signal Frequency and Amplitude As the frequency increased from 100 Hz to 10 kHz, the sound waves with increasing amplitude resulted in unendurable sound, but the sound waves were not audible above 10 kHz with amplitudes up to 6 V. All these cases resulted in an output voltage with some sort of distortion to the waves at certain amplitudes depending on the frequency level. Thus, the effect of audio signals of different frequencies at different amplitudes on the NF mat was studied. The output voltage versus frequency at different amplitudes obtained from the NF mat is shown in Figure 6a. For low-amplitude sound waves at input voltages from 0.6 to 1.2 V, the output voltage generated from the NF mat was found to be increasing almost linearly for low-applied voltages with increases in frequency up to 2000 Hz, as shown in Figure 6b, and then from 1 to 20 kHz, the generated output voltage was found to be almost constant. However, this behavior is found only at low-amplitude sound waves. As the input voltage increased from 1.8 to 6 V, the audio signal amplitude increased and the generated output voltage increased linearly as the frequency increased up to 4000 Hz; then, the output voltage started decreasing as the frequency increased to 20 kHz. The maximum generated voltage obtained from the NF mat was around 330 mV and was found at 4000 Hz, which may be correlated to a natural resonance response of our samples to the surface deformation at this frequency value. At this frequency, we plotted the generated output voltage as a function of the input voltage in Figure 6c, where the output voltage was linearly changing with the input voltage. Figure 6. (a) Measured voltage versus frequency at different input applied voltages. (b) Measured voltage versus low frequency (up to 2 kHz) at different input applied voltages. (c) The output voltage versus applied input voltage at a frequency of 4 kHz. Figure 6. (a) Measured voltage versus frequency at diﬀerent input applied voltages. (b) Measured voltage versus low frequency (up to 2 kHz) at diﬀerent input applied voltages. (c) The output voltage versus applied input voltage at a frequency of 4 kHz. 3.3.2. Acoustic Signal Retracing In this section, we measure the retracing of the acoustic signal detected by the NF mat. The retracing focuses on both signal distortion and frequency detection. Examples of signal proﬁle distortion are shown in both Figures 7 and 8. Sensors 2020, 20, x FOR PEER REVIEW 9 of 13 In this section, we measure the retracing of the acoustic signal detected by the NF mat. The retracing focuses on both signal distortion and frequency detection. Examples of signal proﬁle distortion are shown in both Figures 7 and 8. Sensors 2020, 20, x FOR PEER REVIEW 9 of 13 Figure 7. Examples of retraced acoustic wave signals at different input frequencies at an applied voltage of 1.2 volts. The used frequencies are (a) 300 Hz, (b) 500 Hz, (c) 4 kHz, (d) 5 kHz, (e) 15 kHz and (f) 20 kHz. All wave signals graphs have a y-axis of voltage (in volts) and an x-axis of time (in seconds) Figure 7. Examples of retraced acoustic wave signals at diﬀerent input frequencies at an applied voltage of 1.2 volts. The used frequencies are (a) 300 Hz, (b) 500 Hz, (c) 4 kHz, (d) 5 kHz, (e) 15 kHz and (f) 20 kHz. All wave signals graphs have a y-axis of voltage (in volts) and an x-axis of time (in seconds). Figure 7. Examples of retraced acoustic wave signals at different input frequencies at an applied voltage of 1.2 volts. The used frequencies are (a) 300 Hz, (b) 500 Hz, (c) 4 kHz, (d) 5 kHz, (e) 15 kHz and (f) 20 kHz. All wave signals graphs have a y-axis of voltage (in volts) and an x-axis of time (in d ) Figure 7. Examples of retraced acoustic wave signals at diﬀerent input frequencies at an applied voltage of 1.2 volts. The used frequencies are (a) 300 Hz, (b) 500 Hz, (c) 4 kHz, (d) 5 kHz, (e) 15 kHz and (f) 20 kHz. All wave signals graphs have a y-axis of voltage (in volts) and an x-axis of time (in seconds). ) For relatively low-amplitude sound waves, the output signal was not distorted by much compared to higher amplitudes. However, at lower frequencies in the range of 100 to 800 Hz and for higher frequencies ranging from 10 to 20 kHz, the output signal was aﬀected by distortion even at lower amplitudes. at a rate of 5 Hz. The shown y-axis presents electric voltage (in vo seconds). 3.3.1. Impact of Acoustic Signal Frequency and Amplitude stic Sensing Measurements pact of Acoustic Signal Frequency and Amplitude he frequency increased from 100 Hz to 10 kHz, the sound waves with increasing amplit n unendurable sound, but the sound waves were not audible above 10 kHz with amplitu . All these cases resulted in an output voltage with some sort of distortion to the wave mplitudes depending on the frequency level. Thus, the effect of audio signals of diffe es at different amplitudes on the NF mat was studied. The output voltage versus freque nt amplitudes obtained from the NF mat is shown in Figure 6a. For low-amplitude so input voltages from 0.6 to 1.2 V, the output voltage generated from the NF mat was fo easing almost linearly for low-applied voltages with increases in frequency up to 2000 n in Figure 6b, and then from 1 to 20 kHz, the generated output voltage was found to nstant. However, this behavior is found only at low-amplitude sound waves. As the in ncreased from 1.8 to 6 V, the audio signal amplitude increased and the generated ou ncreased linearly as the frequency increased up to 4000 Hz; then, the output voltage sta g as the frequency increased to 20 kHz. The maximum generated voltage obtained from was around 330 mV and was found at 4000 Hz, which may be correlated to a nat e response of our samples to the surface deformation at this frequency value. At y, we plotted the generated output voltage as a function of the input voltage in Figure e output voltage was linearly changing with the input voltage. , x; doi: FOR PEER REVIEW www.mdpi.com/jo Figure 6. (a) Measured voltage versus frequency at different input applied voltages. (b) Measured voltage versus low frequency (up to 2 kHz) at different input applied voltages. (c) The output voltage versus applied input voltage at a frequency of 4 kHz. Figure 6. (a) Measured voltage versus frequency at diﬀerent input applied voltages. (b) Measured voltage versus low frequency (up to 2 kHz) at diﬀerent input applied voltages. (c) The output voltage versus applied input voltage at a frequency of 4 kHz. 8 of 12 Sensors 2020, 20, 3111 3.3.2. Acoustic Signal Retracing While at low-amplitude sound waves in the medium range, frequencies from 1 to 8 kHz had no distortion compared to other frequencies. Here in the case of medium range frequency, the output signals were aﬀected by distortion only at the highest amplitude level, but the distortion level was very low compared to all other frequencies involved in our studies. 9 of 12 Sensors 2020, 20, 3111 Figure 8. Examples of retraced acoustic wave signals at different input frequency at an applied voltage of 4.8 Volt. The used frequencies are, (a) 300 Hz, (b) 500 Hz, (c) 4 kHz and (d) 5 kHz. (e) represents the output signal voltage obtained for a frequency of 15 kHz and (f) 20 kHz. All wave signal graphs have a y-axis of voltage (in volts) and x-axis of time (in seconds). For relatively low-amplitude sound waves, the output signal was not distorted by much Figure 8. Examples of retraced acoustic wave signals at diﬀerent input frequency at an applied voltage of 4.8 Volt. The used frequencies are, (a) 300 Hz, (b) 500 Hz, (c) 4 kHz and (d) 5 kHz. (e) represents the output signal voltage obtained for a frequency of 15 kHz and (f) 20 kHz. All wave signal graphs have a y-axis of voltage (in volts) and x-axis of time (in seconds). Both Figures 7 and 8 clearly depict the distortion level of output signals for diﬀerent inpu gure 8. Examples of retraced acoustic wave signals at different input frequency at an applied voltage 4.8 Volt. The used frequencies are, (a) 300 Hz, (b) 500 Hz, (c) 4 kHz and (d) 5 kHz. (e) represents e output signal voltage obtained for a frequency of 15 kHz and (f) 20 kHz. All wave signal graphs ave a y-axis of voltage (in volts) and x-axis of time (in seconds). Figure 8. Examples of retraced acoustic wave signals at diﬀerent input frequency at an applied voltage of 4.8 Volt. The used frequencies are, (a) 300 Hz, (b) 500 Hz, (c) 4 kHz and (d) 5 kHz. (e) represents the output signal voltage obtained for a frequency of 15 kHz and (f) 20 kHz. All wave signal graphs have a y-axis of voltage (in volts) and x-axis of time (in seconds). For relatively low-amplitude sound waves, the output signal was not distorted by much compared to higher amplitudes. 3.3.2. Acoustic Signal Retracing However, at lower frequencies in the range of 100 to 800 Hz and for higher frequencies ranging from 10 to 20 kHz, the output signal was affected by distortion even at ower amplitudes. While at low-amplitude sound waves in the medium range, frequencies from 1 to 8 kHz had no distortion compared to other frequencies. Here in the case of medium range frequency, Both Figures 7 and 8 clearly depict the distortion level of output signals for diﬀerent input frequencies (300 Hz, 500 Hz, 4 kHz, 5 kHz, 15 kHz and 20 kHz) at an input applied voltage of 1.2 and 4.8 V, respectively. As the input applied voltage increased to 4.8 V, the distortion rate increased a lot at low and high frequencies except in the medium range frequency, which is 4 kHz, as shown in the ﬁgure. In the case of low frequencies (100 to 800 Hz) and for high frequencies (10 to 20 kHz), the distortion level was very high and the distortion level was found to increase linearly with increases Sensors 2020, 20, 3111 10 of 12 in input sound wave amplitudes. Therefore, based on studies in a range of frequencies with diﬀerent amplitudes, the NF mat had an optimum performance in the range of 1 to 8 kHz with maximum output voltage without much distortion. g For frequency retracing, Table 1 shows the detected frequencies compared to the input acoustic signal frequency with the calculated percentage error. It can be shown that our synthesized NF mat shows a relatively low-retracing frequency error for the whole acoustic frequency spectrum. The maximum absolute error in frequency detection or retracing was found to be 1.6%. Table 1. Measured frequency of the generated signals from the PVDF NF mat compared to input acoustic signals with percentage error calculations. Input Frequency (Hz) Output Frequency (Hz) Absolute Percentage Error (%) 800 787 1.6 1000 990 1 3000 3012 0.4 6000 5924 1.2 8000 8012 0.15 10,000 9920 0.8 15,000 15,197 1.3 20,000 19,960 0.2 Input Frequency (Hz) Output Frequency (Hz) Absolute Percentage Error (%) 4. Conclusions In this paper, we have presented a new application of PVDF NFs as a sensor for acoustic signals. The piezoelectric PVDF NF membrane, synthesized by electrospinning, has been used as a target for acoustic excitation waves at diﬀerent amplitudes and frequencies. The synthesized NFs transduced the acoustic signals into electric potential based on their piezoelectric properties. In addition, the ﬁbers can retrace the input acoustic signals with less distortion at a certain acoustic spectrum region with relatively low-amplitude acoustic excitations. This work is promising for harvesting acoustic signals and noise and transducing them into electrical energy. Author Contributions: Conceptualization, A.H.H., N.S.; Data curation, E.E., S.A.B., R.N.; Formal analysis, N.S., I.K.; Investigation, A.H.H., N.S., I.K.; Methodology, E.E., R.N., S.A.B.; Supervision, N.S., A.H.H.; Visualization, E.E., I.K.; Writing–original draft, R.N., E.E.; Writing–review & editing, I.K., N.S., A.H.H. All authors have read and agreed to the published version of the manuscript. Funding: The project was funded “partially” by the Kuwait Foundation for the Advancement of Sciences un project code: PN17-35EE-02. Acknowledgments: The authors would like to express their appreciation for the valuable eﬀort of Abdulrazaq Masrani in adjusting the voltage-force analysis facility. Additionally, the authors would like to thank both John Avaritsiotis and Murugappan from Kuwait College of Science and Technology (KCST) for their help in acoustic sensing setup. 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. . Panwar, N.; Kaushik, S.; Kothari, S. Role of renewable energy sources in environmental protection: A rev Renew. Sustain. Energy Rev. 2011, 15, 1513–1524. [CrossRef] . 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https://openalex.org/W3201079950	https://serval.unil.ch/resource/serval:BIB_588CC6F9758B.P001/REF.pdf	English	null	Clinical data for paediatric research: the Swiss approach	BMC proceedings	2,021	cc-by	12,210	Proceedings of the National Symposium in Bern, Switzerland, Dec 5-6, 2019 Milenko Rakic1†, Manon Jaboyedoff1,2†, Sara Bachmann3, Christoph Berger4, Manuel Diezi2, Philipp do Canto5, Christopher B. Forrest6, Urs Frey3, Oliver Fuchs7, Alain Gervaix8, Amalia Stefani Gluecksberg9, Michael Grotzer4, Ulrich Heininger3, Christian R. Kahlert10, Daniela Kaiser11, Matthias V. Kopp7, Roger Lauener10, Thomas J. Neuhaus11, Paolo Paioni4, Klara Posfay-Barbe8, Gian Paolo Ramelli9, Umberto Simeoni2, Giacomo Simonetti9, Christiane Sokollik7, Ben D. Spycher1† and Claudia E. Kuehni1† From Clinical Data for Paediatric Research: the Swiss Approach Bern, Switzerland. 5-9 December 2019 From Clinical Data for Paediatric Research: the Swiss Approach Bern, Switzerland. 5-9 December 2019 Correspondence: claudia.kuehni@ispm.unibe.ch * Correspondence: claudia.kuehni@ispm.unibe.ch †Milenko Rakic, Manon Jaboyedoff, Ben D. Spycher and Claudia E. Kuehni contributed equally to this work. 1Institute of Social and Preventive Medicine (ISPM), University of Bern, Mittelstrasse 43, 3012 Bern, Switzerland Full list of author information is available at the end of the article * Correspondence: claudia.kuehni@ispm.unibe.ch †Milenko Rakic, Manon Jaboyedoff, Ben D. Spycher and Claudia E. Kuehni contributed equally to this work. 1Institute of Social and Preventive Medicine (ISPM), University of Bern, Mittelstrasse 43, 3012 Bern, Switzerland Full list of author information is available at the end of the article * Correspondence: claudia.kuehni@ispm.unibe.ch †Milenko Rakic, Manon Jaboyedoff, Ben D. Spycher and Claudia E. Kuehni contributed equally to this work. 1Institute of Social and Preventive Medicine (ISPM), University of Bern, Mittelstrasse 43, 3012 Bern, Switzerland Full list of author information is available at the end of the article Rakic et al. BMC Proceedings 2021, 15(Suppl 13):19 https://doi.org/10.1186/s12919-021-00226-3 Rakic et al. BMC Proceedings 2021, 15(Suppl 13):19 https://doi.org/10.1186/s12919-021-00226-3 BMC Proceedings BMC Proceedings Open Access Abstract Background and purpose: Continuous improvement of health and healthcare system is hampered by inefficient processes of generating new evidence, particularly in the case of rare diseases and paediatrics. Currently, most evidence is generated through specific research projects, which typically require extra encounters with patients, are costly and entail long delays between the recognition of specific needs in healthcare and the generation of necessary evidence to address those needs. The Swiss Personalised Health Network (SPHN) aims to improve the use of data obtained during routine healthcare encounters by harmonizing data across Switzerland and facilitating accessibility for research. The project “Harmonising the collection of health-related data and biospecimens in paediatric hospitals throughout Switzerland (SwissPedData)” was an infrastructure development project funded by the SPHN, which aimed to identify and describe available data on child health in Switzerland and to agree on a standardised core dataset for electronic health records across all paediatric teaching hospitals. Here, we describe the results of a two-day symposium that aimed to summarise what had been achieved in the SwissPedData project, to put it in an international context, and to discuss the next steps for a sustainable future. The target audience included clinicians and researchers who produce and use health-related data on children in Switzerland. © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Page 2 of 15 Rakic et al. BMC Proceedings 2021, 15(Suppl 13):19 Key highlights: The symposium consisted of state-of-the-art lectures from national and international keynote speakers, workshops and plenary discussions. This manuscript summarises the talks and discussions in four sections: (I) a description of the Swiss Personalized Health Network and the results of the SwissPedData project; (II) examples of similar initiatives from other countries; (III) an overview of existing health-related datasets and projects in Switzerland; and (IV) a summary of the lessons learned and future prospective from workshops and plenary discussions. Implications: Streamlined processes linking initial collection of information during routine healthcare encounters, standardised recording of this information in electronic health records and fast accessibility for research are essential to accelerate research in child health and make it affordable. Ongoing projects prove that this is feasible in Switzerland and elsewhere. International collaboration is vital to success. The next steps include the implementation of the SwissPedData core dataset in the clinical information systems of Swiss hospitals, the use of this data to address priority research questions, and the acquisition of sustainable funding to support a slim central infrastructure and local support in each hospital. This will lay the foundation for a national paediatric learning health system in Switzerland. Keywords: Electronic health record, Terminology harmonization, Learning health system, Paediatric research, Conference proceedings Health Network (SPHN). The increasing use of elec- tronic health records (EHRs) to document clinical and administrative information about patient’s encounters in hospitals makes such an approach technically feasible. EHRs contain information on the patients’ complaints, the clinical examination by the physician, results from investigations such as blood tests, imaging or functional tests, diagnoses and treatments. They carry the promise of a faster, cheaper and more meaningful way to evaluate and improve the quality of healthcare [2, 3]. EHRs are rarely standardized within and between institutions and data are often entered in open text fields resulting in un- structured data. These limitations could largely be cir- cumvented if the original data were recorded in a structured and standardized way [4, 5]. A common architecture of EHRs allowing structured data capture during routine medical encounters could allow rapid an- alytics of healthcare data followed by fast feedback of generated knowledge, a process called a learning health system [6, 7]. The SPHN is an initiative of the Swiss Fed- eral Government and aims to contribute to a nationwide interoperability, accessibility and use of health relevant data for evidence-based healthcare research and data- driven precision medicine [8]. SPHN follows a decen- tralized approach whereby standardized electronic health data remain with the healthcare provider but are shareable for research. It should allow data shar- ing for the purpose of data aggregation but also for distributed analysis. During the past 3 years, SPHN has set up nationwide coordinated infrastructures, prepared legal and ethical guidelines and templates to facilitate data sharing and issued calls for research projects that develop infrastructures and start using the generated data [8]. Building a national infrastructure for personalized health research One of the core objectives of the SPHN is to build a na- tional data infrastructure that will enable large scale, high-quality, national projects. Urs Frey, the Chair of the SPHN steering board, presented the vision and guiding principles, the practical implementation and the current status of this initiative. The vision is that future re- searchers can submit a query through a central portal to quickly identify a specific cohort from among all patients at university hospitals in Switzerland and, provided the legal and ethical requirements are met, can access and analyse data from these patients in a project-specific space in a secure computing environment. In reaching this objective SPHN follows a decentralized approach whereby the data remain at the individual hospitals, but are shared on a project-specific basis through a secure network maintained by SPHN Data Coordination Centre (DCC). The SPHN is committed to the FAIR principle, whereby patient data must be Findable, Accessible, Interoperable, and Reusable. The practical implementa- tion requires key changes at the technical and govern- ance level in the different phases of the data flow such as finding, requesting, receiving and using the data. A federated query system and data catalogue will be imple- mented to allow the quick and reliable estimation of availability of data and samples for a specific research project. On the data-provider side, governance processes and compatible IT infrastructure solutions need to be in place to allow extraction and pseudonymization and en- sure semantic interoperability for data exchange. Stan- dards and templates for Common Data Transfer and Use Agreements (DTUA) are being developed to facili- tate data exchange and define key regulatory require- ments regarding the rights, responsibilities and obligations of all parties involved. This manuscript summarises the results of a two-day symposium with the title “Clinical data for Paediatric Re- search: the Swiss approach” held in Bern on December 5 and 6, 2019. The symposium was organised in the con- text of the SwissPedData project by the project PIs (CEK and BS) and research team (MJ and MR). The overall objective was to form a common vision for a paediatric learning health system in Switzerland. Introduction Continuous improvements in patient care are hampered by slow and inefficient processes of generating evidence [1]. Ideally, healthcare systems should be self-correcting and self-improving: information routinely collected from patients should be directly used to improve patient care in a rapid feed-back cycle. Such systems have been de- scribed under the term ‘learning healthcare systems’ [2]. Although large amounts of patient data are collected routinely by healthcare providers, these data are typically unstructured, unstandardised, incomplete and not read- ily available in a format useful for research purposes. In- stead, most data used for healthcare research are obtained in labour-intensive cross-sectional surveys, clinical trials and longitudinal studies that are often set up in a somewhat artificial research setting and do not fully reflect the circumstances of everyday clinical care, i.e. the “real world data”. While the results may help to improve patient care, the learning cycle is slow and costly. Each project typically involves acquiring new re- search grants, recruiting new staff, obtaining ethical ap- proval, setting up the study methodology and infrastructure, recruiting participants, collecting and analysing data, writing up results and getting them pub- lished. This process can take years and generates results at a high cost. The results may be outdated by the time they are published and are potentially biased. All this limits the possibility of continuous and timely, evidence- based improvements in healthcare. This system-wide learning process could be signifi- cantly accelerated if researchers had direct access to standardized data from the health records generated during routine healthcare, a vision shared by various ini- tiatives in Switzerland including the Swiss Personalized Rakic et al. BMC Proceedings 2021, 15(Suppl 13):19 Rakic et al. BMC Proceedings 2021, 15(Suppl 13):19 Page 3 of 15 One of the infrastructure projects funded by the SPHN focused on the collection of clinical data in the paediat- ric setting (SwissPedData [9]), where the need for evidence-based support of treatment decisions and im- provements of care is particularly important. Paediatric medicine lags behind adult medicine in this respect [10]. Evidence in paediatrics is generated slowly and at high costs because many paediatric health conditions are rare [11]. In most settings, no single research institution has enough patients to produce generalizable data that can inform clinical decisions on rare diseases. Consequently, results from studies in adults are extrapolated to chil- dren and off label use of medicines in child healthcare is common [10]. Introduction The clinical research network of Swiss Children’s hospitals, SwissPedNet [12] proposed a paediatrics-specific infrastructure development project “Harmonising the collection of health-related data and biospecimens in paediatric hospitals throughout Switzerland” (SwissPedData). The project was approved and funded by the SPHN in 2017 and was conducted during the period 2018–2020. It aimed to harmonize data and biomaterials across paediatric hospitals in Switzerland with the vision to facilitate high quality clin- ical research in child health. The core task of the project was to create and approve a standardized paediatric dataset for EHRs across Switzerland. Once implemented, this should facilitate paediatric multicentre research based on EHR data for healthcare, public health and causal research, taking a longitudinal life course approach. countries and c) specific existing health-relevant datasets in Switzerland d) and finally, we summarise the lessons learned and e) discussions on future opportunities from workshops and plenary discussions. “Harmonising the collection of health-related data and biospecimens in paediatric hospitals throughout Switzerland” (SwissPedData), an infrastructure project funded by the Swiss personalized health network (SPHN) The presentations summarised in this section describe the aims and results of the SwissPedData project and place it in the context of the broader initiative supported by the Swiss Personalized Health Network (SPHN). Building a national infrastructure for personalized health research Specifically, it aimed to a) summarise the results of the project, b) de- scribe models of paediatric learning health systems in other countries, c) give an overview of available data and successful initiatives in Switzerland, d) showcase what has been reached by SwissPedData and e) discuss pros- pects for the future. The target audience of the sympo- sium included hospital-based clinician-researchers in Paediatrics, allied health professionals, and scientists en- gaged in clinical and epidemiological paediatric research. The program included state-of-the-art lectures by na- tional and international keynote speakers, workshops and plenary discussions. This manuscript is structured into four section where we a) summarise presentations and discussions relating to the Swiss Personalized Health Network (SPHN) and the SwissPedData project, b) next we provide examples of parallel initiatives from other Much has already been achieved. Through various in- frastructure implementation and development projects SPHN has developed standards, procedures and tech- nical infrastructure to enable the nationwide Rakic et al. BMC Proceedings 2021, 15(Suppl 13):19 Page 4 of 15 Page 4 of 15 interoperability and exchange of molecular and clinical patient data. Examples of such infrastructure develop- ment projects are E-General Consent, which aims to de- velop a nationwide harmonized interactive electronic general consent to use data generated in hospitals for re- search, Swiss BioRef, which aims to generate accurate in- dividualized reference ranges for results of blood tests, and SwissPKcdw, which aims to optimize paediatric dos- ing regimens by applying pharmacokinetic modelling to data from routine drug monitoring in clinical data ware- houses [13]. In parallel, so-called driver projects started to use the data generated in the warehouses to answer specific research questions. Examples are a) the Person- alized Swiss Sepsis Study, which uses machine learning to analyse continuous intensive care unit (ICU) monitor- ing, laboratory, microbiology, and -omics data for per- sonalized sepsis management, b) IMAGINE, which proposes to build a Swiss-wide infrastructure for image- based biomarker research & analysis, and c) The Swiss Heart Failure Network that will create a standardized data infrastructure for a Swiss Heart Failure Registry and integrate multi-dimensional features from patient data into machine learning-based diagnostic and risk scores [14]. Building a national infrastructure for personalized health research Throughout the SPHN processes, a number of gen- eral gaps and challenges have been identified including specific organizational, legal and regulatory aspects of data transfer between hospitals, the need for more pa- tient involvement, for specific concepts for governance, processes and infrastructures for -omics data, and for a strategy for engagement with the industry and inter- national consortia. Center and the Biomedical Information Technology (BioMedIT) Network Project [15], provided details about the organisation of the infrastructure network and how researchers will be able to set up a specific project (Fig. 1). A secure IT infrastructure for high performance computing and processing of sensitive, biomedical data has been set up through a network of three interoper- able BioMedIT nodes, one in Lausanne (operated by SIB), one in Basel (operated by the University of Basel), and one in Zurich, operated by Swiss Federal Institute of Technology Zurich (ETHZ). The BioMedIT portal is the single entry point for registered researchers providing access to the network, which runs under one common Information Security Policy. If the data sharing and data usage requirements for a specific project are in place (patient consent, ethical approval, DTUA), the patient data can be assembled from the five university hospitals’ Clinical Data Warehouses, biobanks, −omics platforms, cohort studies, or other data sources. Data packages for a specific project are transferred (end-to-end encrypted) to an isolated project space on one of the BioMedIT nodes for the researcher to access. Within the project space, BioMedIT provides researchers with cutting edge technology for cloud computing, big data storage and high-performance computing. SwissPedNet, the Swiss research network of clinical Paediatric hubs Klara Posfay-Barbe, current president of SwissPedNet, explained how the network was founded in 2012 by the five university children’s hospitals in Switzerland (Basel, Bern, Geneva, Lausanne and Zurich) and the three paediatric teaching hospitals in Aarau, Lucerne, and St. Gallen [12]. In 2015 the department of paediatrics of the Italian speaking canton Ticino joined as a ninth clinical hub. All nine hubs provide age-specific infrastructures, paediatric-trained personnel, paediatric guidance docu- ments and specialized quality management systems for clinical trials in children. SwissPedNet promotes clinical research in paediatrics by supporting paediatric clinician-scientists in their commitment to high-quality clinical research, by facilitating national and inter- national collaboration, providing training opportunities for young researchers and increasing acceptance of clin- ical trials in children. It promotes, facilitates, coordinates and conducts clinical trials in children, from newborns to adolescents, in all paediatric disciplines. SwissPedNet has two associated technology platforms: SwissPedPha, which provides services in paediatric pharmacology, and SwissPedRegistry, which coordinates national registries and cohort studies and advises on the conduct of regis- tries and observational studies in children. SwissPedNet receives direct federal funding to support the infrastruc- ture of the nine clinical hubs, SwissPedPha and SwissPe- dRegistry. It broadly shares the vision of the Swiss Personalized Health Networks and contributes to its de- velopments with a Paediatric focus. Achieving data interoperability Some Swiss hospitals already implemented the use of LOINC codes [17] for laboratory data. The 2018–2020 eHealth strategy takes this issue into account and recommends that semantic standards should be used whenever possible. p Ensuring interoperability of clinical data is a particular challenge as both Crameri and Leichtle highlighted. These data are typically not standardized and often highly unstructured. In principle, two complementary approaches are followed by the SPHN. The first one is to harmonize content and format of clinical data as it is collected or entered into a clinical information system. This approach is being followed by specific SPHN pro- jects such as the paediatric SPHN project “Harmonising the collection of health-related data and biospecimens in paediatric hospitals throughout Switzerland”. The other approach is to map already collected data into a com- mon data model. A common data model specifies the structure and standards for a set of data domains. For unstructured data Natural Language Processing may be necessary to structure the data. The Clinical Data Se- mantic Interoperability Working Group of the DDC is leading the process of defining common data models within the SPHN. This includes a core dataset of basic data on all patients, which is devoted to support distrib- uted queries and an extended dataset covering the needs of specific projects. Achieving data interoperability The specific challenges of reaching interoperability for laboratory data were highlighted by Alexander Leichtle, who is the principal investigator (PI) of the SPHN infra- structure development project Swiss BioRef, which aims to build a nation-wide resource for generating personal- ized reference ranges for laboratory tests. These data come from numerous different sources in heterogeneous Katrin Crameri, Director of the Swiss Institute of Bio- informatics’ (SIB) Personalized Health Informatics Group, which manages the SPHN Data Coordination Fig. 1 The researcher’s new path to nationwide clinical data ([16], page 5) Fig. 1 The researcher’s new path to nationwide clinical data ([16], page 5) Rakic et al. BMC Proceedings 2021, 15(Suppl 13):19 Page 5 of 15 Page 5 of 15 formats. Moreover, some data types like microbiology data are difficult to map. Leichtle highlighted the fact that when working with routine data, continuous moni- toring tools must be in place. Some Swiss hospitals already implemented the use of LOINC codes [17] for laboratory data. The 2018–2020 eHealth strategy takes this issue into account and recommends that semantic standards should be used whenever possible. are expected to produce substantial findings beneficial for people with the same problem may be conducted on children (with or without capacity of judgement) or on adolescents without capacity of judgement. However, this should not prevent minors from being included in studies as they also have the right to receive evidence- based medicine tailored to their age group. Further spe- cific requirements mainly concern principles for consent. Minors must be involved as far as possible in consent giving, with increasing involvement as they grow older. If it is unclear whether a minor has the capacity of judgement, the latter must be presumed. All minors must be given the possibility to revoke consent after reaching maturity. The decentralized political system of Switzerland with 26 cantonal data protection acts greatly complicates the exchange of data. Specific contracts be- tween institutions have to be adapted for each situation. Detailed legal standards for biobanks and big data appli- cations are still lacking. Do Canto emphasized, that while studies must comply with legal standards, the legal standards themselves should be adapted to societal and technological developments for the benefit of patients. formats. Moreover, some data types like microbiology data are difficult to map. Leichtle highlighted the fact that when working with routine data, continuous moni- toring tools must be in place. The aspects of ethical, legal and social implications (ELSI) - the particular case of minors The SPHN recognizes that working toward efficient pro- duction of scientific knowledge as a societal good must go hand in hand with promoting the rights, interests and well-being of the research participants and generating public trust. The SPHN has therefore produced ethical guidelines built on four general principles: respect for persons, privacy, data fairness and accountability [18]. Philipp do Canto, an expert of public health law and legal advisor to the SPHN project SwissPedData, gave an overview on ethical and legal aspects specifically related to collection and use for research of health-related data and bio-specimens from minors. Under Swiss law, mi- nors (children < 14 years and adolescents < 18 years old) are considered vulnerable persons. For these groups, specific requirements apply in addition to general re- quirements for human research on adults. In particular, the Swiss constitution requires the principle of subsidiar- ity, under which research that involves minors is only le- gitimate if findings of equal scientific value cannot be achieved with adults. Furthermore, only projects that im- pose minimal risks and burden on participants and that Rakic et al. BMC Proceedings 2021, 15(Suppl 13):19 Page 6 of 15 Page 6 of 15 SwissPedData - Harmonising the collection of health-related data and biospecimens in paediatric hospitals throughout Switzerland SwissPedData - Harmonising the collection of health-related data and biospecimens in paediatric hospitals throughout Switzerland well as junior physicians who enter the data into the hospital information system and use it for daily clinical work. The dataset that was developed in this effort in- cludes a core module with general paediatric informa- tion that is relevant for all patients, and subspecialty modules that are relevant for subsets of patients. It is compatible with international datasets, in particular the dataset from PEDSnet (chapter 3), but has also add- itional variables that are relevant for the Swiss context or for specific specialties or research interests. Version 1 of SwissPedData has been finalised and is ready for im- plementation into the clinic information software of par- ticipating hospitals. While this takes place, efforts will need to be taken to secure sustainable funding for local support in each hospital, and for central core structures. Based on templates from the SPHN, DTUA are being developed for use by all hospitals. The aspects of ethical, legal and social implications (ELSI) - the particular case of minors Claudia Kuehni, primary investigator of SwissPedData, gave an overview about this SPHN infrastructure devel- opment project, which was funded in 2017 and ran from 2018 to 2020. SwissPedData’s goal was to develop an in- frastructure for affordable, high-quality prospective clin- ical data on every child in Switzerland. Its three work packages (WP) aimed, 1) to create an inventory of re- sources and ongoing research with routine data on child health in Switzerland; 2) to develop a national standard- ized dataset for children visiting paediatric hospitals; and 3) to evaluate whether parents would be willing to ac- tively contribute information on their child’s health to SwissPedData (Fig. 2). The inventory of work package 1 has been com- pleted and consists of routine data collected by the Swiss Federal Statistical Office, such as birth, cause of death and hospital episode statistics, and of non-health related contextual data such as migration statistics and census data. Limited datasets are available from the primary care setting and specific initiatives, such as the Swiss electronic vaccination record [19]. In addition, numer- ous observational studies and registries collect data on specific diseases. Some of these resources were discussed during the symposium and are summarised in section 3 of this paper. Work package 3 aimed to investigate, whether par- ents are willing to provide additional information into the hospital information systems, which can be used for clinical care and for research. This is particularly import- ant for information that is relevant for child health and included when physicians take a complete medical his- tory, but is often not documented consistently in med- ical records due to time constraints, particularly in acute care: family history, environmental exposures and life- style such as active and passive smoking or physical ex- ercise, socioeconomic information and data on social contacts, education and schooling. Work package 3 aimed to pilot paper-based and electronic questionnaires of different length that could be completed by parents in the case of in- or outpatient visits. Evaluation of such parental questionnaires for respiratory outpatients is Work package 2 was the core element of the project and involved an extended Delphi process with all paedi- atric hospitals and representatives from almost all paedi- atric subspecialties in Switzerland. We involved, from all hospitals, senior consultants with expert knowledge, as Fig. The aspects of ethical, legal and social implications (ELSI) - the particular case of minors 2 Schematic outline of the future use of clinical and population-based data relevant for child health for paediatric research in Switzerland, as proposed in the project SwissPedData (own illustration) Fig. 2 Schematic outline of the future use of clinical and population-based data relevant for child health for paediatric research in Switzerland, as proposed in the project SwissPedData (own illustration) Rakic et al. BMC Proceedings 2021, 15(Suppl 13):19 Page 7 of 15 Page 7 of 15 ongoing as part of the SPAC Study [20]. Together, stan- dardized electronic patient records and parental ques- tionnaires should in future boost hospital-based paediatric clinical research in Switzerland. traditional research cycle, which can take 15 years from the formulation of the research idea to the implementa- tion of the findings into healthcare, PEDSnet aims to be a learning health system (LHS) where clinical care, infor- mation technology and research are closely interlinked, research is done at the point of care and feeds directly back into internal improvements within a short time scale (Fig. 3). The international landscape - parallel initiatives in other countries The symposium opened a view on the international landscape, with three examples of how data obtained during clinical care can feed into research, which in turn allows to improve healthcare. These included the PED- Snet initiative in the US, the use of linked clinical in- patient data for observational research and nested trials in the UK, and a multicenter electronic management tool for asthma care in the Netherlands. The Steering Board and committees for research, data and engagement include senior representatives from every participating Children’s Hospital. Other stake- holders closely involved in the organization of PEDSnet are healthcare organizations, researchers, clinicians, pa- tients and families. Currently, PEDSnet hosts analysis- ready standardised longitudinal data from primary, sec- ondary and tertiary care for over 6.8 million patients. Experiences from linking routine hospital datasets in the UK, and their use for observational studies and nested clinical trials Katie Harron, from the University College London, showed how administrative data, including routine hos- pital data, can be used to support clinical trials, for in- stance by capturing outcome information or helping to assess the generalisability of results. In one study, long term educational outcomes on the participants of seven historical infant formula trials were extracted from ad- ministrative education records [37]. In another example, routine datasets from paediatric intensive care units (PICUs) and laboratory surveillance were used to gener- alise the results from a large randomised clinical trial on the use of antibiotic-impregnated central venous cathe- ters to prevent bloodstream infections in PICUs [38]. These examples rely strongly on the ability to match the records that belong to the same person from different datasets (record linkage). This can be done in a deter- ministic manner, which requires an exact match on one (e.g. social security number) or more key identifiers con- tained in both datasets, or in a probabilistic record link- age approach, which allows for some degree of mismatch in the common variables caused by errors or omissions during data entry [39]. Linkage errors includ- ing false matches (records of different persons assigned to the same person) or missed matches (records of the same person assigned to different IDs) can bias the re- sults of a study. This can, for instance, occur through the misclassification of outcomes, over- or undercount- ing of individuals and by excluding unlinked cases. It is therefore important to assess the quality of data linkage and the potential for biases [40]. Katie Harron highlighted several ways in which this can be done, for instance, through the use of positive and negative Descriptive epidemiology: The prevalence of obesity and overweight in 2–17 year olds was similar in EHR-derived data from PEDSnet (18 and 17% re- spectively) and in the National Health and Nutrition Examination Survey (NHANES; 18 and 16%). PED- Snet estimates were more stable because the dataset was much larger [28]. Computable phenotyping: PEDSnet data is used to develop and evaluate computable phenotypes for the identification of patients with rare diseases, such as cancer and glomerular disorders [29, 30]. PEDSnet: a learning health system for Paediatrics Examples are the BACK-OFF trial in Juven- ile Spondyloarthritis [24] or a surgical trial compar- ing three approaches to treat kidney stones [25]. Pseudo-trials: This type of design was used to show the superiority of infliximab versus standard care for Crohn’s disease in patients with new-onset moderate or severe episodes of illness. Infliximab in Crohn’s disease had previously been prioritized over standard of care in paediatrics following positive results in adults, although no RCT was ever conducted in chil- dren. Conducting a RCT today would not have been acceptable from both a clinical and ethical perspec- tive. In collaboration with the learning network ImproveCareNow [26], PEDSnet conducted a “pseudo-trial” study that used available data and compared episodes of care that used infliximab with episodes with standard treatment [27]. Pseudo-trials: This type of design was used to show the superiority of infliximab versus standard care for Crohn’s disease in patients with new-onset moderate or severe episodes of illness. Infliximab in Crohn’s disease had previously been prioritized over standard of care in paediatrics following positive results in adults, although no RCT was ever conducted in chil- dren. Conducting a RCT today would not have been acceptable from both a clinical and ethical perspec- tive. In collaboration with the learning network ImproveCareNow [26], PEDSnet conducted a “pseudo-trial” study that used available data and compared episodes of care that used infliximab with episodes with standard treatment [27]. PEDSnet: a learning health system for Paediatrics The data coordinating centre updates the structure and semantics of the data model and network (the pipeline operations), automates the cross checks to maintain high quality, and provides guidance on how to use complex data to improve research. The centre spe- cific teams extract, transform and upload the data and give site level guidance and implementation. Every 3 months, new data are uploaded and crosschecks are done to test the validity and assure high quality. This is automated by staff with a background in health research and informatics and the tools are publicly available for use in R. flows, and the data quality program. Data quality was an overarching concern during the design of the PEDSnet data network. The goal was to create data of high qual- ity, which are easily accessible. PEDSnet has three rele- vant teams of data experts. The data committee oversees the data model and leads the strategic development of network. The data coordinating centre updates the structure and semantics of the data model and network (the pipeline operations), automates the cross checks to maintain high quality, and provides guidance on how to use complex data to improve research. The centre spe- cific teams extract, transform and upload the data and give site level guidance and implementation. Every 3 months, new data are uploaded and crosschecks are done to test the validity and assure high quality. This is automated by staff with a background in health research and informatics and the tools are publicly available for use in R. collecting data directly from patients in cohort studies or clinical trials. The data network is used to identify specific patients who meet eligibility criteria for a study. Patients’ encrypted identifiers are then sent to each local institution, where a reidentification process is undertaken before chart review to ensure that they meet the selection criteria. Examples are the BACK-OFF trial in Juven- ile Spondyloarthritis [24] or a surgical trial compar- ing three approaches to treat kidney stones [25]. collecting data directly from patients in cohort studies or clinical trials. The data network is used to identify specific patients who meet eligibility criteria for a study. Patients’ encrypted identifiers are then sent to each local institution, where a reidentification process is undertaken before chart review to ensure that they meet the selection criteria. PEDSnet: a learning health system for Paediatrics Key characteristics of the PEDSnet LHS are, (1) that it uses a common interoperable, prospectively designed platform, (2) builds trust among stakeholders, (3) opti- mizes electronic health records to support that data are entered once, but are used many times – for care, im- provement, registries, and research, (4) continuously ex- pands and strengthens its scalable infrastructure, (5) links electronic health record data with other data sources, and (6) secures a sustainable future. PEDSnet: a learning health system for Paediatrics Christopher Forrest, principal investigator of the US PEDSnet initiative [21], discussed different aspects of PEDSnet. A first talk, entitled “From concept to proto- type to national scaling” focused on history, organization, and governance. PEDSnet is a national Paediatric Learning Health System that was founded in the U.S. in 2014 by eight U.S. Children’s Hospitals [22, 23]. It was motivated by the need to obtain child-specific data on the effectiveness and safety, particularly long- term events, of new and already approved drugs and other types of medical, surgical, and behavioral interven- tions. Complementary to expensive clinical trials in small groups of selected patients, use of electronic health record data on a large-scale has potential to improve the sparse evidence base. The overall objectives of PEDSnet are to scale-up a collaboration platform that enables rapid improvement in the outcomes of ill children and of the health systems that serve them. Instead of the In his second talk, Christopher Forrest gave specific ex- amples and case studies, to show how PEDSnet conducts research based on data from routine care, and how re- sults are fed back to improve healthcare in participating hospital. PEDSnet allows to conduct different types of studies: Pragmatic clinical trials: the PEDSnet infrastructure can be leveraged for prospective clinical research, including studies that involve Fig. 3 Improving outcomes with a learning healthcare system: PEDSnet model (by Christopher Forrest) Fig. 3 Improving outcomes with a learning healthcare system: PEDSnet model (by Christopher Forrest) Rakic et al. BMC Proceedings 2021, 15(Suppl 13):19 Page 8 of 15 flows, and the data quality program. Data quality was an overarching concern during the design of the PEDSnet data network. The goal was to create data of high qual- ity, which are easily accessible. PEDSnet has three rele- vant teams of data experts. The data committee oversees the data model and leads the strategic development of network. Lessons learned from online paediatric asthma management g Peter Merkus, from the Radboud University Medical centre (UMC) Amalia Children’s Hospital in Nijmegen, Netherlands, presented the Luchtbrug® (air lift) study, which uses an online platform for the medical manage- ment of children with asthma, which enables patients and their parents to monitor their disease and communi- cate directly with paediatricians. Between 2011 and 2014, children with asthma were randomized to usual care with clinical visits every four months or to a novel management approach with clinical visits every eight months plus monthly online asthma monitoring encounters. Children reported their asthma symptoms and medications to the online platform, which was checked by a respiratory spe- cialist nurse. The nurse gave feedback to patients and in- formed clinicians in case of asthma exacerbations. The online management program reduced outpatient visits by 50% while maintaining asthma control. There was no dif- ference in asthma exacerbations, hospital admissions, and daily medication doses between the two groups [42, 43]. The Luchtbrug® study serves as a new concept how patients with a chronic disease can contribute to a clinical data plat- form. In a follow-up study, Luchtbrug Connect®, self- reported data is complemented by results on pulmonary function from smart inhalers in selected patients with poor adherence, low symptom perception or severe asthma. Routine administrative data collected in the Swiss Federal Statistical Office and how they can be used for research Adrian Spoerri, head of SwissRDL – Medical Registry and Data Linkage, a centre of excellence for registries at the University of Bern – gave an overview of health- related administrative datasets available at the Federal Statistical Office (FSO) and the possibilities for record linkage with clinical datasets, cohort studies or registries for research purposes. He emphasized that researchers should attempt to make use of the available administra- tive datasets. Record linkage of these datasets with each other and with other datasets for research purposes has a firm legal basis and is often technically feasible. Such record linkages are generally performed by the FSO and detailed instructions and an application form are avail- able from the FSO website [45]. For instance, a re- searcher might want to use the Hospital episode statistics (HES) to identify multiple hospitalisations or hospitalisations in another hospital for a given set of pa- tients. The HES has collected sociodemographic vari- ables, characteristics of hospitalizations, diagnoses and interventions on all patients from all hospitals and birth centres since 1998. Experiences from linking routine hospital datasets in the UK, and their use for observational studies and nested clinical trials Longitudinal observational studies: PEDSnet data provided quantitative evidence of the effect of obesity on incident asthma (30% increase in risk [31]), of an association between antibiotics use before 2 years of age and body mass index at age 5 [28, 32, 33] and of an association between oral thrush by age 1 year and development of childhood caries at the age of 2 to 5 years [34]. Comparative effectiveness study: Gastric banding was shown to be inferior regarding reduction of body mass index compared to gastric bypass and sleeve gastrectomy in youth [35]. Covid-19: A recent example is that PEDSnet used paediatric patients’ EHRs to describe the epidemiology of infected patients in the United States [36]. The third talk focused on the PEDSnet data network, its common data model, the system architecture, data Rakic et al. BMC Proceedings 2021, 15(Suppl 13):19 Page 9 of 15 Page 9 of 15 controls, comparisons with external references and com- parison of characteristics of linked and unlinked records [41]. usages of routine datasets for clinical research in Switzerland. The case studies spanned a variety of infor- mation resources including national administrative data- sets collected by the Swiss Federal statistical office, expert (Orphanet) and surveillance (Swissnoso) net- works, registries (NeoNet, Swiss Childhood Cancer Registry), clinical cohorts (Swiss Paediatric Airway Co- hort) and primary care electronic medical records (FIRE project). Lessons learned from online paediatric asthma management An anonymous code allows tracking multiple hospitalizations from the same patient and link- age of different statistics within the FSO. As another ex- ample, a researcher may want to use national mortality records to update vital status in a clinical cohort study. Such a linkage requires re-identification of a social se- curity number based on personal identifying information (PII) such as names, dates of birth, and address. After linkage, the linked data can only be used to answer the specific research question and must be destroyed and cannot be used to update the cohort dataset perman- ently. In the future, the establishment of linkage centres might greatly facilitate the linkage of personal data. In these centres, the data from different sources are linked using PII and the linked data (without PII) is then made accessible to researchers in a data hub via secure remote access. A successful example of how record linkages be- tween multiple administrative datasets can be used for health research is the Swiss National Cohort study (SNC) [46]. The SNC is research platform linking na- tional censuses since 1990 with each other and with Lessons learnt from the study are: to develop the tool from the patient’s perspective; to aim for sustainable im- plementation from the start and anticipate on what is needed; to involve patients and patient organisations in developing and improving the platform; to create obvious personal benefits for patients, aiming for a WIN – WIN situation; to address barriers for implementation (financial and interoperability) adequately; to provide convincing evidence for added value and cost-effectiveness to health insurance companies, hospitals and governments; to invest in a team with common ambition; to ask for professional help in particular with relation to privacy, information technology, legal contracts and similar; to get out of the comfort zone as a researcher and connect with the na- tional governmental institutions and bodies; and, finally, to expect that the processes will take some time [44]. The current landscape in Switzerland: overview of available routine data and examples of successful research based on real world, standardized healthcare data The symposium included a series of presentations highlighting successful examples and potential future Page 10 of 15 Page 10 of 15 Page 10 of 15 Rakic et al. BMC Proceedings 2021, 15(Suppl 13):19 national datasets on migration and mortality, and has led to a large body of peer-reviewed publications. Orphanet, a tool for healthcare and research on rare diseases Loredana D’Amato Sizonenko, coordinator of Orphanet Switzerland [47], gave an overview of the resources pro- vided by Orphanet to improve the visibility of rare dis- eases, provide high-quality information and expertise on rare diseases and contribute to generate knowledge on rare diseases. Orphanet Switzerland is a partner of the International ORPHANET Network, the reference knowledge base for information on rare diseases and or- phan drugs. Orphanet has created a nomenclature for rare diseases which allows assigning each disease to a unique and stable ORPHA-code, which is cross- referenced to other international nomenclatures and translated into nine languages. It is multi-dimensional and multi-hierarchical. ORPHA-codes allow for seman- tic interoperability of rare disease diagnosis, in health- information systems but also in research. In Switzerland, some hospitals already embed ORPHA-codes in their clinical information system in order to allow for quick identification of patients with a rare disease. [ ] Rami Sommerstein, Head of Research & Development at Swissnoso, presented Swissnoso, a research program aiming to reduce infections in hospitals and long-term care facilities in Switzerland [50]. Swissnoso acts as a na- tional non-profit association of experts in hospital hy- giene and develops research projects and guidelines aiming to reduce healthcare-associated infections in hos- pitals or long-term care facilities. The national program “Swiss Clean Care” includes several modules, which sup- port hospitals in the prevention of healthcare-associated infections. The Surgical Sites Infection (SSI) Surveillance module monitors the frequency of postoperative wound infections, with 174 clinics contributing data [51]. The SSI Intervention module aims to reduce post-operative wound infection rates by implementing three interven- tions: correct hair removal, preoperative skin disinfection and antibiotic prophylaxis. When these measures are ad- hered to in 90% of cases, the wound infection rate may be reduced by at least 10%. The “CleanHands” module allows electronic recording of hand hygiene adherence in health care workers by using a mobile device during observations of hand hygiene. The tool provides auto- mated analysis and national benchmarking that will allow immediate feedback of the results. Currently, over 90 clinics are using the instrument. For the project StAR (Strategy against Antibiotic Resistance), Swissnoso de- velops together with the Swiss Society for Infectious Dis- eases SSI and the Swiss Society for Microbiology SGM prescription guidelines for antibiotics, guidelines for the prevention and control of outbreaks, and a concept for the proper handling of antibiotics. Lessons learned from online paediatric asthma management extract their own data, combine them with follow-up data on a local level and use them for local research. Analysis on a national level needs agreement by all par- ticipating units. Second, the project performs regular unit-to-unit comparison and thus provides the possibil- ity of quality control and national and international benchmarking. Results are used for quality improve- ments. Third, the collected data allow the comparison of outcome data between Switzerland and other countries [49]. Research based on routinely obtained data from paediatric hospitals in Switzerland Research projects in different paediatric disciplines already use data generated in routine care and harmo- nized on a national level for health-care research. This includes registries and cohort studies in paediatric on- cology, neonatology and infectious diseases (using data from inpatients) and paediatric pulmonology (using data from outpatient encounters). p ) Mark Adams, coordinator of SwissNeoNet, presented this learning health system for neonatology in Switzerland. SwissNeoNet [48] is a population-based registry hosting the official medical quality register for the neonatal intensive care units (9 units) and intermedi- ate care units (11 units). SwissNeoNet aims to maintain and improve medical care for high-risk newborns and their families. A minimal dataset is collected for all Swiss infants born below 34 weeks of gestational age, those with a birth weight of less than 1500 g and those with neonatal asphyxia. Follow-up data are collected after 2 and 5 years in 16 neuro-developmental paediatric units for infants below 28 weeks gestation and those with as- phyxia. Data are either imported automatically from the clinic information system in SwissNeoNet or entered by the units through a web form. All data are checked for plausibility and reliability. Suspicious entries have to be corrected by the units. Mark Adams explained that par- ticipation in SwissNeoNet has several advantages for the units: first it provides a data repository, where units can p p g Ben Spycher, head of the research group Environmen- tal and Spatial Epidemiology at the ISPM Bern, de- scribed how data from the Swiss Childhood Cancer Registry can be combined with various other datasets in- cluding clinical and administrative data for research pur- poses. The Swiss Childhood Cancer Registry (formerly SCCR, as of 2020 Childhood Cancer Registry, ChCR, [52]) was founded in 1976 and is a population-based registry with nationwide coverage including all children and adolescents under the age of 21 who are diagnosed with cancer in Switzerland. Data completeness has been more than 95% since 1995 for children between 0 and 15 years of age. Vital status is updated through a Page 11 of 15 Rakic et al. Workshops Th The symposium included two workshops in small groups of heterogeneous composition including junior and senior participants, clinicians and non-clinicians. Each group was asked to (1) brainstorm and collect clin- ical questions, where evidence is lacking or contradict- ory; (2) sketch a short research proposal describing the type of data needed and the most appropriate study de- sign; (3) discuss the feasibility of their research project, including sample size and possibility of recruitment; and (4) suggest suitable funding for the hypothetical pro- posal. The underlying aim of the exercise was to raise awareness about uses of a standardized national health record dataset for research and healthcare. p p The Swiss Paediatric Airway Cohort (SPAC) was pre- sented by Carmen de Jong. SPAC is a national longitu- dinal study using data from outpatient encounters for respiratory problems in paediatric hospitals, i.e. children seen in respiratory outpatient clinics [20]. SPAC is an observational multipurpose cohort study embedded in routine care, which aims to describe the spectrum of re- spiratory problems leading to outpatient visits; to distin- guish clinical phenotypes of wheeze and cough [58]; to describe prognosis and predict the long term course of respiratory disease, and to investigate, compare and har- monise diagnostic practices, treatment strategies, and preventive measures used in healthcare in Switzerland [59]. SPAC serves also as a sampling frame for recruiting children for nested studies. Data are collected from med- ical records and via questionnaires to parents. Seven hospitals and 2 private practices have invited over 3000 patients in 28 months. The overall participation rate is 63%, but varying between clinics from 48 to 89%. In order to increase the participation rate, local conditions need be taken into account during recruitment so that it corresponds to routine care procedures. For example, pro-active monitoring and regular reminders, especially in the beginning, improve participation rate. SPAC shows that conduction of a study embedded in routine care but involving active contributions for participants is feasible in Paediatric Outpatient care, achieves relatively high response rates, and can run almost automatically after procedures are well established. The short research proposals spanned a broad range of medical topics and study designs. Implications, sustainability and steps forward Implications, sustainability and steps forward The plenary discussions during the symposium and at its end focused on the next steps in the process of set- ting up a paediatric learning health system in Switzerland. Research based on routinely obtained data from paediatric hospitals in Switzerland BMC Proceedings 2021, 15(Suppl 13):19 combination of passive and active follow-up and record linkage with the mortality records The ChCR forms the basis of the Swiss Childhood Cancer Survivor Study (SCCSS), which collects long-term follow-up informa- tion from 5-year survivors through questionnaires and allows investigating a wide range of somatic and psycho- social outcomes including second primary malignancies and late effect of cancer treatments, educational achieve- ment, and health-related quality of life [53]. Further- more, probabilistic record linkages of the ChCR records with the SNC allowed to investigate the spatial distribu- tion of a range of potential risk factors of cancer inci- dence in children including natural background radiation and proximity of residence to highways and to nuclear power plants [54–57]. general practitioners (GPs). Data collected within FIRE include vital parameters, date of consultation, age, gen- der, medications, diagnoses, and laboratory values. Data are anonymous and automatically transferred every 2 months to the data server hosted at the University of Zurich. This dataset can be used for cross-sectional studies, for the recruitment and follow-up of cohorts, but also to embed new clinical trials. Research based on data obtained in primary Care in Switzerland Obtaining standardized digital data from primary care poses a particular challenge. Sven Streit, Associate Pro- fessor at the Institute of Primary Healthcare (BIHAM), University of Bern, presented the FIRE project (Family medicine ICPC Research using Electronic medical re- cords [60]), a database currently containing primary care data of over 862′990 patients from more than 500 Workshops Th Examples included a diagnostic study on the validity of the tests used for auditory screening in newborns; a benchmarking study assessing quality of treatment for bronchiolitis across different Children’s hospitals; a cohort study on the inci- dence of hearing loss after treatment with aminoglyco- sides in infancy; a cohort study on kidney injury after treatment with acyclovir; and a randomized clinical trial comparing the effectiveness of different treatment regi- mens for type 1 diabetes. Several of the proposed studies aimed to complement the hospital dataset with available data from other sources such as the federal statistical of- fice, or with the collection of additional data through questionnaires or specific examinations. Strengths and weaknesses of the proposed projects and aspects of im- plementation and feasibility were then critically dis- cussed in the plenum. Overall, this exercise gave a first impression of the possibilities that will open and did whet the appetite for more. Scaling up and integrating other data sources After harmonization of the data generated in university hospitals and other teaching hospitals, the SPHN plans to involve other healthcare providers in the process with the ultimate goal of connecting individual patient data from primary, secondary and tertiary care settings. Initial efforts to expand connectivity will focus on other hospi- tals, and networks of paediatricians and general practi- tioners providing primary healthcare. The idea of integrating data from primary care such as data on immunisations was repeatedly expressed and welcomed by participants of the meeting. However, such an exten- sion will require dedicated funding. Securing sustainable funding For the project to continue, it will need a central coordin- ation centre, which can coordinate and facilitate the paedi- atric research collaboration. The tasks of this central body would include communication with the medical and IT representatives of the different children’s clinics, with the SPHN Data Coordination Centre, with international re- search partners and with funders, assistance with writing grant applications and obtaining ethical approval and the provision of standardized datasets for research. Because relevant aspects are covered by the SPHN data centre, the additional infrastructure required for a paediatric module can be relatively limited, but, as a minimum, will require a senior scientist to lead it, a coordinator with a strong re- search background, ideally in paediatric medicine, who communicates with clinicians, research partners and fund- ing institutions, and another person specialized in data handling and information technology, who can work at the data level and communicate with IT specialists from local hospitals and with the SPHN data centre. The collab- orating clinics will also need financial support for IT staff to deliver local data to in regular intervals or at request. The coordination centre can rely on the wider SPHN net- work for assistance including advice regarding ELSI as- pects, the provision of templates for DTUA, and communication with stakeholders from federal and can- tonal governments. Implementation of SwissPedData in the electronic health records of Paediatric teaching hospitals Implementation of SwissPedData in the electronic health records of Paediatric teaching hospitals Implementation of SwissPedData, Version 1, into the electronic health records of all participating children’s Page 12 of 15 Rakic et al. BMC Proceedings 2021, 15(Suppl 13):19 hospitals is the obvious next step in the process. This will take some time, as it needs IT resources and must be harmonized with other pending or planned changes in the EHRs, and other interventions of the IT special- ists. The children’s hospital of Basel, which is about to renew its IT infrastructure, started to implement Swis- sPedData in 2020, and other hospitals are planning to follow soon. Meeting with IT specialists and senior phys- ician representatives of all collaborating hospitals are be- ing planned. The central SPHN data centre and the IT staff of the five university hospitals, who receive substan- tial annual core funding from the SPHN initiative, will support the process. Given the hierarchical organization of the university hospitals, it was agreed that a top-down process with strong commitment from all directors of the Children’s clinics will be the most effective approach for implementation. The actual data exchange and ac- cess can be implemented as dedicated paediatric module on the SPHN infrastructure. of their hospital as an instrument to assess and improve quality of healthcare delivered and boost their clinic’s research output; – motivate physicians to enter data properly into the EHR, and to actively contribute to improving the dataset by providing constructive comments that will lead to improved versions of SwissPedData. – motivate physicians to enter data properly into the EHR, and to actively contribute to improving the dataset by providing constructive comments that will lead to improved versions of SwissPedData. Availability of data and materials For data and materials relating to this document, please contact the corresponding author, Prof Claudia Kuehni claudia.kuehni@ispm.unibe.ch Abbreviations DTUA D T Abbreviations DTUA: Data Transfer and Use agreement; LHS: Learning health system; FSO: Federal Statistical Office; PEDSnet: A multi-specialty network that con- ducts observational research and clinical trials across multiple children’s hos- pital health systems in the US; SIB: Swiss Institute of Bioinformatics; SPHN: Swiss Personalized Health Network; SPAC: Swiss Paediatric Airway Cohort - an observational research project in Switzerland, which uses data obtained during outpatient encounters in large paediatric hospitals; SwissPedData: “Harmonising the collection of health-related data and bios- pecimens in paediatric hospitals throughout Switzerland”, an infrastructure development project of the SPHN funded in 2017; SwissPedNet: Swiss Research Network of clinical Pediatric Hubs; SBP: Swiss Biobanking Platform; FIRE: Family medicine ICPC Research using Electronic medical records Declaration Ethics approval and consent to participate Not applicable. References 1. Morris ZS, Wooding S, Grant J. The answer is 17 years, what is the question: understanding time lags in translational research. J R Soc Med. 2011;104(12): 510–20. https://doi.org/10.1258/jrsm.2011.110180. 2. Deans KJ, Sabihi S, Forrest CB. Learning health systems. Semin Pediatr Surg. 2018;27(6):375–8. https://doi.org/10.1053/j.sempedsurg.2018.10.005. 3. Cimino JJ. Collect once, use many. Enabling the reuse of clinical data through controlled terminologies. J Ahima. 2007;78(2):24–9 quiz 31-2. 4. Kush R, Alschuler L, Ruggeri R, Cassells S, Gupta N, Bain L, et al. Implementing single source: the STARBRITE proof-of-concept study. J Am Med Inform Assoc. 2007;14(5):662–73. https://doi.org/10.1197/jamia.M2157. g j 5. Breil B, Semjonow A, Müller-Tidow C, Fritz F, Dugas M. HIS-based Kaplan- Meier plots - a single source approach for documenting and reusing routine survival information. BMC Med Informa Decis Making. 2011;11(1):11. https://doi.org/10.1186/1472-6947-11-11. Acknowledgements The authors would like to thank the speakers of the symposium and all the people who helped with the organization and coordination: Katrin Crameri, Alex Leichtle, Katie Harron, Peter Merkus, Loredana d’Amato Sizonenko, Adrian Spoerri, Alexander Leichtle, Mark Adams, Rami Sommerstein, Sven Streit, Matthias Baumgartner, Christina Schindera, Maria Otth, Carmen de Jong, Christina Mallet, Antonella Mazzei Abba, Christophe Folly, Eugénie Collaud, Sven Strebel, and Katharina Flandera. 6. Seid M, Margolis PA, Opipari-Arrigan L. Engagement, peer production, and the learning healthcare system. JAMA Pediatr. 2014;168(3):201–2. https://doi. org/10.1001/jamapediatrics.2013.5063. 7. Institute of Medicine Roundtable on Evidence-Based, M., The National Academies Collection: Reports funded by National Institutes of Health, in The Learning Healthcare System: Workshop Summary, L. Olsen, D. Aisner, and J.M. McGinnis, Editors. 2007, National Academies Press (US) copyright © 2007, National Academy of Sciences.: Washington (DC). Competing interests ll h d l h All authors declare that they have no competing interests. Authors’ contributions All authors attended the symposium and contributed to the writing of this report. Milenko Rakic and Manon Jaboyedoff coordinated the activity and led the editing, Ben Spycher and Claudia Kuehni did most of the writing. All authors approved the final document and consent to the publication of the manuscript. 8. Swiss Personalized Health Network (SPHN). Available from: https://sphn.ch/, access date: 23.04.2021. 9. Jaboyedoff, M., et al., SwissPedData: Standardising hospital records for the benefit of paediatric research. medRxiv, 2021: p. 2021.06.16.21258848. 9. Jaboyedoff, M., et al., SwissPedData: Standardising hospital records for the benefit of paediatric research. medRxiv, 2021: p. 2021.06.16.21258848. 10. Kern SE. Challenges in conducting clinical trials in children: approaches for improving performance. Expert Rev Clin Pharmacol. 2009;2(6):609–17. https://doi.org/10.1586/ecp.09.40. 10. Kern SE. Challenges in conducting clinical trials in children: approaches for improving performance. Expert Rev Clin Pharmacol. 2009;2(6):609–17. https://doi.org/10.1586/ecp.09.40. Consent for publication Not applicable. Consent for publication Not applicable. Taken together, these actions should lay the foundations for a national paediatric LHS in Switzerland and help improve healthcare for children. Once implemented, SwissPedData can serve as core dataset on which further data layers could in future be added. Such data could in- clude primary care data, immunisations, school examina- tions, administrative data (birth and mortality records, socio-economic and demographic information) or infor- mation on lifestyle factors and environmental exposures collected directly from families, for instance, through mobile devices or web-based applications. Furthermore, as children grow older, their electronic health records will capture information on long term health outcomes in adult life. Such extensions would greatly increase the scope of possible research and allow large-scale epi- demiological investigations of children’s health, poten- tially over their life course and contribute to reducing disease burden in adult life. Using the data The following possibilities for securing future funding were discussed during the meeting: It is important that concrete research proposals and pro- jects begin building on the harmonized dataset immedi- ately, even though the quality of the data that will be available at the time of implementation will not be per- fect. As demonstrated in PEDSnet, missingness of data, coding errors and bugs are most easily spotted when the data are being used to answer actual research questions, and concise projects give the opportunity to improve the data batch by batch [61]. As also shown in PEDSnet, first projects can demonstrate that improving data quality and shareability is a win-win for all stakeholders. Early use of the data for research can: – Participate in suitable calls for proposals from funding bodies at the national (e.g. next SPHN call and the Swiss National Science Foundation) or international level (such as EU funding or NIH) and in multinational consortia; – Charge cost-covering fees for data delivery and other services provided by the SwissPedData coordination centre. These costs can be included in budgets of re- search grants from conventional funders, such as the Swiss National Science Foundation or smaller fund- ing agencies; – boost awareness of new opportunities for improving healthcare afforded by the availability of large, standardized, national dataset among clinicians, researchers, and patient representatives; g g – Build up collaborations with industry, for instance for post-marketing studies. International collaboration will be essential for achiev- ing necessary sample sizes for certain research projects, – improve the willingness of local hospital directors to speed up implementation of the dataset in the EHR Page 13 of 15 Rakic et al. BMC Proceedings 2021, 15(Suppl 13):19 Page 13 of 15 particularly those relating to rare diseases and for studies that draw added value from comparisons between differ- ent healthcare systems. Such collaboration will also in- crease the national and international visibility and recognition of SwissPedNet and improve chances of se- curing sustainable funding. Availability of data and materials Author details 1 1Institute of Social and Preventive Medicine (ISPM), University of Bern, Mittelstrasse 43, 3012 Bern, Switzerland. 2Service of Pediatrics, Department Women-Mother-Child, Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland. 3University of Basel Children’s Hospital Basel (UKBB), University of Basel, Basel, Switzerland. 4University Children’s Hospital Zurich, University of Zurich, Zurich, Switzerland. 5Public Sector Law, Zurich, Switzerland. 6Children’s Hospital of Philadelphia, Philadelphia, PA, USA. 7Department of Paediatrics, Inselspital, Bern University Hospital, University of Bern, Bern, Switzerland. 8Department of Woman, Child and Adolescent, Children’s Hospital, Geneva University Hospitals and Faculty of Medicine, Geneva, Switzerland. 9Paediatric Department of Southern Switzerland, Ente Ospedaliero Cantonale, Bellinzona, Switzerland and Università della Svizzera Italiana, Lugano, Switzerland. 10Children’s Hospital of Eastern Switzerland, St. Gallen, Switzerland. 11Children’s Hospital of Lucerne, Cantonal Hospital Lucerne, Lucerne, Switzerland. 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https://openalex.org/W4361894810	https://aacr.figshare.com/articles/journal_contribution/Supplementary_Figure_6_from_Tankyrase_and_the_Canonical_Wnt_Pathway_Protect_Lung_Cancer_Cells_from_EGFR_Inhibition/22391499/1/files/39837039.pdf	unk	null	Supplementary Figure 6 from Tankyrase and the Canonical Wnt Pathway Protect Lung Cancer Cells from EGFR Inhibition	null	2,023	cc-by	5	S6C S6D S6C S6D S6D
https://openalex.org/W3098018905	https://link.springer.com/content/pdf/10.1140/epjc/s10052-018-5515-8.pdf	English	null	Holographic Van der Waals phase transition of the higher-dimensional electrically charged hairy black hole	European physical journal. C, Particles and fields	2,018	cc-by	8,998	Holographic Van der Waals phase transition of the higher-dimensional electrically charged hairy black hole Hui-Ling Li1,2,a, Zhong-Wen Feng3, Xiao-Tao Zu1 1 School of Physical Electronics, University of Electronic Science and Technology of China, Chengdu 610054, China 2 College of Physics Science and Technology, Shenyang Normal University, Shenyang 110034, China 3 College of Physics and Space Science, China West Normal University, Nanchong 637002, China Received: 7 May 2017 / Accepted: 30 December 2017 / Published online: 20 January 2018 © The Author(s) 2018. This article is an open access publication Abstract With motivation by holography, employing black hole entropy, two-point connection function and entangle- ment entropy, we show that, for the higher-dimensional Anti- de Sitter charged hairy black hole in the ﬁxed charged ensem- ble, a Van der Waals-like phase transition can be observed. Furthermore, based on the Maxwell equal-area construction, we check numerically the equal-area law for a ﬁrst order phase transition in order to further characterize the Van der Waals-like phase transition. Waals phase transition in the canonical ensemble (ﬁxed elec- tric charge), i.e. the isocharge in the temperature–entropy plane has an unstable branch and two stable ones when the charge below a critical value. And there exists a second order critical point at a critical charge. Recently, the research on a Van der Waals-like phase transition has been generalized to the extended phase space [5–10]. In this framework, the cos- mological constant is taken as a thermodynamical pressure, and its conjugate quantity is treated as the thermodynamical volume. Consequently, this extended phase space makes the Van de Waals description more precise. Very recently, entanglement entropy has also been used to detect phase structures in different AdS backgrounds. John- son [11] proposed that, like black hole entropy, entanglement entropy also exhibited a Van der Waals-like phase transition in the temperature-entanglement entropy plane in both the ﬁxed potential ensemble and the charge ensemble. It was subsequently generalized to supergravity STU black holes that involve four charges by Caceres et al. [12]. The result showed that Van der Waals behavior was observed in the cases of three charge and four charge, however, for the one- charge and two-charge cases, the STU black holes did not present this phase transition. Caceres et al. also veriﬁed that, for a charge conﬁguration that presented a Van der Waals-like phase transition, the entanglement entropy indeed exhibits a similar phase transition at the same critical temperature and the same critical exponents as the ones obtained in the black hole entropy. a e-mail: LHL51759@126.com Holographic Van der Waals phase transition of the higher-dimensional electrically charged hairy black hole Furthermore, the equal-area law of entangle- ment entropy was also checked by Nguyen [13] for an AdS Reissner–Nordström black hole in the canonical ensemble. In addition, Zeng et al. have investigated phase structures of holographic entanglement entropy in massive gravity [14], in the Born–Infeld Anti-de Sitter background [15] and in the quintessence Reissner–Nordström AdS background [16], andall theresults showedthat thereexists aVandeWaals-like phase transition in these gravity backgrounds [14–18]. Eur. Phys. J. C (2018) 78:49 https://doi.org/10.1140/epjc/s10052-018-5515-8 Regular Article - Theoretical Physics 1 Introduction According to the Anti-de Sitter space/Conformal Field The- ory (AdS/CFT) correspondence [1], the thermodynamics of AdS black holes plays a key role in comprehending the ther- mal properties of the holographically dual ﬁeld theories. To the compelling phenomena of thermodynamics belong phase transitions in AdS spacetime. The pioneer discussion of phase transitions was presented by Hawking and Page [2]. In 1983, in a four-dimensional non-charged AdS back- ground, Hawking and Page proved that there exists a ﬁrst order phase transition between the AdS and Schwarzschild AdS black hole. Speciﬁcally speaking, the thermal AdS is unstable, and it has to undergo a phase transition to the sta- ble Schwarzschild AdS black hole at last, which is the best- known Hawking–Page phase transition. Later, in 1999, in a charged AdS background, Chamblin et al. [3,4] explored the phase structure of an Reissner– Nordström AdS black hole. Compared to the non-charged AdS case, the charged black hole’s phase structure becomes richer and is associated with the chosen statistical ensem- ble. It was found that the charged AdS black hole in the temperature–entropy plane presented an analogous Van der 123 123 123 49 Page 2 of 11 49 Page 2 of 11 Eur. Phys. J. C (2018) 78 :49 where In the framework of holography, in higher-dimensional AdS spacetime, it is interesting to detect the phase struc- ture of hairy black holes. Since there exist hair parameters, hairy black hole solutions become far richer than in General Relativity [19,20]. The model for higher-dimensional hairy black holes was ﬁrst introduced by Oliva and Ray in 2011. They developed a novel construction of conformal couplings of a scalar ﬁeld to arbitrary higher order Euler densities, and further solvedtheequations of motionunder sphericallysym- metric conditions [21]. Although hairy black holes with van- ishing cosmological constant are known in four dimensions, the scalar ﬁeld conﬁguration of these black holes diverges at the horizon [22,23]. This may be seen as a natural con- sequence of the well-known no-hair theorems. However, by introducing a cosmological constant and a conformal cou- pling, no-hair theorems can be circumvented. In 2014, in higher-dimensional AdS spacetime, Giribet et al. proved that analytic solutions to higher-dimensional hairy black holes do exist and the scalar conﬁguration is regular everywhere out- side and on the horizon. 1 Introduction It turned out that the hairy solution asymptotically goes to (Anti-) de Sitter spacetime at large distance and admits a spherical horizon as well as a hori- zon of a different topology [24]. In 2015, the thermodynam- ics of higher-dimensional hairy black holes and the different phases of hairy black holes in AdS5 space were also studied in detail [25,26]. At the same time, for these hairy solutions in a ﬁve-dimensional gravitational system, Hennigar and Mann have ﬁrst revealed a reentrant phase transition in the case of demanding the positivity of entropy [7]. In 2017, Hennigar, Mann and Tjoa have found that, for a class of asymptoti- cally AdS hairy black holes in Lovelock gravity where a real scalar ﬁeld is conformally coupled to gravity, a novel form of phase transition akin to a superﬂuid phase transition can be observed [27]. Here, employing the black hole entropy, two-point correlated function and entanglement entropy, we attempt to study whether the Van der Waals-like phase tran- sition can be observed, and we discuss the hair parameter effect on the phase transition. where κ = 16πG, (2) Lm (φ, ∇φ) = b0φ15 + b1φ7Sμνμν + b2φ−1 Sμγ μγ Sνδνδ −4Sμγ νγ Sνδμδ + Sμνγ δSνμγ δ , (3) Sμνγ δ = φ2Rμνγ δ −12δ[γ [μδδ] ν]∇ρφ∇ρφ −48φδ[γ [μ∇ν]∇δ]φ + 18δ[γ [μ∇ν]φ∇δ]φ. (4) κ = 16πG, (2) (3) (4) Here, b0, b1 and b2 are real coupling constants, which are generated by a real scalar the ﬁeld conformally coupled to gravity. The static spherically symmetric black hole solution coming from the action (1) can be written as ds 2 = −N 2 (r) f (r) dt 2 + dr 2 g (r) + r 2dΩ2 3, (5) (5) where g (r) = f (r) = 1 −m r 2 −q r 3 + e 2 r 4 + r 2 l 2 , N 2 (r) = 1. (6) (6) Here, l is AdS radius l2 = −6/Λ, and dΩ2 3 is the metric of the unit 3-sphere. The integration constants m and e are related with the mass and the electric charge of the hairy black hole, and q is given with respect to the scalar coupling constants by the relation q = 64πG 5 εb1 −18b1 5b0 3/2 , (7) (7) where ε = −1, 0, +1. 1 Introduction For the ﬁve-dimensional black hole solution to exist, the scalar coupling constants must obey the following constraint: 10b0b2 = 9b2 1. (8) 10b0b2 = 9b2 1. (8) The Maxwell potential is Aμ = √ 3 e r 2 δ 0 μ (9) (9) with Fμν = ∂μAν −∂ν Aμ. On the other hand, the scalar ﬁeld conﬁguration takes the form 2 Phase transition and Maxwell’s equal-area law for the thermodynamic entropy To plot the isocharges in the T –S plane, ﬁrstly we should get the critical values of phase transition by using the following relations: f (r) = 1 −8M 3πr 2 −4Q′2 3πr 4 + r 2 l 2 , (15) T ′ = r+ πl2 + 1 2πr+ − 2Q′2 3π2r+5 , (16) S′ = π2 2 r3 +. (17) ∂T ∂S Q = ∂2T ∂2S Q = 0. (22) (15) (22) (16) For the complicated hairy black hole, it is hard to obtain analytical values directly. We can get them numerically. In Table 1, we tabulate the critical charge QC, critical entropy SC and critical temperature TC for different hair parameter q. From this table, we see that the critical entropy becomes smaller as q increases. According to Eq. (14), we know that q must satisfy q ⩽2r 3 +/5 in order for the positivity condition of entropy to hold. S′ = π2 2 r3 +. (17) (17) In this case, the solution reduces to the ﬁve-dimensional Reissner–Nordström AdS black hole. The phase transition of the Reissner–Nordström AdS black hole has been dis- cussed extensively [13,28]. The critical values of the phase transition are given as Note that, for the very small values of the hair parame- ter, one can get a perturbative solution of the critical values. When q is very small, we expand Eq. (21) about the zero point in terms of the hair parameter q, and we ﬁnd SC ′ = π2 6 √ 3 l3, (18) QC ′ = π 6 √ 5 l2, (19) TC ′ = 4 √ 3 5πl . (20) (18) (19) T (S, Q, q) = −32π2Q2 + 321/3π4/3S4/3 + 12S2 l2 622/3π5/3S5/3 +π1/3 30S2 + l2(400π2Q2 −321/3π4/3S4/3) q 3622/3l2S8/3 −5 π7/3(1600l2π2Q2 + 321/3l2π4/3S4/3 + 30S2) q2 432(22/3l2S11/3) (20) Here, we mainly focus on studying the phase structure for the case q ̸= 0. +π1/3 30S2 + l2(400π2Q2 −321/3π4/3S4/3) q 3622/3l2S8/3 −5 π7/3(1600l2π2Q2 + 321/3l2π4/3S4/3 + 30S2) q2 432(22/3l2S11/3) Now, we begin to explore the hairy black hole’s critical behavior and phase transition in the temperature–entropy plane. From Eqs. (13) and (14), we can get the function T (S, Q, q) by eliminating r+, ( ) +O[q]3. (23) +O[q]3. 2 Phase transition and Maxwell’s equal-area law for the thermodynamic entropy φ (r) = n r1/3 , n = ε −18 5 b1 b0 1/6 . (10) (10) Let us start by reviewing the exact solution of an electri- cally charged hairy black hole in ﬁve dimensions. Einstein– Maxwell-Λ theory conformally coupled to a scalar ﬁeld in higher dimensions and see whether it can exhibit analytic solutions [24,25]. Here, we are concerned with the case of ﬁve dimensions, and the action of the theory reads In Ref. [25], Galante et al. have discussed the thermodynamic properties of higher-dimensional black holes in detail. The mass and charge of the hairy black hole are M = 3π 8 m = 3π e2l2 −ql2r+ + l2r4 + −r6 + 8l2r2 + (11) (11) I = 1 κ d 5x√−g R −2Λ −1 4 F2 + κLm (φ, ∇φ) , and Q = − √ 3π 8 e, (12) (12) (1) 123 Eur. Phys. J. C (2018) 78 :49 Page 3 of 11 49 49 respectively, where r+ is the event horizon, which is given by the equation f (r+) = 0. The Hawking temperature and black hole entropy are given by [7,25] respectively, where r+ is the event horizon, which is given by the equation f (r+) = 0. The Hawking temperature and black hole entropy are given by [7,25] Table 1 The critical values of the electric charge, temperature and entropy for different q Table 1 The critical values of the electric charge, temperature and entropy for different q q QC TC SC −0.010 0.0486384 0.439591 1.03476 −0.005 0.0538006 0.440337 0.992571 0.005 0.0629513 0.441770 0.906233 0.010 0.0670977 0.442460 0.862219 T = 1 πl2r4 + −32Q2l2 3π2r+ + ql2 4 + l2 2 r3 + + r5 + , (13) S = π2 2 r3 + −5 2q . (14) (13) (14) Note that, for the hairy black hole solution to exist, q can take three different values namely q = 0, ± \|q\|. For q = 0 e2 = 4Q′2/3π2, we have Note that, for the hairy black hole solution to exist, q can take three different values namely q = 0, ± \|q\|. For q = 0 e2 = 4Q′2/3π2, we have q = ± 0.010 and q = ± 0.005 as examples. 2 Phase transition and Maxwell’s equal-area law for the thermodynamic entropy b Q = 0.0338006 < QC, Q = 0.0538006 = QC, Q = 0.0738006 > QC. c Q = 0.0429513 < QC Q = 0.0629513 = QC, Q = 0.0829513 > QC. d Q = 0.0470977 < QC, Q = 0.0670977 = QC, Q = 0.0870977 > QC 0 1 2 3 4 5 6 S 0.36 0.38 0.40 0.42 0.44 0.46 0.48 0.50 T (a) q 0 010 0 1 2 3 4 5 6 S 0.36 0.38 0.40 0.42 0.44 0.46 0.48 0.50 T (b) q 0 005 0 1 2 3 4 5 6 S 0.36 0.38 0.40 0.42 0.44 0.46 0.48 0.50 T (a) q 0 010 When q = 0 and e2 = 4Q′2/3π2, these questions reduce to Eqs. (18), (19) and (20). Obviously, for these small values, thesecriticalquantitiesaremodiﬁedfromtheRNAdSvalues. According to the relation in Eq. (21) and these critical values, we plot the isocharges in the T –S plane for different q in Fig. 1. Each curve corresponds to a different electric charge. Remarkably, as can be seen from these plots, a Van der Waals-like phase transition is clearly present in the T –S plane. For different q, the phase structure is similar. When Q > QC, the temperature is monotonically larger as entropy increases, and the system is thermodynamically stable. As Q decreases and arrives at the critical value QC, an inﬂec- tion point appears and the heat capacity is divergent at this point, which corresponds to a second order phase transition. In the Q < QC case, in addition to two stable branches, the isocharge has an unstable branch with negative heat capacity, CQ = T ∂S ∂T Q , (28) (28) which corresponds to a ﬁrst order phase transition. Like the Van der Waals phase transition of the liquid–gas system, this unstable portion should be replaced by using an isotherm T = T ∗which obeys Maxwell’s equal-area pre- scription. The subcritical temperature T ∗can be obtained from the plot of the free energy F = M −T S versus the temperature. The plots in Fig. 2 show that the relations between the temperature and free energy for different q, and for Q < QC, we always observe a classic swallowtail struc- ture in each plot, which is responsible for the ﬁrst order phase transition in Fig. 1. 2 Phase transition and Maxwell’s equal-area law for the thermodynamic entropy (23) This corresponds to T (S, Q, q) = 1 3 3√ 2l2π5/3(5π2q + 4S)5/3 75π4q2 −128l2π2Q2 + 120π2Sq + 48S2 +9 3√ 4l2π10/3q(5π2q + 4S)1/3 +6 3√ 4l2π4/3S(5π2q + 4S)1/3 . (2 T = −e2 2πr5 + + 1 2πr+ + r+ l2π + q 4πr4 + + O[q]2. (24) (24) When q = 0 and e2 = 4Q′2/3π2, the temperature becomes thetemperatureoftheRNAdSblackhole,whichisconsistent with Eq. (16). Employing Eqs. (22) and (23), we can get (21) Note that the phase structure of a hairy black hole is not only related to electric charge Q, but also the scalar hair param- eter q. Based on the function T (S, Q, q) above, the phase structure of the charged hairy black hole can be detected. We ﬁnd that, by choosing some proper hair parameters q, the isocharges in the T –S plane can exhibit a Van der Waals-like phase transition. For convenience, we keep the AdS radius l = 1 throughout this paper. Here, we discuss the phase transition in a ﬁxed electric charged ensemble, and we take SC = l3π2 6 √ 3 −7π2q 8 + O[q]2, (25) \|eC\| = 8 √ 3 ⎛ ⎝l2π 24 √ 5 + 3 3 5πq 8l ⎞ ⎠+ O[q]2, (26) T C = 4 √ 3 5lπ + 9q 20l4π + O[q]2. (27) (25) (26) (27) 3 3 49 Page 4 of 11 49 Page 4 of 11 Eur. Phys. J. C (2018) 78 :49 49 Page 4 49 0 1 2 3 4 5 6 S 0.36 0.38 0.40 0.42 0.44 0.46 0.48 0.50 T (a) q 0 010 0 1 2 3 4 5 6 S 0.36 0.38 0.40 0.42 0.44 0.46 0.48 0.50 T (b) q 0 005 0 1 2 3 4 5 6 S 0.36 0.38 0.40 0.42 0.44 0.46 0.48 0.50 T (c) q 0 005 0 1 2 3 4 5 6 S 0.36 0.38 0.40 0.42 0.44 0.46 0.48 0.50 T (d) q 0 010 Fig. 1 Plots of the temperature versus the black hole entropy for differ- ent q. The red dash line corresponds to the temperature of the ﬁrst order phase transition. The values of the electric charge chosen (from top to bottom) are as follows. a Q = 0.0286384 < QC, Q = 0.0486384 = QC, Q = 0.0686384 > QC. 2 Phase transition and Maxwell’s equal-area law for the thermodynamic entropy We indicate the transition temperature T ∗ by a red dashed line in Fig. 2, which corresponds to the hor- izontal coordinate of the junction. Next, in order to further characterize the Van der Waals- like phase transition, we turn to checking the Maxwell equal- area law for the ﬁrst order phase transition and the corre- sponding statement can be written as A1 ≡ Smax Smin T (S, Q, q) dS = T ∗(Smax −Smin) ≡A2, (29 (29) where T (S, Q, q) is deﬁned in Eq. (21), Smin and Smax are the smallest and largest roots of the equation T (S, Q, q) = T ∗. Now, we take q = −0.010 as an example to show how to verify the Maxwell equal-area law. Numerically, using q = −0.010, Q = 0.0286384 and l = 1, we obtain T ∗from the plot (a) in Fig. 2. Then, substituting T ∗= 0.4506 into Eq. (21), we obtain the smallest value Smin = 0.306264 and largest value Smax = 2.628909 by resolving the equation T (S, −0.01, 0.0286384) = 0.4506. Thus,using the values of T ∗, Smin and Smax, we can get A2 = 1.04658 at the right side of Eq. (29), and we obtain A1 = 1.04538 by integrating the left side of Eq. (29). Namely, A1 equals A2 within our Fig. 1 Plots of the temperature versus the black hole entropy for differ- ent q. The red dash line corresponds to the temperature of the ﬁrst order phase transition. The values of the electric charge chosen (from top to bottom) are as follows. a Q = 0.0286384 < QC, Q = 0.0486384 = QC, Q = 0.0686384 > QC. b Q = 0.0338006 < QC, Q = 0.0538006 = QC, Q = 0.0738006 > QC. c Q = 0.0429513 < QC, Q = 0.0629513 = QC, Q = 0.0829513 > QC. d Q = 0.0470977 < QC, Q = 0.0670977 = QC, Q = 0.0870977 > QC 12 123 Eur. Phys. J. 2 Phase transition and Maxwell’s equal-area law for the thermodynamic entropy C (2018) 78 :49 Page 5 of 11 49 49 0.42 0.44 0.46 0.48 0.50 T 0.15 0.10 0.05 0.05 0.10 0.15 F (a) q 0 010 0.40 0.42 0.44 0.46 0.48 0.50 T 0.15 0.10 0.05 0.05 0.10 0.15 F (b) q 0 005 0.38 0.40 0.42 0.44 0.46 0.48 0.50 T 0.10 0.05 0.05 0.10 0.15 0.20 F (c) q 0 005 0.40 0.42 0.44 0.46 0.48 0.50 T 0.10 0.05 0.05 0.10 0.15 0.20 F (d) q 0 010 Fig. 2 Plots of the temperature versus the free energy for different in the case of Q < QC. In each graph, the red dash line indicate a ﬁrst order phase transition temperature T ∗. a Q = 0.0286384 an T ∗= 0.4506. b Q = 0.0338006 and T ∗= 0.4524. c Q = 0.042951 and T ∗= 0.4552. d Q = 0.0470977 and T ∗= 0.4561 0.42 0.44 0.46 0.48 0.50 T 0.15 0.10 0.05 0.05 0.10 0.15 F (a) q 0 010 0.40 0.42 0.44 0.46 0.48 0.50 T 0.15 0.10 0.05 0.05 0.10 0.15 F (b) q 0 005 0.38 0.40 0.42 0.44 0.46 0.48 0.50 T 0.05 0.10 0.15 0.20 F Table 2 Check of the Maxwell equal-area construction in the T –S plane q T ∗ Smin Smax A1 A2 −0.010 0.4506 0.3062640 2.6289090 1.04538 1.04658 −0.005 0.4524 0.2371192 2.6989999 1.11250 1.11375 0.005 0.4552 0.1098390 2.7624530 1.20665 1.20747 0.010 0.4561 0.0488927 0.7663060 1.22943 1.22924 Table 2 Check of the Maxwell equal-area construction in the T –S plane numeric accuracy with these values. Repeating the procedure above, we can obtain A1 and A2 for other q, and we tabulate these values in Table 2. From this table, it is obvious that the Maxwell equal-area construction holds in the T –S plane. 3 Phase transition and Maxwell’s equal-area law for two-point correlation function (31) from zero to UV cutoff θC ≲θ 0. Then, by turning off the hair parameter q, mass M and electric charge Q of the charged hairy black hole background, we get a pure AdS in global coordinates, ds 2 = − 1 + r 2 l 2 dt2 + 1 + r 2 l 2 −1 dr 2 + r 2dΩ2 3, (35) (35) with this metric, repeating the same procedure as calculation L, numerically, we obtain L′. Thus, subtracting this quantity from the hairy black hole, we get the renormalized geodesic length δL = L −L′. Here, we take θ 0 = 0.45 and θ 0 = 0.50 as an example to discuss the charged hairy black hole’s phase structure, and the corresponding cutoffs are chosen to be θC = 0.449 and 0.499 in the numerical computations. For the θ 0 = 0.45 case, in Fig. 3, we present the plots of the isocharges generated for the two-point correlation function for different hair parameters q. In each panel, the isocharges in the T –δL plane from top to bottom correspond to Q < QC, Q = QC and Q > QC, respectively. From these plots, we see that the T versus δL plots are qualitatively similar to the ones in Fig. 1. We conﬁrm that there exists indeed a Van der Waals-like phase transition in the T –δL plane, and the critical temperature and critical charge are the same as the ones obtained from Fig. 1. ( ) 0.0000 0.0005 0.0010 0.0015 0.0020 0.38 0.40 0.42 0.44 0.46 0.48 0.50 T (d) q 0 010 δL In order to further characterize a Van der Waals-like phase transition for the two-point correlation function, we choose two different θ0 to verify the equal-area law in the T –δL plane, similarly deﬁned as A1 ≡ δLmax δLmin T (δL, q, Q) dδL = T ∗(δLmax −δLmin) ≡A2, (36) (36) Fig. 3 Plots of isocharges in the T –δL plane for θ 0 = 0.45. The blue dash line corresponds to the temperature of the ﬁrst order phase transition. The values of the electric charge chosen (from top to bottom) are the same as the ones in Fig. 1 where δLmin and δLmax are the smallest and largest roots of the equation T (δL, q, Q) = T ∗. 3 Phase transition and Maxwell’s equal-area law for two-point correlation function In this section, we proceed to discuss on the phase structure of two-point correlation function. In recent years, the two- point correlation function has appeared to be a useful tool which can be used to explore some physical phenomena such as holographic singularities [29,30], holographic thermal- ization [31,32], holographic CFTs on maximally symmetric spaces [33], holographic butterﬂy effect [34–37] and quan- tum phase transition [38]. Motivated by the above-mentioned issues, we attempt to detect whether the two-point correla- tion function can present a Van der Waals-like behavior for the charged hairy black hole. According to the Anti-de Sitter space/Conformal Field Theory dictionary, in the large △limit, the equal-time two- point correlation function can be holographically expressed as [39] O(t0, xi) O(t0, x j) ≈e−△L, (30) (30) where △is the conformal dimension of the scalar operator O, and L is the length of the buck geodesic between the point (t0, xi) and (t0, x j) on the Anti-de Sitter boundary. Due to the charged hairy black hole’s symmetry in ﬁve-dimensional AdS spacetime, we can simply set (θ = θ 0, ϕ = π/2, ψ = 0) and (θ = θ 0, ϕ = π/2, ψ = π) as the two boundary points. (t0, xi) and (t0, x j) on the Anti-de Sitter boundary. Due to the charged hairy black hole’s symmetry in ﬁve-dimensional AdS spacetime, we can simply set (θ = θ 0, ϕ = π/2, ψ = 0) and (θ = θ 0, ϕ = π/2, ψ = π) as the two boundary points. By utilizing θ to parameterize the trajectory, the proper length takes the form L = θ0 0 L(r(θ), θ) dθ, L = r ′ 2(θ) g(r) + r 2(θ); (31) (d) q 0 010 (31) Fig. 2 Plots of the temperature versus the free energy for different q in the case of Q < QC. In each graph, the red dash line indicates a ﬁrst order phase transition temperature T ∗. a Q = 0.0286384 and T ∗= 0.4506. b Q = 0.0338006 and T ∗= 0.4524. c Q = 0.0429513 and T ∗= 0.4552. d Q = 0.0470977 and T ∗= 0.4561 here r′ ≡dr/dθ. Taking L as the Lagrangian and imagining θ as time, with the Euler–Lagrange equation ∂L ∂r = d dθ ∂L ∂r ′(θ) , (32) (32) 12 3 Eur. Phys. J. 3 Phase transition and Maxwell’s equal-area law for two-point correlation function C (2018) 78 :49 49 Page 6 of 11 49 0.0000 0.0005 0.0010 0.0015 0.0020 δL 0.36 0.38 0.40 0.42 0.44 0.46 0.48 0.50 T (a) q 0 010 0.0000 0.0005 0.0010 0.0015 0.0020 0.36 0.38 0.40 0.42 0.44 0.46 0.48 0.50 T (b) q 0 005 0.0000 0.0005 0.0010 0.0015 0.0020 0.36 0.38 0.40 0.42 0.44 0.46 0.48 0.50 T (c) q 0 005 0.0000 0.0005 0.0010 0.0015 0.0020 0.38 0.40 0.42 0.44 0.46 0.48 0.50 T (d) q 0 010 δL δL δL Fig. 3 Plots of isocharges in the T –δL plane for θ 0 = 0.45. The lue dash line corresponds to the temperature of the ﬁrst order phase ransition. The values of the electric charge chosen (from top to bottom) re the same as the ones in Fig. 1 0.0000 0.0005 0.0010 0.0015 0.0020 δL 0.36 0.38 0.40 0.42 0.44 0.46 0.48 0.50 T (a) q 0 010 0.0000 0.0005 0.0010 0.0015 0.0020 0.36 0.38 0.40 0.42 0.44 0.46 0.48 0.50 T (b) q 0 005 0.0000 0.0005 0.0010 0.0015 0.0020 0.36 0.38 0.40 0.42 0.44 0.46 0.48 0.50 T (c) q 0 005 0.48 0.50 T δL δL 0.0000 0.0005 0.0010 0.0015 0.0020 δL 0.36 0.38 0.40 0.42 0.44 0.46 0.48 0.50 T (a) q 0 010 0.0000 0.0005 0.0010 0.0015 0.0020 0.36 0.38 0.40 0.42 0.44 0.46 0.48 0.50 T (b) q 0 005 δL 0.0000 0.0005 0.0010 0.0015 0.0020 δL 0.36 0.38 0.40 0.42 0.44 0.46 0.48 0.50 T (a) q 0 010 we arrive at the equation of motion of r(θ), we arrive at the equation of motion of r(θ), 0.50 T g ′(r)r ′(θ) 2 −2g(r)r′′(θ) + 2g 2(r)r(θ) = 0 (33) with the boundary condition g ′(r)r ′(θ) 2 −2g(r)r′′(θ) + 2g 2(r)r(θ) = 0 (33) g ′(r)r ′(θ) 2 −2g(r)r′′(θ) + 2g 2(r)r(θ) = 0 (33) with the boundary condition r(0) = r0, r ′(0) = 0. (34) (34) By using the condition above and resolving Eq. (33), we obtain the numeric result of r(θ). Notice that the geodesic length is divergent for a ﬁxed θ0, therefore, it needs to be regularized. Here, we do it by subtracting the geodesic length of the minimal surface in pure AdS with the same boundary θ = θ0 (denoted by L′). In order to accomplish this, we ﬁrst obtain L by integrating the length function in Eq. 3 Phase transition and Maxwell’s equal-area law for two-point correlation function d Q = 0.0470977 < QC Table 3 Check of the Maxwell equal-area construction in the T –δL plane θ 0 = 0.45 θ 0 = 0.50 q = −0.010 T ∗= 0.4506 δLmin = 0.0001181430 A1 = 0.00027069 δLmin = 0.0001788345 A1 = 0.00040808 δLmax = 0.0007194292 A2 = 0.00027094 δLmax = 0.0010853080 A2 = 0.00040846 q = −0.005 T ∗= 0.4524 δLmin = 0.0001099755 A1 = 0.00028904 δLmin = 0.0001664403 A1 = 0.00043580 δLmax = 0.0007494337 A2 = 0.00028929 δLmax = 0.0011305550 A2 = 0.00043617 q = 0.005 T ∗= 0.4552 δLmin = 0.00009738140 A1 = 0.00031608 δLmin = 0.0001473907 A1 = 0.00047660 δLmax = 0.00079196240 A2 = 0.00031617 δLmax = 0.0011947060 A2 = 0.00047674 q = 0.010 T ∗= 0.4561 δLmin = 0.00009229728 A1 = 0.00032276 δLmin = 0.0001397911 A1 = 0.00048686 δLmax = 0.00079951970 A2 = 0.00032256 δLmax = 0.0012065893 A2 = 0.00048657 Table 3 Check of the Maxwell equal-area construction in the T –δL plane θ 0 = 0.45 θ 0 = 0.50 = −0.010 T ∗= 0.4506 δLmin = 0.0001181430 A1 = 0.00027069 δLmin = 0.0001788345 A1 = 0.00040808 δLmax = 0.0007194292 A2 = 0.00027094 δLmax = 0.0010853080 A2 = 0.00040846 = −0.005 T ∗= 0.4524 δLmin = 0.0001099755 A1 = 0.00028904 δLmin = 0.0001664403 A1 = 0.00043580 δLmax = 0.0007494337 A2 = 0.00028929 δLmax = 0.0011305550 A2 = 0.00043617 = 0.005 T ∗= 0.4552 δLmin = 0.00009738140 A1 = 0.00031608 δLmin = 0.0001473907 A1 = 0.00047660 δLmax = 0.00079196240 A2 = 0.00031617 δLmax = 0.0011947060 A2 = 0.00047674 = 0.010 T ∗= 0.4561 δLmin = 0.00009229728 A1 = 0.00032276 δLmin = 0.0001397911 A1 = 0.00048686 δLmax = 0.00079951970 A2 = 0.00032256 δLmax = 0.0012065893 A2 = 0.00048657 0.0000 0.0005 0.0010 0.0015 0.0020 0.0025 0.42 0.43 0.44 0.45 0.46 0.47 0.48 0.49 T (a) q 0 010 0.0000 0.0005 0.0010 0.0015 0.0020 0.0025 0.0030 0.0035 0.42 0.43 0.44 0.45 0.46 0.47 0.48 0.49 T (b) q 0 005 δL δL Table 3 Check of the Maxwell equal-area construction in the T –δL plane θ 0 = 0.45 θ 0 = 0.50 q = −0.010 T ∗= 0.4506 δLmin = 0.0001181430 A1 = 0.00027069 δLmin = 0.0001788345 A1 = 0.0004080 δLmax = 0.0007194292 A2 = 0.00027094 δLmax = 0.0010853080 A2 = 0.0004084 q = −0.005 T ∗= 0.4524 δLmin = 0.0001099755 A1 = 0.00028904 δLmin = 0.0001664403 A1 = 0.0004358 δLmax = 0.0007494337 A2 = 0.00028929 δLmax = 0.0011305550 A2 = 0.0004361 q = 0.005 T ∗= 0.4552 δLmin = 0.00009738140 A1 = 0.00031608 δLmin = 0.0001473907 A1 = 0.0004766 δLmax = 0.00079196240 A2 = 0.00031617 δLmax = 0.0011947060 A2 = 0.0004767 q = 0.010 T ∗= 0.4561 δLmin = 0.00009229728 A1 = 0.00032276 δLmin = 0.0001397911 A1 = 0.0004868 δLmax = 0.00079951970 A2 = 0.00032256 δLmax = 0.0012065893 A2 = 0.0004865 Table 3 Check of the Maxwell equal-area construction in the T –δL plane ( ) 0.0000 0.0005 0.0010 0.0015 0.0020 0.0025 0.42 0.43 0.44 0.45 0.46 0.47 0.48 0.49 T (d) q 0 010 δL Fig. 3 Phase transition and Maxwell’s equal-area law for two-point correlation function T (δL, q, Q) is an inter- polating function which can be given by our numeric result. Besides θ 0 = 0.45, in Fig. 4, for the case θ 0 = 0.50, we also plot the isocharge in the T –δL plane when the charge where δLmin and δLmax are the smallest and largest roots of the equation T (δL, q, Q) = T ∗. T (δL, q, Q) is an inter- polating function which can be given by our numeric result. Besides θ 0 = 0.45, in Fig. 4, for the case θ 0 = 0.50, we also plot the isocharge in the T –δL plane when the charge 123 Page 7 of 11 49 Page 7 of 11 49 Eur. Phys. J. C (2018) 78 :49 0.0000 0.0005 0.0010 0.0015 0.0020 0.0025 0.42 0.43 0.44 0.45 0.46 0.47 0.48 0.49 T (a) q 0 010 0.0000 0.0005 0.0010 0.0015 0.0020 0.0025 0.0030 0.0035 0.42 0.43 0.44 0.45 0.46 0.47 0.48 0.49 T (b) q 0 005 0.0000 0.0005 0.0010 0.0015 0.0020 0.0025 0.42 0.43 0.44 0.45 0.46 0.47 0.48 0.49 T (c) q 0 005 0.0000 0.0005 0.0010 0.0015 0.0020 0.0025 0.42 0.43 0.44 0.45 0.46 0.47 0.48 0.49 T (d) q 0 010 δL δL δL δL Fig. 4 Plots of isocharges in the T –δL plane for θ 0 = 0.50. The blue dash line corresponds to the temperature of the ﬁrst order phase transition for Q < QC. a Q = 0.0286384 < QC. b Q = 0.0338006 < QC. c Q = 0.0429513 < QC. d Q = 0.0470977 < QC 0.0000 0.0005 0.0010 0.0015 0.0020 0.0025 0.42 0.43 0.44 0.45 0.46 0.47 0.48 0.49 T (a) q 0 010 0.0000 0.0005 0.0010 0.0015 0.0020 0.0025 0.0030 0.0035 0.42 0.43 0.44 0.45 0.46 0.47 0.48 0.49 T (b) q 0 005 0.0000 0.0005 0.0010 0.0015 0.0020 0.0025 0.42 0.43 0.44 0.45 0.46 0.47 0.48 0.49 T (c) q 0 005 0.0000 0.0005 0.0010 0.0015 0.0020 0.0025 0.42 0.43 0.44 0.45 0.46 0.47 0.48 0.49 T (d) q 0 010 δL δL δL δL Fig. 4 Plots of isocharges in the T –δL plane for θ 0 = 0.50. The blue dash line corresponds to the temperature of the ﬁrst order phase transition for Q < QC. a Q = 0.0286384 < QC. b Q = 0.0338006 < QC. c Q = 0.0429513 < QC. 3 Phase transition and Maxwell’s equal-area law for two-point correlation function d Q = 0.0470977 < QC 0.000 0.005 0.010 0.015 0.020 0.025 0.030 0.035 S 0.36 0.38 0.40 0.42 0.44 0.46 0.48 0.50 T (a) q 0 010 0.000 0.005 0.010 0.015 0.020 0.025 0.030 0.035 S 0.36 0.38 0.40 0.42 0.44 0.46 0.48 0.50 T (b) q 0 005 0.000 0.005 0.010 0.015 0.020 0.025 0.030 0.035 S 0.36 0.38 0.40 0.42 0.44 0.46 0.48 0.50 T (c) q 0 005 T 0.00 0.01 0.02 0.03 0.04 S 0.40 0.41 0.42 0.43 0.44 0.45 0.46 0.47 0.48 T (a) q 0 010 0.00 0.01 0.02 0.03 0.04 0.40 0.42 0.44 0.46 0.48 0.50 T (b) q 0 005 δS 0.000 0.005 0.010 0.015 0.020 0.025 0.030 0.035 S 0.36 0.38 0.40 0.42 0.44 0.46 0.48 0.50 T (a) q 0 010 0.000 0.005 0.010 0.015 0.020 0.025 0.030 0.035 S 0.36 0.38 0.40 0.42 0.44 0.46 0.48 0.50 T (b) q 0 005 0.000 0.005 0.010 0.015 0.020 0.025 0.030 0.035 S 0.36 0.38 0.40 0.42 0.44 0.46 0.48 0.50 T (a) q 0 010 0.00 0.01 0.02 0.03 0.04 S 0.40 0.41 0.42 0.43 0.44 0.45 0.46 0.47 0.48 T (a) q 0 010 0.00 0.01 0.02 0.03 0.04 0.40 0.42 0.44 0.46 0.48 0.50 T (b) q 0 005 0.00 0.01 0.02 0.03 0.04 S 0.40 0.42 0.44 0.46 0.48 0.50 T (c) q 0 005 δS 0.00 0.01 0.02 0.03 0.04 S 0.40 0.41 0.42 0.43 0.44 0.45 0.46 0.47 0.48 T (a) q 0 010 0.000 0.005 0.010 0.015 0.020 0.025 0.030 0.035 S 0.36 0.38 0.40 0.42 0.44 0.46 0.48 0.50 (a) q 0 010 0.000 0.005 0.010 0.015 0.020 0.025 0.030 0.035 S 0.36 0.38 0.40 0.42 0.44 0.46 0.48 0.50 T (b) q 0 005 0.000 0.005 0.010 0.015 0.020 0.025 0.030 0.035 S 0.36 0.38 0.40 0.42 0.44 0.46 0.48 0.50 T (c) q 0 005 0.000 0.005 0.010 0.015 0.020 0.025 0.030 0.035 S 0.36 0.38 0.40 0.42 0.44 0.46 0.48 0.50 T (d) q 0 010 Fig. 5 Plots of isocharges in the T –△S plane for ϕ 0 = 0.45. The red dash line corresponds to the temperature of the ﬁrst order phase transition. The values of the electric charge chosen (from top to bottom) are the same as the ones in Fig. 3 Phase transition and Maxwell’s equal-area law for two-point correlation function 4 Plots of isocharges in the T –δL plane for θ 0 = 0.50. The blue dash line corresponds to the temperature of the ﬁrst order phase transition for Q < QC. a Q = 0.0286384 < QC. b Q = 0.0338006 < QC. c Q = 0.0429513 < QC. d Q = 0.0470977 < QC 12 49 Page 8 of 11 Eur. Phys. J. C (2018) 78 :49 49 Page 8 of 11 0.000 0.005 0.010 0.015 0.020 0.025 0.030 0.035 S 0.36 0.38 0.40 0.42 0.44 0.46 0.48 0.50 T (a) q 0 010 0.000 0.005 0.010 0.015 0.020 0.025 0.030 0.035 S 0.36 0.38 0.40 0.42 0.44 0.46 0.48 0.50 T (b) q 0 005 0.000 0.005 0.010 0.015 0.020 0.025 0.030 0.035 S 0.36 0.38 0.40 0.42 0.44 0.46 0.48 0.50 T (c) q 0 005 0.000 0.005 0.010 0.015 0.020 0.025 0.030 0.035 S 0.36 0.38 0.40 0.42 0.44 0.46 0.48 0.50 T (d) q 0 010 Fig. 5 Plots of isocharges in the T –△S plane for ϕ 0 = 0.45. The red dash line corresponds to the temperature of the ﬁrst order phase transition. The values of the electric charge chosen (from top to bottom) are the same as the ones in Fig. 1 0.00 0.01 0.02 0.03 0.04 S 0.40 0.41 0.42 0.43 0.44 0.45 0.46 0.47 0.48 T (a) q 0 010 0.00 0.01 0.02 0.03 0.04 0.40 0.42 0.44 0.46 0.48 0.50 T (b) q 0 005 0.00 0.01 0.02 0.03 0.04 S 0.40 0.42 0.44 0.46 0.48 0.50 T (c) q 0 005 0.00 0.01 0.02 0.03 0.04 S 0.40 0.42 0.44 0.46 0.48 0.50 T (d) q 0 010 δS Fig. 6 Plots of isocharges in the T–△S plane for ϕ 0 = 0.50. The red dash line corresponds to the temperature of the ﬁrst order phase transition for Q < QC. a Q = 0.0286384 < QC. b Q = 0.0338006 < QC. c Q = 0.0429513 < QC. 5 Discussion and conclusion (37) Note that recent work has indicated that the equal-area law is only valid very near the critical point [42]. Here, for the equal-area law of the entanglement entropy and two-point correlation function, by employing the same equal-area law as that used in Ref. [42], we perform numerical computations to explore this problem with the different ratios Q/QC = 0.9 and 0.5. The relevant numerical results have been put in Tables 5, 6, 7 and 8. Here, the relative error is taken to be the difference between A (I) and A (II) divided by their average. From these tables, it is obvious that Maxwell equal- area law is only valid near the critical point. Away from the critical point, the relative error becomes signiﬁcantly large, and the Maxwell equal-area law cannot hold on these planes, which supports the point in the light of [42]. As far as the entanglement entropy and the two-point correlation function are concerned, a holographic equal-area law is still an open question. where ΓA is a codimension-2 minimal surface with boundary condition ∂ΓA = ∂A. Here, we choose ϕ = ϕ0 as the hairy black hole’s entangling surface and employ r(ϕ) to parame- terize the minimal surface. With the symmetry, in this static AdS background, the entanglement entropy is given by S = π ϕ 0 0 r 2 sin2 ϕ r ′ 2(ϕ) f (r) + r 2(ϕ) dϕ, (38) (38) in which r ′(ϕ) ≡dr/dϕ. Adopting a similar procedure to the two-point correlation function case, we ﬁrst arrive at the equation of motion of r(ϕ) by utilizing the Euler–Lagrange equation, and with the boundary condition, the numerical result of r(ϕ) is obtained. Then we integrate the entropy function S in Eq. (38) up to the UV cutoff ϕ C (that is, ϕ C ≈ϕ 0). Thus, subtracting the pure AdS entanglement entropy (which is denoted by S0), we are abletogettheregularizedentanglemententropy△S = S−S0 of a charged hairy black hole. In this paper, in the framework of holography, we discuss the phase structure of a charged hairy black hole in ﬁve- dimensional AdS background (in the ﬁxed electric charge ensemble). The result shows that a Van der Waals-like phase transition can be observed in the T –S plane, T –δL plane and T –△S plane, and the critical charge and critical temperature are equal. 3 Phase transition and Maxwell’s equal-area law for two-point correlation function C (2018) 78 :49 Page 9 of 11 49 where △Smin and △Smax are the smallest and largest roots of T (△S, q, Q) = T ∗, T (△S, q, Q) is an interpolating func- tion which is given by our numeric result, and T ∗is a transi- tion temperature, which is equal to the ﬁrst order phase tran- sition temperature T ∗found for black hole entropy in Sect. 2. In order to verify the equal-area law, we take ϕ 0 = 0.45 and ϕ 0 = 0.50 as example, we also present the isocharges in the T –△S plane for ϕ 0 = 0.50 in Fig. 6. For different q and ϕ0, the numeric results of △Smin, △Smax, A1 and A2 are listed in Table 4. According to this table, we conclude that the Maxwell equal-area construction in the T –△S plane is valid within a reasonable error. Again, the result shows that like the black hole entropy, the entanglement entropy can indeed present a Van der Waals-like phase transition for the charged hairy black hole. satisﬁes Q < QC. In Table 3, for different q and θ 0, we tabu- late the values of δLmin, δLmax, A1 and A2. Obviously, for the two-point correlation function, the equal-area law hods for the ﬁve-dimensional charged hairy black hole. This strength- ens our conclusion that, indeed, the isocharges in the T –δL plane can present the same Van der Waals-like phase transi- tion as the black hole entropy. 4 Holographic phase transition for entanglement entropy Now, we move on to take into account the entanglement entropy case. According to the Ryu–Takayanagi description, the holographic entanglement entropy for a region A can be expressed as [40,41] S = Area(ΓA) 4 , (37) 3 Phase transition and Maxwell’s equal-area law for two-point correlation function 1 0.00 0.01 0.02 0.03 0.04 S 0.40 0.41 0.42 0.43 0.44 0.45 0.46 0.47 0.48 (a) q 0 010 0.00 0.01 0.02 0.03 0.04 0.40 0.42 0.44 0.46 0.48 0.50 T (b) q 0 005 0.00 0.01 0.02 0.03 0.04 S 0.40 0.42 0.44 0.46 0.48 0.50 T (c) q 0 005 0.00 0.01 0.02 0.03 0.04 S 0.40 0.42 0.44 0.46 0.48 0.50 T (d) q 0 010 δS Fig. 6 Plots of isocharges in the T–△S plane for ϕ 0 = 0.50. The red dash line corresponds to the temperature of the ﬁrst order phase transition for Q < QC. a Q = 0.0286384 < QC. b Q = 0.0338006 < QC. c Q = 0.0429513 < QC. d Q = 0.0470977 < QC 0.000 0.005 0.010 0.015 0.020 0.025 0.030 0.035 S 0.36 0.38 0.40 0.42 0.44 0.46 0.48 0.50 T (c) q 0 005 0.000 0.005 0.010 0.015 0.020 0.025 0.030 0.035 S 0.36 0.38 0.40 0.42 (c) q 0 005 0.000 0.005 0.010 0.015 0.020 0.025 0.030 0.035 S 0.36 0.38 0.40 0.42 0.44 0.46 0.48 0.50 T (d) q 0 010 ( ) 0.00 0.01 0.02 0.03 0.04 S 0.40 0.42 0.44 0.46 0.48 0.50 T (d) q 0 010 0.000 0.005 0.010 0.015 0.020 0.025 0.030 0.035 S 0.36 0.38 0.40 0.42 0.44 0.46 0.48 0.50 T (d) q 0 010 Fig. 6 Plots of isocharges in the T–△S plane for ϕ 0 = 0.50. The red dash line corresponds to the temperature of the ﬁrst order phase transition for Q < QC. a Q = 0.0286384 < QC. b Q = 0.0338006 < QC. c Q = 0.0429513 < QC. d Q = 0.0470977 < QC Fig. 5 Plots of isocharges in the T –△S plane for ϕ 0 = 0.45. The red dash line corresponds to the temperature of the ﬁrst order phase transition. The values of the electric charge chosen (from top to bottom) are the same as the ones in Fig. 1 123 Eur. Phys. J. 5 Discussion and conclusion Notice that, for some q value, where the grav- ity background exhibits a negative entropy, there no longer exists a reasonable phase transition. In order to guarantee that the entropy is positive, the scalar hair parameter q must satisfy the condition q ⩽2r 3 +/5. Since the corresponding expression for the critical values are too complicated in this charged hairy AdS background, here, we proceed to compute numerically. Speciﬁcally, choosing ϕ 0 = 0.45, we present the isocharges in the T –△S plane for different hair parameters q in Fig. 5. Comparing with Figs. 1 and 3 again, we ﬁnd that, like the black hole entropy and two-point correlation function, the entanglement entropy also exhibits a Van der Waals-like phase transition; moreover, the critical charge and critical temperature are also identiﬁed with them. We go on to verify whether the Maxwell construction works for the entanglement entropy in the T –△S plane. The analogous equal-area law becomes A1 ≡ △Smax △Smin T (△S, q, Q) d△S = T ∗(△Smax−△Smin) ≡A2, A1 ≡ △Smax △Smin T (△S, q, Q) d△S = T ∗(△Smax−△Smin) ≡A2, (39) It is interesting to note that, for the charged hairy black hole, in Ref. [7], Hennigar and Mann have ﬁrst revealed a reentrant phase transition, and they have carefully studied (39) 123 12 3 Eur. Phys. J. 5 Discussion and conclusion C (2018) 78 :49 49 Page 10 of 11 Table 4 Check of the Maxwell equal-area construction in the T –△S plane ϕ 0 = 0.45 ϕ 0 = 0.50 q = −0.010 T ∗= 0.4506 △Smin = 0.002343243 A1 = 0.0052170 △Smin = 0.00321284 A1 = 0.0081764 △Smax = 0.013932195 A2 = 0.0052220 △Smax = 0.02136945 A2 = 0.0081814 q = −0.005 T ∗= 0.4524 △Smin = 0.00227335 A1 = 0.0055859 △Smin = 0.00302369 A1 = 0.0087256 △Smax = 0.01463187 A2 = 0.0055910 △Smax = 0.02232081 A2 = 0.0087300 q = 0.005 T ∗= 0.4552 △Smin = 0.00201383 A1 = 0.0061112 △Smin = 0.00273341 A1 = 0.0095445 △Smax = 0.01544459 A2 = 0.0061137 △Smax = 0.02369990 A2 = 0.0095440 q = 0.010 T ∗= 0.4561 △Smin = 0.00193166 A1 = 0.0062515 △Smin = 0.00261655 A1 = 0.0097623 △Smax = 0.01563169 A2 = 0.0062486 △Smax = 0.02400015 A2 = 0.0097531 Table 5 Check of the Maxwell’s area law in the T –δL plane for q = 0.010 and θ0 = 0.45 T ∗ Q Q/Qc A (I) A (II) Relative error 0.447060 0.0603879 0.9 2.42670 × 10−7 2.61054 × 10−7 0.0182480 0.464856 0.0335489 0.5 9.12172 × 10−6 6.55055 × 10−6 0.0820291 Table 6 Check of the Maxwell equal-area law in the T–δL plane for q = −0.010 and θ0 = 0.50 T ∗ Q Q/Qc A(I) A (II) Relative error 0.442300 0.0437746 0.9 1.24830 × 10−7 1.35112 × 10−7 0.0197772 0.451123 0.0243192 0.5 3.55393 × 10−6 2.44831 × 10−6 0.0921010 Table 7 Check of the Maxwell equal-area law in the T–△S plane for q = −0.010 and ϕ0 = 0.45 T ∗ Q Q/Qc A (I) A (II) Relative error 0.442300 0.0437746 0.9 1.54531 × 10−6 1.70203 × 10−6 0.0241305 0.451123 0.0243192 0.5 4.50689 × 10−5 3.12496 × 10−5 0.0905374 Table 8 Check of the Maxwell equal-area law in the T–△S plane for q = 0.010 and ϕ0 = 0.50 T ∗ Q Q/Qc A (I) A (II) Relative error 0.447060 0.0603879 0.9 8.04075 × 10−6 7.81503 × 10−6 0.00711788 0.464856 0.0335489 0.5 2.73991 × 10−4 1.93884 × 10−4 0.08560700 Table 4 Check of the Maxwell equal-area construction in the T –△S plane the criticality and the Van der Waals behavior in the P–V plane. 5 Discussion and conclusion Here, in the T –S plane, by choosing some proper val- ues of q, we also present the Van der Waals-like phase tran- sition, thereby strengthening the conclusion of Ref. 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W3160320150.txt	https://www.int-res.com/articles/ame_oa/a087p079.pdf	en		The microbiology of isoprene cycling in aquatic ecosystems	Aquatic microbial ecology	2,021	cc-by	14,009	Vol. 87: 79–98, 2021 https://doi.org/10.3354/ame01972 AQUATIC MICROBIAL ECOLOGY Aquat Microb Ecol Contribution to AME Special 7 ‘SAME 16: Progress and perspectives in aquatic microbial ecology’ Published online July 15 OPEN ACCESS REVIEW The microbiology of isoprene cycling in aquatic ecosystems R. A. Dawson1,, A. T. Crombie1, P. Pichon2, M. Steinke2, T. J. McGenity2, J. C. Murrell1, 1 University of East Anglia, School of Environmental Sciences, Norwich Research Park, Norwich NR4 7TJ, UK 2 University of Essex, School of Life Sciences, Wivenhoe Park, Colchester CO4 3SQ, UK ABSTRACT: Isoprene (2-methyl-1, 3-butadiene) is emitted in vast quantities (> 500 Tg C yr−1). Most isoprene is emitted by trees, but there is still incomplete understanding of the diversity of isoprene sources. The reactivity of isoprene in the atmosphere has potential implications for both global warming and global cooling, with human health implications also arising from isopreneinduced ozone formation in urban areas. Isoprene emissions from terrestrial environments have been studied for many years, but our understanding of aquatic isoprene emissions is less complete. Several abundant phytoplankton taxa produced isoprene in the laboratory, and the relationship between chlorophyll a and isoprene production has been used to estimate emissions from marine environments. The aims of this review are to highlight the role of aquatic environments in the biological cycling of isoprene and to stimulate further study of isoprene metabolism in marine and freshwater environments. From a microbial ecology perspective, the isoprene metabolic gene cluster, first identified in Rhodococcus sp. AD45 (isoGHIJABCDEF ) and subsequently found in every genome-sequenced isoprene-degrader, provides the ideal basis for molecular studies on the distribution and diversity of isoprene-degrading communities. Further investigations of isopreneemitting microbes, such as the influence of environmental factors and geographical location, must also be considered when attempting to constrain estimates of the flux of isoprene in aquatic ecosystems. We also report isoprene emission by the scleractinian coral Acropora horrida and the degradation of isoprene by the same coral holobiont, which highlights the importance of better understanding the cycling of isoprene in marine environments. KEY WORDS: Isoprene · Biogenic volatile organic compounds · BVOC · Phytoplankton · Coral · Marine · Freshwater Isoprene is one of the most abundant atmospheric trace gases, and yet, compared to methane, the biogeochemical cycle for isoprene is relatively poorly understood. In particular, comparatively little is known about the biological consumption of isoprene in the biosphere before it enters the atmosphere. Iso- prene, like methane, can be a source of carbon and energy for aerobic bacteria (Van Ginkel et al. 1987, van Hylckama Vlieg et al. 2000, Crombie et al. 2015). The majority of isoprene is produced by land plants (Guenther et al. 2006, 2012), but there are many marine and freshwater sources of isoprene. The aims of this review are to summarize what is known about aquatic isoprene cycling and to highlight the poten- Corresponding authors: j.c.murrell@uea.ac.uk, and r.dawson@uea.ac.uk © The authors 2021. Open Access under Creative Commons by Attribution Licence. Use, distribution and reproduction are unrestricted. Authors and original publication must be credited. 1. INTRODUCTION Publisher: Inter-Research · www.int-res.com 80 Aquat Microb Ecol 87: 79–98, 2021 tial for further study of the ecology of isoprene-degrading microbes and the biological consumption of isoprene in aquatic environments. Our understanding of the physiology, biochemistry, molecular biology and ecology of isoprene-degrading bacteria has focussed mostly on terrestrial environments (reviewed by McGenity et al. 2018, Carrión et al. 2020b, Murrell et al. 2020), and so we summarise what is known about the biology of isoprene consumption using examples mainly from the terrestrial environment and then focus on aquatic environments. Isoprene is released into the biosphere at around 500 Tg C yr−1, a flux approximately equal in magnitude to that of methane in terms of global emissions (Fig. 1) (Atkinson & Arey 2003, Guenther et al. 2012). Isoprene is highly volatile and reactive, and it has a variety of effects on the Earth’s climate depending on the levels of other compounds, including nitrogen oxides, in the atmosphere (Atkinson & Arey 2003). Isoprene increases the longevity of methane in the atmosphere by reacting with hydroxyl radicals and thus, indirectly, acts as a global warming gas (Collins et al. 2002, Folberth et al. 2006). Oxidation products of isoprene can also promote aerosol formation, leading to increased cloud albedo and global cooling. Reactions of isoprene with atmospheric compounds also affect the levels of ozone in the lower atmosphere (Trainer et al. 1987, Atkinson & Arey 2003, Carlton et al. 2009). Increased planting of major isoprene-producing crop plants such as oil palm, poplar and willow has stimulated considerable interest in the effects of isoprene on air quality (Sharkey et al. 2008, Hewitt et al. 2009, Karlsson et al. 2020, Monson et al. 2020). 2. EMISSION OF ISOPRENE FROM TERRESTRIAL ENVIRONMENTS The majority (> 90%) of isoprene released into the atmosphere is from terrestrial plants, primarily trees (Fig. 1). Not all trees produce isoprene, and there is still considerable debate as to the exact role of isoprene in species that do produce it. Isoprene is produced from dimethylallyl diphosphate (DMAPP), a precursor molecule for many building blocks of cells including sterols, carotenoids, chlorophyll, quinones and hormones. DMAPP biosynthesis occurs via the mevalonate (MVA) pathway (in animals, fungi, archaea, in a minority of bacteria and in the plant cytosol) or the methylerythritol 4-phosphate (MEP) pathway (in chloroplasts, the major site of isoprene Fig. 1. Estimated annual net fluxes of isoprene from different environments (shown in brackets), both biogenic and anthropogenic, where each component is shown in Tg C yr−1 (adapted from Murrell et al. 2020). Where isoprene emissions are poorly constrained, a question mark has been included in place of values for net flux Dawson et al.: Aquatic cycling of isoprene production, and also in bacteria) (Rohmer 1999, Zhao et al. 2013). In trees that are substantial producers of isoprene, 1−20% of the carbon fixed through photosynthesis and the Calvin cycle can be released to the atmosphere as isoprene, mainly via the leaves (Guenther et al. 1995, Harley et al. 1996, Sharkey & Yeh 2001, Lantz et al. 2019). Isoprene biosynthesis is a costly process in terms of reducing power and energy, and so it represents a substantial metabolic drain on the plant. This metabolic ‘bleed’ is therefore presumably of significant benefit to plants, and the current dogma is that there are multiple roles for isoprene in protecting the plant from thermal and oxidative stress (Singsaas et al. 1997, Sharkey et al. 2001, Vickers et al. 2009), stabilizing membranes (Velikova et al. 2011), protecting against predation by grazing insects, and acting as a signalling molecule (Lantz et al. 2019, Zuo et al. 2019). These possible roles for isoprene production by plants have been reviewed in detail (Sharkey et al. 2008, Vickers et al. 2009, Lantz et al. 2019). Other sources of isoprene in the biosphere include some bacteria, archaea, fungi, and especially microalgae and macroalgae, which are thought to be the main producers of isoprene in the marine environment, contributing up to around 11.6 Tg C yr–1 (Broadgate et al. 1997, Luo & Yu 2010, Shaw et al. 2010, Conte et al. 2020). Biological sources of isoprene in wetlands, and freshwater, coastal and marine environments are discussed in Sections 7-10 and detailed in Table 1. Although likely to be small in comparison with biological sources, anthropogenic isoprene emissions may be important in urban areas. Vehicle exhaust is likely to be a large anthropogenic source of isoprene (Reimann et al. 2000, Khan et al. 2018), although no definitive global annual emissions data have been reported. Industrial production of polyisoprene rubber generates an estimated 0.8 Tg C yr−1 isoprene (Greve 2000), with other processes likely producing additional emissions each year. 3. IDENTIFICATION OF SOILS AS A SINK FOR ISOPRENE Given that trees are the main sources of isoprene to the atmosphere, it is perhaps not surprising that most attention has focussed on biological consumption in terrestrial environments. Landmark experiments by Cleveland & Yavitt (1997, 1998) demonstrated that a variety of soils could consume isoprene down to very low levels (< 5 ppbv), indicating that microbes that could degrade isoprene were present in soil. The first 81 reports of the isolation of bacteria that could consume isoprene were in the 1980s and 1990s (Van Ginkel et al. 1987, Ewers et al. 1990, Cleveland & Yavitt 1998, van Hylckama Vlieg et al. 1998), and these were mainly Actinobacteria of the genera Nocardia, Arthrobacter and Rhodococcus. Related strains such as Gordonia and Mycobacterium and also proteobacterial strains were subsequently isolated from estuarine sediments (Acuña Alvarez et al. 2009, Johnston et al. 2017). Isoprene-degrading strains of Pseudomonas, Klebsiella and Alcaligenes have also been isolated from rubber-contaminated soils (Srivastva et al. 2015). More recently, the leaf environment (the phyllosphere) has yielded a number of isoprene-degraders, including further Rhodococcus strains, and a Gramnegative Variovorax strain from the leaves of the high isoprene-emitting poplar tree (Crombie et al. 2018). The leaves of willow, and soils in the vicinity of these isoprene-emitting trees, have provided isoprene-degrading strains from the genera Nocardioides, Gordonia, Rhodococcus, Ramlibacter, Variovorax and Sphingopyxis, several of which have been characterised in detail (Larke-Mejía et al. 2019, 2020, Dawson et al. 2020, Gibson et al. 2020). Isoprene degraders have also been isolated from tropical tree species (Singh et al. 2019). All of these isolates are aerobes, and thus far there has only been one report of anaerobic degradation of isoprene, describing an enrichment of homoacetogens that can use isoprene as an electron acceptor, forming a mixture of methylbutenes in the process (Kronen et al. 2019). 4. BACTERIAL METABOLISM OF ISOPRENE Benchmark studies in the field of isoprene degradation by van Hylckama Vlieg et al. (1998) described the utilization of isoprene by Rhodococcus sp. AD45, isolated from freshwater sediment. In this actinobacterium, they proposed that isoprene oxidation was initiated via a soluble diiron-centre oxygenase, isoprene monooxygenase (IsoMO), and the toxic intermediate 1, 2-epoxyisoprene formed was then conjugated to glutathione by a glutathione S-transferase (van Hylckama Vlieg et al. 1998). Rhodococcus strain AD45 subsequently became the ‘workhorse’ bacterium for the study of isoprene degradation. The genome sequence and expression studies of this isoprene degrader revealed 22 genes involved in isoprene metabolism, which are all located on a 300 kbp plasmid (Fig. 2) (Crombie et al. 2015). IsoMO, consisting of a 3-subunit oxygenase, a reductase, a ferredoxin and a coupling protein, is encoded by 6 genes, 82 Aquat Microb Ecol 87: 79–98, 2021 Fig. 2. Isoprene (iso) metabolic gene clusters from representative Gram-positive isoprene-degrading bacteria from aquatic ecosystems (Johnston et al. 2017) and representative Gram-negative isoprene-degrading bacterium Variovorax sp. WS11 from soils (adapted from Dawson et al. 2020). Arrows represent the direction of transcription of the iso metabolic genes. Conserved iso metabolic genes are presented in consistent colours. The sizes of the genes represent approximate scaling, where isoA from Rhodococcus sp. AD45 is 1524 bp isoABCDEF, which are adjacent to isoGHIJ that encode enzymes involved in downstream steps in isoprene metabolism (Figs. 2 & 3), including the glutathione S-transferase (IsoI). The plasmid also contains genes involved in biosynthesis of the glutathione necessary to detoxify epoxyisoprene (Crombie et al. 2015). The use of glutathione is an uncommon trait in Gram-positive bacteria (Johnson et al. 2009). Other possible intermediates include 1-hydroxy-2glutathionyl-2-methyl-3-butene (HGMB) and 2-glutathionyl-2-methyl-3-butenoate (GMBA), followed by β-oxidation to enable strain AD45 to grow on isoprene as the sole carbon and energy source (Fig. 3) (van Hylckama Vlieg et al. 2000). The isoprene metabolic pathway has previously been reviewed by Murrell et al. (2020). Loss of the 300 kbp plasmid resulted in loss of the ability of strain AD45 to grow on isoprene. Isoprene degradation is induced by either epoxyisoprene or a subsequent intermediate product of isoprene metabolism in strain AD45, and expression studies have shown that in induced cells, iso genes are highly expressed, accounting for around 25% of all transcripts observed (Crombie et al. 2015). We are also studying putative transcriptional regulators in the vicinity of the iso gene cluster to determine how isoprene degradation is regulated. Fig. 3. Isoprene metabolic pathway, initially proposed by van Hylckama Vlieg et al. (adapted from van Hylckama Vlieg et al. 2000, Crombie et al. 2015). Asterisks: enzyme-catalysed reactions. Predicted metabolic reactions for which the enzyme/ enzymes are unknown are shown in a hyphenated list. IsoMo: isoprene monooxygenase; HGMB: 1-hydroxy-2-glutathionyl-2-methyl-3-butene; GMB:2-glutathionyl-2-methyl-3-butenal; GMBA: 2-glutathionyl-2-methyl-3-butenoic acid; CoA: coenzyme A; AldH: aldehyde dehydrogenase Dawson et al.: Aquatic cycling of isoprene The biochemistry of isoprene monooxygenase is also an interesting area of current study. Purification of the various components of the IsoMO and reconstitution assays reveal that all components, the dimeric oxygenase (IsoA2B2E2), a Rieske-type ferredoxin (IsoC), coupling protein (IsoD) and a flavoprotein NADH reductase (IsoF), are essential for activity (Sims 2020, L. Sims et al. unpubl. data). Preliminary data also suggest that the IsoMO has a smaller substrate range than other related soluble diiron-centre monooxygenases such as the soluble methane monooxygenase (sMMO), which can co-oxidise isoprene to 1, 2-epoxyisoprene (Patel et al. 1982). It will be interesting to explore the biotechnological potential of IsoMO, particularly with respect to the production of chiral epoxides from alkenes (Sims 2020). 5. CHARACTERISATION OF OTHER ISOPRENE-DEGRADING BACTERIA The other well-characterised isoprene-degrader is the Gram-negative Variovorax sp. WS11, isolated from soil in the vicinity of a willow tree (Larke-Mejía et al. 2019). This isoprene-degrader also contains the iso metabolic gene cluster on a plasmid, and these genes are significantly upregulated in the presence of isoprene and epoxyisoprene (Dawson et al. 2020). Two putative LysR-type transcriptional regulators, encoded by genes located 5’ and 3’ of the iso metabolic gene cluster in Variovorax sp. WS11, are also the subject of further study (R. A. Dawson, A. T. Crombie & J. C. Murrell unpubl.). The development of robust molecular genetics methods for this isoprene-degrader, such as targeted mutagenesis, is now enabling the dissection of the downstream regions of the isoprenedegradation pathway. The IsoMO of Variovorax sp. WS11 oxidises a variety of alkenes, and its activity is inhibited by several alkynes, with inhibition increasing significantly with increasing chain length (Dawson et al. 2020). Octyne was the most potent inhibitor tested, and in contrast, acetylene (at 50 μM), a strong suicide inhibitor of the related enzyme soluble methane monooxygenase, was a poor inhibitor of IsoMO. This finding paves the way for the use of specific inhibitors to determine if isoprene oxidation in environmental samples is mainly due to bona fide isoprene-degraders (most likely) or possibly due to co-oxidation reactions by the catalytically versatile sMMO present in methaneoxidising bacteria (methanotrophs), which are widely distributed in the environment. Characterisation of other newly isolated isoprenedegraders such as Gordonia, Mycobacterium, Nocar- 83 dioides and Sphingopyxis has increased our baseline knowledge of the metabolism of isoprene by bacteria by providing important information on the physiology, biochemistry and molecular biology of diverse genera of isoprene-degraders (Johnston et al. 2017, Larke-Mejía et al. 2019, 2020, Gibson et al. 2020). The availability of representatives across several genera has also enabled comparative genomics of isoprene-degraders, revealing that they all contain isoprene degradation gene clusters as shown in Fig. 2 (Larke-Mejía et al. 2019, Carrión et al. 2020a), which are invariably found together, either on the genome or, as in the case of Variovorax sp. WS11 and Rhodococcus strain AD45, on large plasmids (Crombie et al. 2015, Dawson et al. 2020). The ability to make glutathione also appears to be a common feature of these bacteria due to the role of glutathione in the detoxification of the reactive epoxide intermediate, 1, 2-epoxyisoprene, formed by the oxidation of isoprene by IsoMO. Comparative analysis of genes encoding IsoMO has also enabled us to differentiate this key enzyme, which initiates the metabolism of isoprene by bacteria, from other soluble diironcentre monooxygenases (SDIMOs) such as sMMOs, alkene monooxygenases and toluene monooxygenases (Carrión et al. 2018). 6. MOLECULAR ECOLOGY OF ISOPRENE-DEGRADERS It is now clear that iso genes fall into distinct clades when analysed phylogenetically in combination with other SDIMO-encoding genes (Leahy et al. 2003, Carrión et al. 2018). This molecular and biochemical information on isoprene-degraders has enabled the development of molecular ecology tools to determine the distribution, diversity and activity of these bacteria in the environment. Following the methodological strategies of Dumont & Murrell (2005) and Farhan Ul Haque et al. (2019) to study the microbial ecology of aerobic methanotrophs, DNA-based detection methods have been developed to investigate isoprenedegraders in the environment (Carrión et al. 2020b). Analysis of the genomes of isoprene-degraders and alignment of iso genes has revealed a consistent organisation of isoprene degradation genes. We have used the isoA gene, encoding the putative active site-containing α-subunit of the oxygenase component of IsoMO, as a phylogenetic marker to determine the presence of isoprene-degraders in DNA extracted from a number of different (mostly terrestrial) environments, including various soils, sedi- 84 Aquat Microb Ecol 87: 79–98, 2021 ments, and leaf samples. isoA-specific PCR primer sets were designed which do not ‘cross-react’ with the corresponding homologues encoding the α-subunits of other SDIMOs, for example the large subunit of sMMO (mmoX) (El Khawand et al. 2016, Carrión et al. 2018). These isoA PCR primer sets have provided valuable data on the distribution and diversity of isoprene-degraders in various environments, and isoA sequences have complemented data on isoA from cultivated isoprene-degraders to assist in mapping the phylogeny of these IsoMO enzymes (Crombie et al. 2018, Carrión et al. 2020a, Dawson et al. 2020, Murrell et al. 2020). For example, screening of soil and leaf samples not only revealed the presence of actinobacterial isoprene-degraders such as Rhodococcus, Gordonia, Mycobacterium and Nocardioides and proteobacterial representatives Sphingopyxis and Variovorax, but also the presence of sequences representing uncultivated isoprene-degraders (Carrión et al. 2020a). A quantitative PCR assay based on isoA has also been developed which, when normalised against 16S rRNA gene copies in DNA from environmental samples, revealed the relative abundance of putative isoprene-degraders. For example, isoA copies appeared to be most abundant in soil collected from within the vicinity of high isoprene-emitting trees such as oil palm, poplar and willow with between ~75 and 300 copies of isoA genes per million 16S rRNA genes (Carrión et al. 2018, 2020a). Surveys such as these now need to be carried out in other environments (see Section 11). Amplification of isoA sequences by PCR reveals the presence of isoprene-degraders in an environmental sample but this approach does not necessarily retrieve the most active isoprene-oxidisers and, hence, as with methanotrophs, DNA-stable isotope probing (DNA-SIP) (Dumont & Murrell 2005, Kröber & Eyice 2019) was used to identify the most active isoprene-degraders in a variety of soil, leaf and estuarine sediment samples. Early DNA-SIP experiments with 13C-labelled isoprene used sediment from the Colne Estuary, UK, originally investigated for isoprene-degradation potential by Acuña Alvarez et al. (2009). This led to the identification of actinobacterial isoprene-degraders such as Gordonia, Mycobacterium and Rhodococcus (Johnston et al. 2017). Soil from the vicinity of willow trees also revealed the presence of similar Actinobacteria, but also of Betaproteobacteria, such as Comamonas and Variovorax, which are capable of consuming isoprene (El Khawand et al. 2016, Larke-Mejía et al. 2019). El Khawand et al. (2016) showed that the isoA sequences from marine and estuarine iso- prene-degrading bacteria cluster together in a phylogenetic tree (as in Fig. 4), while the isoA sequences from isoprene-degraders isolated from terrestrial and freshwater samples lacked a clear environment-phylotype relationship. Cultivationindependent studies, which combined functional gene probing with DNA-SIP, provided further isoA sequences to use alongside isoA sequences from cultivated representatives, thus improving considerably the robustness of isoA phylogenetic trees and enabling the detection of uncultivated isoprenedegraders in the environment (Carrión et al. 2018). By combining DNA-SIP with metagenomics, using a ‘focussed metagenomics’ strategy, the near-complete genomes of key, active isoprene-degrading bacteria have been retrieved from several environments (Carrión et al. 2018, 2020a, Crombie et al. 2018, Larke-Mejía et al. 2019). Study of these genomes has yielded not just individual iso genes but also complete iso metabolic gene clusters containing isoABCDEF and isoGHIJ (Fig. 2), providing valuable data for comparative genomics, aiding phylogenetic analysis and revealing the presence of new isoprene-degraders without the need for cultivation. For example, leaf washings of the high isoprene-emitter white poplar Populus alba were incubated in microcosms with 13C-labelled isoprene. After assimilation of labelled isoprene, heavy DNA from active isoprene-degraders in these environmental samples was isolated by CsCl density-gradient centrifugation and sequenced. Near-complete genomes of putative isoprene-degrading Rhodococcus and Variovorax were obtained (Crombie et al. 2018). The power of this focussed metagenomics approach was demonstrated by cloning the iso genes from the Variovorax genome into a mutant Rhodococcus strain AD45 that had been cured of the 300 kbp plasmid containing all of its iso genes (also a convenient expression system for future sitedirected mutagenesis studies on IsoMO), which therefore did not oxidise isoprene. The isoABCDEF gene cluster from the Variovorax genome conferred on this Rhodococcus mutant the ability to oxidise isoprene, thus proving that the iso genes retrieved through DNA-SIP-enabled metagenomics were indeed from a bona fide isoprene-degrader (Crombie et al. 2018). Informed by these molecular data, enrichment and isolation strategies were used to isolate representative isoprene-degraders of the genus Variovorax, including Variovorax sp. WS11 described earlier, which has now become the Gramnegative model isoprene-degrader of choice (along with the actinobacterial Rhodococcus strain AD45) Dawson et al.: Aquatic cycling of isoprene 85 Fig. 4. Phylogeny based on ratified IsoA amino acid sequences (colour-coded) from aquatic isoprene-degraders and from the soil-derived isoprene-degrader Variovorax sp. WS11. : genome-sequenced isoprene-degrading bacterium which contains a verified iso metabolic gene cluster. Included for reference: SDIMO α-oxygenase subunits from the soluble methane monooxygenase (MmoX) and toluene 4-monooxygenase (TmoA). The closest relative to IsoA, the α-oxygenase subunit from the alkene monooxygenase of Xanthobacter sp. PY2 (XamoA) was also included for reference. This tree was drawn using the maximum likelihood programme in Mega7 (Kumar et al. 2016), with bootstrap values (500 replications) shown at the nodes. Branch lengths measured in the number of substitutions per site to study the metabolism and pathways of isoprene degradation in bacteria (Dawson et al. 2020). We now have a good understanding of the types of isoprene-degraders present in the terrestrial environment and also of the biology of isoprene degradation, with the majority of isolates from soils, leaves and sediments. Here, we review the state of knowledge on isoprene in marine and freshwater environments and highlight the potential for future research on this topic. 7. EMISSION OF ISOPRENE BY TERRESTRIAL WETLANDS Wetlands are an interesting but understudied environment when considering the cycling of biogenic volatile organic compounds (BVOCs). Temperaturedependent shifts in volatile organic compound (VOC) emissions from terrestrial plants are well-established (Lantz et al. 2019), but wetlands may respond differently than drier ecosystems due to buffering of extremes by the high specific heat capacity of water and the cooling effects of flowing and evaporating water. Few measurements of isoprene emissions from fenland plants are available (Seco et al. 2020), and the isoprene-degrading communities of these ecosystems are currently unstudied. Several studies have identified the isoprene-emitting potential of moss species in arctic and sub-arctic wetlands (Janson & De Serves 1998, Ekberg et al. 2009, 2011, Lindwall et al. 2016, Hellén et al. 2020, Seco et al. 2020). Swedish Sphagnum and graminoid-rich fens emit isoprene at 219.8 ± 125.8 and 93 ± 22 μmol m−2 d−1, respectively, exhibiting diurnal regulation due to light and temperature (Janson & De Serves 1998, Seco et al. 2020). In contrast to leaves, which heat and cool rapidly due to a large surface area in direct contact with the air, bodies of water react much more slowly. This difference may contribute to the observation that the temperature history of the previous 48 h is a significant influencer of wetland isoprene emission (Ekberg et al. 2009, 2011). Moisture content was also a potent controller of isoprene emission, with plants from dry and wet fenland sites producing 70 and 2000 μg C m−2 h−1, respectively (Janson & De Serves 1998), with Sphagnum balticum producing 30% more isoprene under moist conditions compared to dry conditions (Ekberg et al. 2011). More direct comparisons of isoprene emission from plants in dry and wetland sites are required to adequately define the relative contributions of each to the iso- 86 Aquat Microb Ecol 87: 79–98, 2021 prene budget. Algae may also be key contributors to isoprene emissions from wetlands, but their role will be discussed in Section 10. Although global isoprene emission from wetlands is difficult to quantify, due to insufficient measurements and because estimates of global wetland coverage range from 0.54−21.26 million km2 (Hu et al. 2017), the contribution of wetlands to the global isoprene budget is low compared to dryland terrestrial plants in warm climates, with oil palm plantations alone producing an estimated 7800 μg C m−2 h−1 (Misztal et al. 2011). The inclusion of isoprene flux measurements in future studies, as well as studies of the isoprene-degrading communities that colonise isoprene-emitting mosses and wider wetland ecosystems, would greatly improve our understanding of the contribution of these ecosystems to isoprene cycling. 8. EMISSION OF ISOPRENE BY FRESHWATER SOURCES While lakes and other fresh waters are significant sources of VOCs such as methane (Wik et al. 2016), the contribution of freshwater ecosystems to isoprene emissions is still a point of much debate. Isopreneproducing species of phytoplankton (both marine and freshwater) have been reported from diverse taxa (Exton et al. 2013, Steinke et al. 2018), with isoprene emission potentially linked to stress responses, as in plants (Zuo et al. 2012a,b, Meskhidze et al. 2015, Dani & Loreto 2017, Xu et al. 2017). The evolutionary basis for isoprenoid biosynthesis by algae is complicated; while plants use a combination of the cytosolic mevalonate (MVA) pathway and the plastid-localised methylerythritol 4-phosphate (MEP) pathway (Zhao et al. 2013), most green algae only use the MEP pathway, and some red algae and charophyte green algae use the MVA pathway (Lohr et al. 2012, Bentlage et al. 2016). Steinke et al. (2018) reported the flux of both isoprene and dimethyl sulfide (DMS) from the oligotrophic Lake Constance, Central Europe. Of 10 phytoplankton strains isolated from this lake, 5 emitted isoprene, at rates between 12.6 and 174.3 nmol (g chl a)−1 h−1, lower than the overall estimated lake flux of 304 nmol (g chl a)−1 h−1. This discrepancy was attributed to their screening technique which may have excluded some isoprene-emitting taxa, or due to the variability of isoprene emission with changing environmental conditions. In another study, it was shown that the Villasjön Lake (northern Sweden) emitted 20 ± 10 μmol m−2 d−1 in the summer (Seco et al. 2020). Isoprene emission by lakes was suggested to rival terrestrial plants in areas with high lake coverage and low plant coverage, although this is yet to be explored (Steinke et al. 2018). 9. ISOPRENE EMISSIONS BY COASTAL AND OPEN MARINE ENVIRONMENTS Little information is available with regards to the emission of isoprene from estuarine environments. According to a simple model which calculated the annual rate of isoprene production for the Colne Estuary, UK, using data from benthic incubations, intertidal sediments from the Colne Estuary were responsible for an estimated 681 μmol m−2 yr−1 (Exton et al. 2012). Comparisons with other freshwater and marine sources of isoprene are difficult to conduct due to the scarcity of flux measurements. Long-term studies of isoprene flux are required from a variety of aquatic environments in order to fully quantify the relative contributions of each type of ecosystem to annual isoprene emissions. Sediment samples from the Colne Estuary emitted an average of 8.38 ± 1.18 nmol isoprene m−2 h−1, with little variation between the head and mouth of the estuary (Exton et al. 2012). The dissolved concentration of isoprene in the estuary was higher at low tide which, coupled with the high isoprene production from sediments, led to the conclusion that the microphytobenthos was primarily responsible for the emission of isoprene (Exton et al. 2012). Estimates of annual marine emissions of isoprene range from 0.1 to 11.6 Tg C yr−1 (Broadgate et al. 1997, Luo & Yu 2010, Shaw et al. 2010, Conte et al. 2020). The disparity between estimates is a result of the differing measurement or estimation techniques used and of the effects of seasonality and corresponding productivity of the measurement period in question. Bottom-up estimates couple laboratory-based measurements of isoprene emission by phytoplankton with satellite-based measurements of phytoplankton biomass, based on the abundance of chlorophyll a (Broadgate et al. 1997, Shaw et al. 2003, Luo & Yu 2010, Halsey et al. 2017, Davie-Martin et al. 2020, Li et al. 2020). Top-down approaches combine measurements of isoprene flux from seas with inversion modelling and global scaling (Arnold et al. 2009, Luo & Yu 2010, Shaw et al. 2010). Luo & Yu (2010) calculated annual aquatic isoprene emission rates of 0.32 and 11.6 Tg C yr−1 by bottom-up and top-down approaches, respectively, demonstrating the need for improvements in our understanding of the factors constraining estimates of isoprene emission. Dawson et al.: Aquatic cycling of isoprene Various studies have reported in situ measurements of isoprene from seawater (Milne et al. 1995, Broadgate et al. 1997, Tran et al. 2013, Kameyama et al. 2014, Ooki et al. 2015), often relying on purgeand-trap or cryogenic enrichment systems (Table 1). Measurements of isoprene concentrations in seawater range from <1 to 541 pmol l−1, with the highest concentrations typically reported in productive coastal zones or regions of phytoplankton blooms (Milne et al. 1995, Exton et al. 2012, Tran et al. 2013, Kameyama et al. 2014, Zindler et al. 2014, Ooki et al. 2015, Booge et al. 2016). The requirement for large samples and interference by other BVOCs may result in overestimations of isoprene concentrations, prompting Hrebien et al. (2020) to develop the cumulative headspace injection technique. The limit of detection of this technique was suitable for use in productive tropical coastal regions. Also, Li et al. (2020) reported a novel technique which relied on adsorption and desorption at room temperature, with measurements comparable to those of previous studies. Direct measurements of marine isoprene concentrations and fluxes have been gathered from only a small number of areas, with little spatiotemporal coverage (Table 1) (Hackenberg et al. 2017, Conte et al. 2020, Li et al. 2020). While this relative lack of data precludes large-scale modelling of marine isoprene cycling, it has aided our understanding of various biotic and abiotic drivers of isoprene emission. As in plants, isoprene emission may play a role in the resistance of algae to abiotic stresses (Zuo et al. 2012a,b, Meskhidze et al. 2015, Xu et al. 2017). Isoprene is synthesised within the plastid (Bentlage et al. 2016, Zuo 2019) in a manner which is strongly influenced by temperature and photosynthetically active radiation (PAR) (Broadgate et al. 2004, Halsey et al. 2017, Booge et al. 2018, Conte et al. 2020, Davie-Martin et al. 2020). This relationship is due to the influence of these factors on phytoplankton growth, and also the requirement of photosynthetic electron transport for the production of isoprenoid precursors (Broadgate et al. 1997, Rasulov et al. 2009, Niinemets & Sun 2015, Conte et al. 2020, Tripathi et al. 2020). Phytoplankton blooms are a seasonal driver of increasing isoprene concentrations, meaning that drivers of phytoplankton growth must be taken into account when considering the annual flux of isoprene (Kameyama et al. 2014, Ooki et al. 2019, Davie-Martin et al. 2020, Rodríguez-Ros et al. 2020, Tripathi et al. 2020). Ooki et al. (2015) identified a maximum in isoprene concentration between 10 and 20°C, consistent with phytoplankton blooming temperatures. Measurements of isoprene have 87 typically identified a diel cycle of increasing isoprene concentration with increasing light and temperature (Matsunaga et al. 2002, Broadgate et al. 2004, Liakakou et al. 2007, Acuña Alvarez et al. 2009, Shaw et al. 2010, Exton et al. 2012). Exton et al. (2012) reported peaks of isoprene concentration in the Colne Estuary in the daytime and night-time, although the night-time peak was significantly smaller and influenced by the aforementioned tidal changes and proximity to the sediment. A diel pattern of isoprene concentration/emission was also demonstrated in Lake Constance (Steinke et al. 2018). Daytime and night-time peaks were also identified in the western North Pacific, with a larger peak in the daytime (Matsunaga et al. 2002). However, the concentration of isoprene in the air was greater at night (Matsunaga et al. 2002). Similar observations were made in the Yellow Sea/East China Sea as isoprene concentrations peaked at midday but were still detected at night at a lower concentration (Wu et al. 2021). The highest marine and freshwater concentrations of isoprene were reported in the afternoon rather than morning, with seasonal studies reporting higher concentrations in summer than at other times of year (Broadgate et al. 1997, Seco et al. 2020). Contrary to these observations, Bonsang et al. (1992) and Hackenberg et al. (2017) identified no significant diel variation in isoprene concentration, although the latter study suggested that the lack of diel variation may have been due to sampling in remote regions with little biological activity. However, a consistent correlation between surface isoprene concentrations and the sum of photoprotective carotenoids was found, providing an alternative metric to chlorophyll a content (Hackenberg et al. 2017). Oligotrophic zones, which are often limited in nutrients such as iron, have reported lower concentrations of isoprene than more productive areas closer to coastlines (Hackenberg et al. 2017, Rodríguez-Ros et al. 2020), while Davie-Martin et al. (2020) reported that net production of isoprene was higher in the open ocean in the eastern parts of the North Atlantic Ocean and decreased with proximity to the coastal shelf in March in the northern Atlantic. The sea-surface microlayer (SML) concentrates organic matter on the ocean’s surface, where photochemical reactions produce organic vapours, including isoprene. Initial estimates of the photochemical production of isoprene ranged from 14.4 to 33 120 nmol m−2 d–1 (Ciuraru et al. 2015). Brüggemann et al. (2018) updated these predictions using parameters such as biological net productivity and Aquat Microb Ecol 87: 79–98, 2021 88 Table 1. Reported concentrations of isoprene and flux measurements according to sampling location and type. Values in brackets were derived from models. NA: not available; GBR: Great Barrier Reef Sampling area Isoprene flux (nmol m−2 d—1) Isoprene conc. (pmol l—1) Sample type 181−313.1 (28.8−1238.4) NA 14.5 0.2−395 (3−13) 1−94 5.6 22−46 1−541 23.8 9.5 NA 4.8−57.1 48−96 (36−216) 9.51−101.2 (5.8−244.8) 158.4 13.7−60.8 NA 42.6 10.1−28.3 32−300 (14.4−158.4) 1.3−20.2 2−12 12−94 In situ, seawater, shipboard measurement Seawater, purge trap system In situ, seawater, shipboard measurement In situ, seawater, shipboard measurement In situ, seawater, shipboard measurement Seawater, purge-and-trap system Seawater, laboratory measurement In situ, seawater and atmospheric measurements Seawater, laboratory measurement In situ, room temperatureenriched thermal desorption Seawater, purge-and-trap system Marine air and seawater 21.4−143.8 12.4−114.1 1.4−67.7 0.7−54.3 52.4 17.1 NA 27.54 7.3−232.3 6.5−51.9 201.1 ± 28.3 0.7−451.8 Estuary water, cryo-trap system. Sediment sampling Intertidal rock pool Mace Head, Ireland 974.9 24.9−865.5 Seawater and seaweed, cryo-trap system. In situ rock pool study with stainless steel bucket Reef sediments Heron Island (GBR) Lizard Island (GBR) 194.4−290.4 69.6−139.2 NA NA Sediment, benthic chambers Sediment, benthic chambers Hrebien et al. (2020) Hrebien et al. (2020) Lake Lake Constance Lake Villasjön 350.4 600−20 000 183−722 NA Lake water, purge-and-trap system Eddy covariance measurements Steinke et al. (2018) Seco et al. (2020) 300−93 000 NA Eddy covariance measurements Marine Southern Ocean Arctic Ocean Southern Atlantic Ocean Eastern Atlantic Ocean Florida Straits North Pacific/ Mediterranean Sea North Pacific Northeast Pacific Northwest Pacific North Sea Yellow Sea/ East China Sea Coastal Japan (Hokkaido) Indian Ocean Estuary Colne Estuary, UK Fenland Northern Sweden, Graminoid a 9.8−50.8 6.7−25 NA MODIS chlorophyll observations coupled with phytoplankton study In situ, seawater, shipboard measurement (1986) Seawater, in situ, cryo-trap system. Estimation by Liss and Merlivat relationship Seawater and atmospheric samples. Laboratory analysis In situ, room temperatureenriched thermal desorption In situ, seawater, shipboard measurement Reference Meskhidze & Nenes (2007)a, Kameyama et al. (2014) Rodríguez-Ros et al. (2020) Ooki et al. (2015) Tran et al. (2013) Ooki et al. (2015) Rodríguez-Ros et al. (2020) Baker et al. (2000), Arnold et al. (2009)b Milne et al. (1995), Arnold et al. (2009)b Bonsang et al. (1992) Li et al. (2020) Moore & Wang (2006) Matsunaga et al. (2002), Arnold et al. (2009)b Palmer & Shaw (2005) Ooki et al. (2015) Liss & Merlivat (1986), Broadgate et al. (1997) Wu et al. (2021) Li et al. (2020) Ooki et al. (2015) Exton et al. (2012) Broadgate et al. (2004) Seco et al. (2020) Remotely sensed data from Moderate Resolution Imaging Spectroradiometer (MODIS) and Sea-viewing Wide Field-ofview Sensor (SeaWiFS); bModelled results were calculated by a combination of phytoplankton functional type, observed by PHYSAT (Alvain et al. 2005), and 3-D global chemical transport model Dawson et al.: Aquatic cycling of isoprene 89 pmol l–1, corresponding with salinity ranging from 4 mean surface wind speeds, estimating 0.7−1.52 Tg C −1 yr isoprene from the SML. More recently, a global to 23%, respectively (Rinnan et al. 2014). Although simulation of isoprene emission estimated 0.39 Tg C the primary isoprene emitters in this environment yr−1 due to photoproduction in the SML (Conte et al. were not identified, the phototrophic communities 2020). Photochemical production of isoprene from tend to be composed of benthic cyanobacteria and the SML may account for a portion of the deviation chlorophytes, such as Dunaliella salina (Rinnan et al. between top-down and bottom-up estimates of 2014). Further studies are required to investigate the aquatic isoprene emissions. The fact that these emisbiological components of isoprene cycling in extreme sions are dependent on sunlight may also contribute environments, particularly hypersaline environments to the observations of a diel pattern of isoprene which constitute a considerable proportion of the emissions. The concentration of isoprene in the SML Earth’s surface. remained enriched at low windspeeds, requiring approximately 10 m s−1 before the depletion of isoprene was observed (Ciuraru et al. 2015); this correlated with 10. ISOPRENE EMISSION BY MICROALGAE, the observations of marine particle nucleation made MACROALGAE AND CORAL-ASSOCIATED by Wen et al. (2006), as they also found that greater SPECIES wind speed increased the likelihood of nucleation occurrence. Enrichments of SML isoprene may also Most reports of aquatic isoprene-emitting organinfluence local communities of isoprene-degrading isms focus on phytoplankton (Table 2), with only one bacteria, providing a rich area for molecular ecology study demonstrating isoprene emission by macroalgae studies. Table 2. Published rates of isoprene emission in different aquatic Isoprene emissions associated with phytoisoprene-emitting organisms, collated by Booge et al. (2016) plankton blooms and other isoprene-emitting species may result in changes in cloud formation Taxonomic Isoprene emission range Reference and local climate due to the influence of isoprene group (μmol (g chl a)−1 d—1) on secondary organic aerosol (SOA) formation (Meskhidze & Nenes 2007, Cropp et al. 2018). Haptophytes 1.24−2.73 Arnold et al. (2009) However, Arnold et al. (2009) estimated that iso2.88−15.36 Exton et al. (2013) prene-derived SOA could only account for 0.5% 5.4−11.45 Colomb et al. (2008) of marine organic carbon aerosols, while Hu et Cyanobacteria 2.40−22.10 Arnold et al. (2009) al. (2013) posited that phytoplankton blooms 2.40−11.76 Exton et al. (2013) 1.4−1.5 Shaw et al. (2003) could produce sudden spikes in SOA over re0.02±0.003–0.16±0.04b Steinke et al. (2018) mote oceans. Alternatively, isoprene may be an Chlorophytes 2.85 Colomb et al. (2008) insignificant contributor to global marine SOA 1.2 Exton et al. (2013) yield but a significant contributor to SOA yield 0.36±0.22 Bonsang et al. (2010) over tropical oceans (Gantt et al. 2009), and SOA 83.28a Broadgate et al. (2004) formation has been correlated with both sea0.16±0.02b Halsey et al. (2017) sonal variation and chlorophyll a (Cui et al. 0.049±0.036b Steinke et al. (2018) 2019). The important impact of isoprene on Diatoms 0.00−10.10 Arnold et al. (2009) 0.72−9.36 Exton et al. (2013) global SOA formation over aquatic ecosystems 28.48 Colomb et al. (2008) cannot be accurately estimated at present due to 0.56−1.32 Bonsang et al. (2010) the incomplete understanding of aquatic iso0.36±0.17–12.38±5.81c Dani et al. (2017) prene emissions. Two examples illustrate the 0.47±0.07b Halsey et al. (2017) uncertainties associated with isoprene in the Dinoflagellates 4.56−27.6 Exton et al. (2013) marine environment. A recent study estimated Cryptophytes 9.36±0.72 Exton et al. (2013) that as much as 4.8 Tg C yr−1 of isoprene was 0.01±0.009b Steinke et al. (2018) emitted solely by diatoms in temperate oceans (Dani et al. 2017), nearly half of the high end of Fluxes are shown as μmol (g chl a)–1 d–1, except aIsoprene emission by the macroalgae Ulva intestinalis was measured in pmol previous total marine estimates (Luo & Yu 2010), g−1 (dw) d−1;; bIsoprene emission reported by Halsey et al. (2017) and some diatom species produce isoprene at and Steinke et al. (2018) measured in μmol (g chl a)−1 h−1; cIsorates as high as 35 μg (g chl a)−1 h−1 (Meskhidze prene emissions by diatoms were normalised to chlorophyll a et al. 2015). Isoprene concentrations in a hyperand chlorophyll c saline saltern (Mallorca) ranged from 209 to 313 90 Aquat Microb Ecol 87: 79–98, 2021 from Indonesia suggested that BVOC production by coral holobionts exceeded that of other primary producers such as macroalgae and seagrasses Major taxoSpecific Isoprene Units Reference nomic group group emission rate (Exton et al. 2015). Isoprene-degrading capacity was demonstrated in tropical Macroalgae Red algae 0.31−5.26 pmol g−1 (dw) h−1 Broadgate et al. coastal seawater microcosms (Acuña (2004) −1 −1 Alvarez et al. 2009), and isoprene proBrown algae 0.69−1.38 pmol g (dw) h Broadgate et al. (2004) duction by the coral Acropora aspera −1 −1 was confirmed by Swan et al. (2016). Corals Acropora 0.01−0.058 ng g (fw) h Swan et al. aspera (2016) Isoprene emissions were highest when Acropora 0.97−2.24 pmol cm−1 h−1 This study corals were stressed by agitation horrida caused by shaking, coinciding with the release of mucus. Conversely, isoprene was not detected, and mucus (Table 3) (Broadgate et al. 2004). All 10 species of seawas not produced, when A. aspera fragments were weed tested, including the abundant macroalga Ulva undisturbed. Symbiodiniaceae that associate with intestinalis, emitted isoprene in a temperature- and the gastrodermis of the coral host were suspected as light-dependent manner. The red alga Asparagopsis the primary source of isoprene, although this was armata was the highest producer, while the brown not investigated. Under stressful conditions, such as alga Ascophyllum nodosum was the lowest producer, elevated temperature or light intensity, coral hosts with isoprene emission rates of 5.26 and 0.46 pmol exude their zooxanthellae symbionts, which are g−1 (dry weight, dw) h−1, respectively (Broadgate et secreted via mucus sloughing (Broadbent & Jones al. 2004). Aquatic isoprene emitters may continue to 2004). A. aspera inhabits shallow bodies of water, produce isoprene in the dark, albeit at a lower rate such as reef flats and lagoons, making this species than the photosynthesis-driven isoprene emission rate particularly susceptible to solar irradiance. This (Shaw et al. 2003, Ooki et al. 2019). Studies in terresstress is thought to induce production of VOCs intrial plants have demonstrated the sudden cessation of cluding DMS and isoprene by coral-associated zooisoprene emission in the absence of light, with only a xanthellae and may result in mucus production by brief spike in emission concurrent with the release of the coral host (Swan et al. 2016). the stored isoprenoid precursor DMAPP (Rasulov et Since comprehensive data on isoprene production al. 2009, Li & Sharkey 2013, Weise et al. 2013). The by corals are lacking, for this work we conducted estimated rate of isoprene production in macroalgae experiments with A. horrida coral fragments using a only decreased from 30 pmol g−1 (dw) h−1 during the static headspace sampling method. A. horrida fragday to 10 pmol g−1 (dw) h−1 at night. Likewise, cyanoments produced isoprene at a mean rate of 0.97 ± bacteria and diatoms retained approximately 10% of 0.12 and 2.24 ± 0.89 pmol cm−2 h−1 during incubations at 27 and 29°C, respectively (Fig. 5). Isoprene protheir daytime rate of isoprene emission when in dark duction rates were significantly different between conditions (Shaw et al. 2003, Ooki et al. 2019). Exton et temperature regimes (p ≤ 0.05), determined by 1-way al. (2012) demonstrated a peak in isoprene emission at ANOVA, suggesting that isoprene is involved in a night in the Colne Estuary, further indicating that thermo-protective response by A. horrida. Cropp et night-time isoprene emission should be considered in al. (2018) suggested that corals may emit BVOCs in aquatic models of isoprene flux. Dani et al. (2020) response to stressors such as irradiance, indicating demonstrated the ability of mixotrophic Chlorella that the relationship between isoprene emission and vulgaris to produce isoprene in the dark only while coral stress requires further investigation. Isoprene supplied with glucose, thus a contribution to aquatic production by Acropora spp. suggested that scleracisoprene production by mixotrophic protists growing tinian coral holobionts are important isoprene proheterotrophically should be considered. Gantt et al. ducers in tropical marine regions and contribute to (2009), however, reported that certain diatom and the total atmospheric flux of isoprene from the marcoccolithophore species did not produce any signifiine environment. However, compared to isoprene cant quantity of isoprene when incubated in the dark. emissions from microalgae (Colomb et al. 2008, BonIsoprene production was reported in laboratory sang et al. 2010, Exton et al. 2013) and macroalgae cultures of coral-associated dinoflagellates, such as (Broadgate et al. 2004) the measurements reported Symbiodiniaceae (Exton et al. 2013). Also, field data Table 3. Emission of isoprene by macroalgae and corals. dw: dry weight; fw: fresh weight Dawson et al.: Aquatic cycling of isoprene Fig. 5. Isoprene production by Acropora horrida coral fragments incubated at 27°C (n = 8) and 29°C (n = 3) (± SE), normalised by surface area. Coral fragments were incubated for 4 h in 100 ml gas-tight bottles containing purged artificial seawater and exposed to a light intensity of 300 μmol photons m−2 s−1. Control bottles containing only purged artificial seawater were incubated under the same conditions. Coral isoprene production was control-corrected by subtracting control-bottle isoprene concentrations from isoprene concentrations measured in bottles containing coral fragments. Using the purge-and-trap method, isoprene was measured via a Shimadzu GC-2014 gas chromatograph coupled with a flame ionisation detector (GC-FID). The GC column was a 1.5 m glass column packed with 10% Apiezon L on CarboWax (60/80 mesh). The statistical test performed on these data was 1-way ANOVA here for A. horrida are lower. The data presented here represent the emission of isoprene by A. horrida normalised per unit of surface area. A. horrida has a large surface area relative to its 2-dimensional footprint. Hence, per sea surface area, coral reefs may add substantially to local isoprene emissions. Additionally, while Swan et al. (2016) primarily attributed isoprene emissions from A. aspera to the symbiotic zooxanthellae, Raina et al. (2013) found that Acropora spp. produced dimethylsulfoniopropionate (DMSP) even in the absence of Symbiodiniaceae symbionts, indicating the possibility that the coral host may be responsible for the emission of other BVOC. Lawson et al. (2020) also recently reported significant changes to the output of BVOCs from Symbiodiniaceaeassociated bacteria when incubated with filtrate derived from the phytoplankton, indicating a role of bacteria in the emission of BVOCs from corals, although this has not been linked to isoprene. The impact of changing environmental conditions on isoprene emission by coral requires further study. A. aspera emits isoprene under ambient conditions (Swan et al. 2016), and no significant difference in isoprene emission by A. intermedia or Pocillopora damicornis occurred in response to acute heat stress 91 (Lawson et al. 2021). The contribution of other coral species, particularly those belonging to more thermally tolerant genera such as Porites spp. (Loya et al. 2001), must be investigated further. Hrebien et al. (2020) reported that coral reefs with a covering of microphytobenthos at Heron Island (southern Great Barrier Reef) generated isoprene at rates between 194.4 and 290.4 nmol m−2 d–1 (Table 1), whereas reef sediments at Lizard Island on the northern end of the Great Barrier Reef generated between 69.6 and 139.2 nmol m−2 d–1. The difference in production was suggested to be due to differences in the composition of the local benthic communities (Hrebien et al. 2020). The greatest values for isoprene flux were recorded during the daytime, while night-time values became negative at times. Fluxes of −1.5 ± 1.0 to 4.8 ± 0.8 nmol m−2 h−1 at Heron Island and 1.2 ± 6.1 to 2.6 ± 0.2 nmol m−2 h−1 at Lizard Island were recorded in the dark, respectively (Hrebien et al. 2020), suggesting that these carbonate sediments and their associated microphytobenthos may become sources or sinks for isoprene according to a diel cycle. 11. MECHANISMS OF ISOPRENE LOSS FROM AQUATIC ECOSYSTEMS Mechanisms of isoprene loss from the ocean are poorly understood, although the greatest loss is likely to be due to abiotic processes such as air−sea gas exchange, driven by wind-speed and local temperature (Palmer & Shaw 2005, Booge et al. 2018). Bacteria were suggested to be the second most important cause of isoprene loss (Palmer & Shaw 2005). Reports have also suggested the importance of reactive oxygen species (ROS) and hydoxyl radicals (OH) in the aqueous phase, also important in atmospheric cycling of isoprene (Seinfeld & Pandis 2006, Wennberg et al. 2018, Berndt et al. 2019), although the turnover of these processes in the ocean has not been fully described (Riemer et al. 2000, Palmer & Shaw 2005, Zinser 2018). The role of isoprene-degrading microorganisms in marine ecosystems has yet to be quantified. Booge et al. (2018) emphasised that calculations of isoprene loss, aside from abiotic processes such as air−sea exchange, must include microbial metabolism. They reported rate-loss constants for microorganisms in equatorial and coastal regions of 0.01 and 0.1 d–1, respectively, indicating greater microbial activity in productive coastal zones, although total bacterial cell numbers did not significantly influence the rate of consumption (Booge et al. 2018). Factors that influence bacterial community composition must 92 Aquat Microb Ecol 87: 79–98, 2021 also be considered. Areas with a high abundance of grazers of haptophytes, a phytoplankton functional type which is also responsible for emission of isoprene (Table 2), may remove the correlation between bacterial abundance and local aquatic isoprene concentration (noted by Booge et al. 2018). Intertidal waters and sediments, including almost fresh, brackish and marine waters, were the subject of some of the first studies into the identification of aquatic isoprene-degraders. Isoprene-enriched microcosms from water in the Colne Estuary (UK) were dominated by Mycobacterium (approximately 90% relative abundance), with a small contribution by Ochrobacterium closer to the mouth of the estuary (Acuña Alvarez et al. 2009). Isoprene-enriched microcosms from sediments taken from the same locations were dominated by Rhodococcus spp. (approximately 65−70%), with smaller contributions by Actinosynnema (Acuña Alvarez et al. 2009). Isoprene-enriched sediments and waters from the Berre Lagoon (southern France) were both dominated by Rhodococcus spp. (80−93% relative abundance), unlike the Colne Estuary in which Rhodococcus spp. were only detected in the sediments (Acuña Alvarez et al. 2009). Isoprene-enriched taxa were linked to the function of isoprene degradation by DNA-SIP. For example, Actinobacteria were the most abundant isoprene-degrading bacteria from the Colne Estuary (Johnston et al. 2017). Members of this phylum have been linked to isoprene degradation in previous studies of freshwater (sediment and water), as well as terrestrial leaves and soils, with Rhodococcus spp. frequently dominating isoprene-enriched communities (Acuña Alvarez et al. 2009, Carrión et al. 2018, Crombie et al. 2018, Larke-Mejía et al. 2019). Rhodococcus sp. AD45, the basis for several studies of the mechanisms of isoprene degradation, was initially isolated from freshwater sediment (van Hylckama Vlieg et al. 1998). Isoprene-degrading bacteria have been enriched and isolated from marine ecosystems. Acuña Alvarez et al. (2009) reported that the microbial community of isoprene-enriched Indonesian seawater was dominated by Stappia spp. (62% relative abundance), with a significant contribution from Rhodococcus spp. (31% relative abundance). Stappia sp. Il2 was also isolated from seawater in the Western Channel, UK (Johnston et al. 2017), indicating the ability of isoprene-degraders from this genus to inhabit environmentally distinct marine ecosystems. Acuña Alvarez et al. (2009) also reported that isoprene produced by Dunaliella tertiolecta and Phaeodactylum tricornutum was consumed at environmentally relevant con- centrations, by a mixture of isoprene-degrading bacteria isolated from estuarine sources. They noted that these concentrations did not support the proliferation of these bacteria but that the cell density initially increased in response to isoprene exposure, suggesting that the nutritional versatility of isoprenedegraders was important to their lifestyle. This versatility was supported by Moore et al. (2020), who noted an increase in intracellular ATP in Pelagibacter sp. HTCC1062 (SAR11) when supplemented with isoprene. Interestingly, when co-cultured with Pelagibacter sp. HTCC1062 (SAR11), the isoprene-emitting Thalassiosira pseudonana displayed a 20% increase in carbon fixation due to VOC removal by the bacterium (Moore et al. 2020). Although few studies are available that demonstrate isoprene degradation by marine microbial communities, the role of microorganisms as a marine isoprene sink has already been suggested (Moore & Wang 2006, Davie-Martin et al. 2020). The isoprene oxidation capacity of microbes associated with corals has also not been tested, and so we measured isoprene degradation in serum bottles containing either ground coral fragments or bacterial cells filtered from aquarium seawater in which the corals were growing (Fig. 6). Compared with the sterile controls, biodegradation of isoprene was clearly demonstrated in both sample types. Irrespective of the incubation temperature, isoprene loss was significantly greater after 22 d in the ground-coral incubations than in incubations with planktonic cells concentrated onto filters (p < 0.01). Data were analysed for statistical significance by the repeated measures ANOVA. Thus, the isoprene-enriched microenvironment associated with the coral holobiont may facilitate the establishment of an isoprene-degrading community, resulting in the observed differences in degradation rates between coral fragments and planktonic bacterial cells. These findings also suggest that gross isoprene production by A. horrida is likely to be considerably higher than net production identified in Table 3 and, in the absence of isoprene-degrading microorganisms, that coralsourced isoprene could have a larger impact on atmospheric chemistry, particularly at a local scale. 12. FUTURE PERSPECTIVES Very few reports of isoprene emission and flux are available from aquatic ecosystems, particularly freshwater, adding to the uncertainty in estimates of global aquatic isoprene cycling. The disparity between top-down and bottom-up measurements of Dawson et al.: Aquatic cycling of isoprene Fig. 6. Microbial isoprene degradation in serum bottles (125 ml) containing ground Acropora horrida coral fragments or 0.22 μM filters containing aquarium water-associated bacterial cells (± SE). Following incubation at 27 or 29°C, coral fragments (0.43 ± 0.14 g, 27°C; 0.34 ± 0.07 g, 29°C) were ground into a course powder using a sterile pestle and mortar and added to serum bottles containing 10 ml of ONR7a seawater-nutrient medium (Dyksterhouse et al. 1995). Gasphase isoprene (100 μl), taken from the headspace of liquid isoprene (≥ 99% purity), was injected into each bottle. Aquarium water (300 ml), maintained at 27 or 29°C, was filtered through 0.22 μM filters to collect bacterial cells. A single filter was added to each serum bottle containing 10 ml of ONR7a seawater-nutrient medium and enriched with 100 μl of gas-phase isoprene. No-inoculum controls (n = 6) were prepared by injecting 100 μl of gas-phase isoprene into serum bottles containing 10 ml of ONR7a seawater-nutrient medium only. Serum bottles were incubated in the dark at 30°C. Headspace isoprene concentrations were measured using an ATI Unicam 610 Series gas chromatograph with a 1.5 m packed column (10% Apiezon L on CarboWax, 60−80 mesh) and a flame ionisation detector marine isoprene emissions demonstrates the need for an improved understanding of the factors controlling isoprene cycling (Arnold et al. 2009, Luo & Yu 2010, Hu et al. 2013). Exceptions to the typical diel cycle of isoprene emission must also be studied. Exton et al. (2012) identified 2 distinct peaks in isoprene concentration in the daytime and night-time in the Colne Estuary, and cyanobacteria and diatoms also retain a 93 portion of the daytime rate of isoprene emission while in the dark (Shaw et al. 2003, Ooki et al. 2019). The freshwater mixotroph, Chlorella vulgaris, produced isoprene at the same rate irrespective of whether it was grown as a photoautotroph under light or as a heterotroph feeding on exogenous glucose in the dark (Dani et al. 2020). It will be interesting to determine the contribution of mixotrophic marine protists to dark isoprene production, especially in the oligotrophic ocean. Moreover, some terrestrial heterotrophic bacteria produce isoprene (Kuzma et al. 1995), so their contribution to dark isoprene production in the aquatic environment merits investigation. There is a need for in situ measurements of isoprene emissions (Palmer & Shaw 2005, Conte et al. 2020) over a wider range of marine environments and time scales, and for an improved understanding of the difference in emission rates between different phytoplankton functional types (PFTs) (Booge et al. 2016, 2018, Dani et al. 2017, Conte et al. 2020). This requirement is evident when considering the differences in isoprene emission reported by Exton et al. (2013). Marine microalgae of the classes Chlorophyceae and Dinophyceae produced isoprene at rates of 0.05 and 0.86 μmol (g chl a) h−1, respectively, indicating that the local composition of the phytoplankton community can cause wide variations in emission rates (Exton et al. 2013, Booge et al. 2016). The need for a greater range of emission data from different phytoplankton functional types has been identified by recent studies (Booge et al. 2018, Conte et al. 2020). There is very little information available regarding the community composition or abundance of isoprenedegraders in marine ecosystems. Given the significant increases in dissolved and emitted isoprene during phytoplankton blooms (Moore & Wang 2006, Ooki et al. 2019, Davie-Martin et al. 2020, Tripathi et al. 2020), blooming events may trigger seasonal changes in microbial isoprene-degrading communities. Systematic surveys of these communities using functional gene probes would begin to identify the interactions between isoprene-emitting and isoprene-degrading microorganisms in aquatic ecosystems. Previous studies used specifically designed probes to target the conserved active site component of isoprene monooxygenase (isoA) in the isoprene metabolic pathway (El Khawand et al. 2016, Carrión et al. 2018). Thus far, only the 16S rRNA gene has been used to distinguish between isoprene-enriched microcosms from estuarine, freshwater, and marine waters and sediments (Acuña Alvarez et al. 2009). Studies using the 16S rRNA gene primarily identified members of the Actinobacteria and Alphaproteobacteria (Acuña Alvarez 94 Aquat Microb Ecol 87: 79–98, 2021 et al. 2009, Johnston et al. 2017). Further studies using isoA-specific probes are required, as this would begin to reveal the distribution, diversity, and abundance of isoprene-degraders in aquatic samples. The interface between aquatic and terrestrial ecosystems also requires further study, as isoprene emissions from terrestrial sources may influence aquatic isoprenedegrading bacteria, and vice versa. Inland aquatic ecosystems such as estuarine water and sediments, rivers, and also partially submerged vegetation such as mangrove swamps may provide valuable insights into the interplay between aquatic and terrestrial isoprene sources and sinks. Given that lakes constitute a large surface area in northern latitudes, their contribution to isoprene production merits further investigation, especially in areas of the Arctic where warming is most rapid. Additional isolates of aquatic isoprene-degrading bacteria are required in order to conduct further laboratory-based studies. At present, isoA serves as the basis for molecular probing of isoprene-degrading communities, but this does not account for alternative metabolic pathways. A recent study by Kronen et al. (2019) demonstrated anaerobic metabolism of isoprene, with isoprene acting as an electron acceptor, highlighting the need for a broader understanding of the diversity of isoprene-utilising microorganisms. The majority of isolated isoprene oxidisers are terrestrial, with the few aquatic isolates sharing the archetypal isoprene metabolic pathway first described in Rhodococcus sp. AD45 (van Hylckama Vlieg et al. 2000). Aside from bona fide isoprene-degraders, some SDIMO are able to catalyse the co-oxidation of isoprene to epoxyisoprene without its subsequent incorporation as a source of carbon or energy. Such SDIMO include the sMMO of Methylococcus capsulatus (Bath) (Colby et al. 1977, Green & Dalton 1989, Dawson et al. 2020) and the 4-component alkene monooxygenase of Xanthobacter autotrophicus Py2 (Small & Ensign 1997, Ensign 2001, Johnston et al. 2017). A recent study demonstrated the use of inhibitory linear alkynes to differentiate between isoprene oxidation by the bona fide isoprene-degrader Variovorax sp. WS11 and the methanotroph M. capsulatus (Bath), wherein acetylene specifically inhibited co-oxidation by the sMMO and octyne specifically inhibited the isoprene monooxygenase (Dawson et al. 2020). This approach could be used to interrogate the active isoprene-degrading communities of various environmental samples. Co-oxidation of isoprene may facilitate cross-feeding of unrelated microorganisms in aquatic ecosystems, which has frequently posed an issue for DNA-SIP studies (Mooshammer et al. 2021). Meta-metabolomics analysis would provide valuable insights into the contributions of isoprenedegrading and co-oxidising microorganisms. At the laboratory scale, this could begin with co-culture work similar to that described by Acuña Alvarez et al. (2009) and Moore et al. (2020). By using labelled CO2, the flow of carbon could be tracked between isoprene-emitting phytoplankton and isoprene-consuming organisms. Isoprene metabolism may not be limited to bacteria; to date there have been no reports of archaeal or fungal isoprene-degraders, although the abundance of the Zygomycota increased during incubations with isoprene in a soil study (Gray et al. 2015). On a broader scale, a combination of omics techniques performed on water and sediment from diverse locations would further clarify the biological underpinnings of isoprene cycling in aquatic environments. Acknowledgements. J.C.M., A.T.C. and R.A.D. were funded through a European Research Council Advanced Grant (IsoMET 694578) to J.C.M. We acknowledge funding through a Natural Environment Research Council grant to J.C.M. (NE/J009725/1) and T.J.M. (NE/J009555/1) and funding via the Earth and Life Systems Alliance (ELSA) at the University of East Anglia. We also acknowledge the award of a Leverhulme Trust Early Career Fellowship (ECF-2016-626) to A.T.C. We thank Russell Smart, Farid Benyahia and John Green for their technical support. LITERATURE CITED Acuña Alvarez L, Exton DA, Timmis KN, Suggett DJ, McGenity TJ (2009) Characterization of marine isoprenedegrading communities. 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W3152684551.txt	https://ejournals.epublishing.ekt.gr/index.php/psychology/article/download/23019/19508	en		Ο ρόλος της βίωσης θετικών συναισθημάτων στην ψυχοθεραπευτική διαδικασία: ανασκόπηση της θεωρητικής και ερευνητικής βιβλιογραφίας	Psychologia: to periodiko tīs Ellīnikīs Psychologikīs Etaireias/Psychologia. To periodiko tīs Ellīnikīs Psychologikīs Etaireias	2,020	cc-by-sa	12,105	Psychology: the Journal of the Hellenic Psychological Society Vol 23, No 1 (2018) Special Section - Emotions: Theory, research and applications The role of experiencing positive emotions in the psychotherapeutic process: a review of the theory and research literature Αναστάσιος Σταλίκας, Αλκμήνη Μπούτρη doi: 10.12681/psy_hps.23019 Copyright © 2018, Αναστάσιος Σταλίκας, Αλκμήνη Μπούτρη This work is licensed under a Creative Commons Attribution-ShareAlike 4.0. To cite this article: Σταλίκας Α., & Μπούτρη Α. (2018). The role of experiencing positive emotions in the psychotherapeutic process: a review of the theory and research literature. Psychology: The Journal of the Hellenic Psychological Society, 23(1), 70–91. https://doi.org/10.12681/psy_hps.23019 https://epublishing.ekt.gr \| e-Publisher: EKT \| Downloaded at: 21/05/2024 20:58:56 Powered by TCPDF (www.tcpdf.org) ΨΥΧΟΛΟΓΙΑ, 2018, 23 (1) ◆ 70-91 PSYCHOLOGY, 2018, 23 (1) ◆ 70-91 Ο ρόλος της βίωσης θετικών συναισθημάτων στην ψυχοθεραπευτική διαδικασία: ανασκόπηση της θεωρητικής και ερευνητικής βιβλιογραφίας Αναστάσιος Σταλίκας1 Αλκμήνη Μπούτρη1 Σε αυτό το άρθρο παρουσιάζονται οι βασικότερες θεωρητικές προτάσεις, μοντέλα και σχετικά ερευνητικά ευρήματα αναφορικά με τον ρόλο της βίωσης των θετικών συναισθημάτων στην ψυχοθεραπεία. Στο πρώτο μέρος παρουσιάζεται ο ρόλος της συναισθηματικής παρουσίας, έκφρασης, και βίωσης στη ψυχοθεραπεία και ο ρόλος του συναισθήματος στις διαφορετικές θεωρητικές προσεγγίσεις. Στο δεύτερο μέρος παρουσιάζονται ο ρόλος του χιούμορ, της χαράς και της αγάπης στη ψυχοθεραπευτική διαδικασία και στο τρίτο μέρος το μοντέλο της Διεύρυνσης και Δόμησης και η εφαρμογή του στη ψυχοθεραπευτική θεωρία και έρευνα. ΠερΙληψη Λέξεις Κλειδιά: Θετικά συναισθήματα, ψυχοθεραπεία Εισαγωγή Στην προσπάθεια κατανόησης της ανθρώπινης ύπαρξης και θεραπείας της ψυχικής ασθέ νειας, ο χώρος της ψυχοθεραπείας επικεντρώθηκε στην ανθρώπινη αδυναμία και την παθολογία. Αυτή η εστίαση επέφερε σημαντική πρόοδο στην ψυχοθεραπεία, οδήγησε στην ταξινόμηση της ψυχοπαθολογίας, την κατασκευή διαγνωστικών εργαλείων, την διαμόρφωση θεραπευτικών τεχνικών, παρεμβάσεων και μεθοδολογιών με αποτέλεσμα την ανάπτυξη και βελτίωση της ψυχοθεραπευτικής διαδικασίας και της αποτελεσματικότητάς της. Ιστορικά, στη μελέτη των συναισθηματικών διεργασιών στην ψυχοθεραπεία το επίκεντρο των ερευνητών αποτέλεσαν συναισθήματα αρνητικά, επώδυνα, απωθημένα, μπλοκαρισμένα, μη εκφρασμένα ή δυσλειτουργικά (Greenberg & Safran, 1987). Όλες οι διαφορετικές ψυχοθεραπευτικές προσεγγίσεις εστίασαν στην θεραπευτική διαχείριση των αρνητικών συναισθημάτων και εμπει ριών. Μέσα σε αυτό το πλαίσιο, η βίωση θετικών συναισθημάτων ήταν στο περιθώριο και συνήθως γινόταν αντιληπτή ως δείκτης θεραπευτικής αλλαγής, και όχι ως γενεσιουργός παράγοντας της αλλαγής. (Korman & Greenberg,1996). Οι θεραπευόμενοι νιώθουν χαρά μετά από μια περίοδο θλίψης, ενδιαφέρον καθώς ανακάμπτουν από την κατάθλιψη, περιέργεια μόλις διαχειρίζονται τον φόβο (Fitzpatrick & Stalikas, 2008). 1. Πάντειο Πανεπιστήμιο Κοινωνικών και Πολιτικών Επιστημών, Τμήμα Ψυχολογίας * Στοιχεία αλληλογραφίας: Α. Σταλίκας, Πάντειο Πανεπιστήμιο Κοινωνικών και Πολιτικών Επιστημών, Τμήμα Ψυχολογίας, Λ. Συγγρού 136, 17671, Αθήνα. Ηλ. Διεύθυνση: anstal@panteion.gr Ο ρόλος της βίωσης θετικών συναισθημάτων στην ψυχοθεραπευτική διαδικασία ◆ 71 O ταχέως αναπτυσσόμενος κλάδος της Θετικής Ψυχολογίας εντόπισε αυτήν την ανισορροπία ανάμεσα στην μελέτη του αρνητικού και του θετικού συναισθήματος και επανέφερε στο προσκήνιο την επιστημονική μελέτη της βέλτιστης ανθρώπινης λειτουργίας, στοχεύοντας στην ανακάλυψη και προώθηση των παραγόντων που επιτρέπουν στα άτομα και τις κοινωνίες να θριαμβεύουν (Seligman & Csikszentmihalyi, 2000). Όπως αναφέρει ο Seligman (1998, σελ.2) «η ψυχολογία δεν είναι μόνο η μελέτη της αδυναμίας και της ζημιάς, είναι επίσης η μελέτη της δύναμης και της αρετής. Η θεραπεία δεν είναι μόνο η επιδιόρθωση αυτού που έχει χαλάσει, είναι και η ανάπτυξη του καλύτερου που έχουμε μέσα μας.» Τα τελευταία 50 χρόνια διαφορετικοί κλάδοι της ψυχολογίας έχουν εκδηλώσει θεωρητικό και ερευνητικό ενδιαφέρον για την διερεύνηση του ρόλου των θετικών συναισθημάτων και των θετικών χαρακτηριστικών σε ποικίλους τομείς της ανθρώπινης ύπαρξης. Στο επίκεντρο βρίσκονται μεταβλητές όπως η ψυχολογική ανθεκτικότητα (Alvarez & Nemeny, 2001; Λακιώτη, 2011; Needles & Abramson, 1990; Tugade & Fredrickson, 2004; Waugh, Fredrickson, & Taylor, 2008), τα θετικά χαρακτηριστικά προσωπικότητας (Δημητριάδου & Ζύμνης, 2011; Mayne & Bonanno, 2001), οι θετικές καταστάσεις (Seligman, 2002a), τα θετικά συναισθήματα (Fredrickson, 2001, 2004; Fredrickson, Coffey, Cohn, & Finkel, 2008; Gloria & Steinhardt, 2016) και η σχέση τους με μια ποικιλία άλλων μεταβλητων, όπως είναι το στρες (Garland, Fredrickson, Kring, Johnson, Meyer, & Penn, 2010; Ong, Bergeman, Bisconti, & Wallace, 2006), η αντιμετώπιση προβλημάτων (Folkman & Moskowitz, 2000; Tugade, Fredrickson, & Barrett, 2004), η ικανοποίηση στη ζωή, την εργασία και τις διαπροσωπικές σχέσεις (Galanakis, Stalikas, Kallia, Karagianni, & Karela, 2009; Lizano & Barak, 2015; Σταλίκας & Μυτσκίδου, 2011; Waldinger, Schulz, Hauser, Allen, & Crowell, 2004;), η μακροβιότητα (Danner, Snowdon, & Friesen, 2001), η προσκόλληση (Feeney, 1999), η σωματική υγεία (Danner et al., 2001) και η εργασιακή εξουθένωση (Cameron, Dutton, & Quinn, 2003). Ο στόχος αυτού του άρθρου είναι η συστη- ματική και ενδελεχής παρουσίαση της θεωρητικής και ερευνητικής βιβλιογραφίας αναφορικά με το ρόλο και τη λειτουργία των θετικών συναισθημάτων στην ψυχοθεραπευτική διαδικασία. Σε ακολουθία με την γενικότερη αρχή του κλάδου της θετικής ψυχολογίας, η παρουσίαση αυτή πραγματοποιείται συμπληρωματικά με την υπάρχουσα ψυχοθεραπευτική βιβλιογραφία και επιχειρεί να προσθέσει και να διευρύνει την επίτευξη της θεραπευτικής αλλαγής. Λαμβάνοντας υπόψη ότι η κάθε ψυχοθεραπευτική προσέγγιση προσεγγίζει την ανθρώπινη φύση, ψυχική δομή, ψυχολογικές λειτουργίες, και την θεραπευτική πρακτική με διαφορετικό τρόπο, στάση και πρίσμα, είναι σαφές ότι η συνεισφορά των θετικών συναισθημάτων στην κλινική πράξη είναι διαφορετική για κάθε ψυχοθεραπευτική προσέγγιση.. Στο πρώτο μέρος του άρθρου πραγματοποιείται μια σύντομη ανασκόπηση της λειτουργίας των αρνητικών συναισθημάτων στις σημαντικότερες θεραπευτικές προσεγγίσεις και των αντίστοιχων ερευνητικών ευρημάτων. Στη συνέχεια παρουσιά ζονται τα αποτελέσματα ερευνών και οι θεωρητικές προτάσεις που έχουν διατυπωθεί από διαφορετικές ψυχοθεραπευτικές προσεγγίσεις για τα θετικά συναισθήματα. Τέλος, το άρθρο επικεντρώνεται στην θεωρία Διεύρυνσης και Δόμησης (Fredrickson, 1998, 2004) για τα θετικά συναισθήματα που προέρχεται από τον κλάδο της Θετικής Ψυχολογίας και στις ερευνητικές εφαρμογές της στην ψυχοθεραπευτική διαδικασία. Το συναίσθημα στην ψυχοθεραπευτική θεωρία Οι συναισθηματικές διεργασίες και διαδικα σίες κατέχουν κεντρική θέση στην ψυχοθεραπευτική θεωρία, έρευνα, και πράξη. Παρόλα αυτά, ο ορισμός, η λειτουργία, ο ρόλος και η εμπλοκή των συναισθημάτων στην ψυχοθεραπευτική αλλαγή, καθώς και ο τρόπος με τον οποίο ο θεραπευτής αξιοποιεί θεραπευτικά το συναισθηματικό βίωμα του πελάτη, όπως και η θέση που αποδίδεται στα συναισθήματα διαφέρουν σε κάθε ψυχοθεραπευτική προσέγγιση. Κάποιες προσεγγίσεις παρουσιάζουν ομοιότητες, κάποιες άλλες αλληλοσυ- 72 ◆ Αναστάσιος Σταλίκας και Αλκμήνη Μπούτρη μπληρώνονται, ενώ άλλες διαφέρουν σημαντικά. Παρότι διαφέρουν στη θεραπευτική αξία του συναισθήματος, όλες οι προσεγγίσεις κάνουν αναφορά στην θεραπευτική χρησιμότητα της βίωσης και έκφρασης συναισθήματος στην ψυχοθεραπευτική διαδικασία και συμφωνούν στο ότι η βίωση και έκφραση συναισθήματος συμβάλλει στην θεραπευτική αλλαγή, άλλοτε άμεσα, έχοντας από μόνη της θεραπευτική αξία, και άλλοτε έμμεσα, ως επικουρική διαδικασία. Η θεραπευτική αξία της βίωσης και έκφρασης συναισθήματος συνοψίζεται σε πέντε βασικούς άξονες (Σταλίκας και Μπούτρη, 2004): (α) το συναίσθημα ως εκφόρτιση, (β) το συναίσθημα ως μέσο ενόρασης, (γ) το συναίσθημα ως μέσο μάθησης, (δ) το συναίσθημα ως μέσο γνωστικής αναδόμησης, και (ε) το συναίσθημα ως προσαρμοστικός οδηγός. Το συναίσθημα ως εκφόρτιση: Μια βασική λειτουργία της βίωσης συναισθήματος είναι η καθαρτική του δράση. Ο πελάτης επικεντρώνεται και εκφράζει συναισθήματα που έχει απωθήσει, καταπιέσει, και αγνοήσει. Τα συναισθήματα αυτά εκφορτίζονται, η ενέργεια που κατασπαταλάται για την συγκράτηση τους απελευθερώνεται και οι έμμεσες παρενέργειες τους στη συμπεριφορά του ατόμου εξαλείφονται. Για κάποιες ψυχοθεραπευτικές προσεγγίσεις, η συναισθηματική εκφόρτιση αρχικά θεωρήθηκε επαρκής για την επαναφορά της ψυχικής ισορροπίας του ατόμου και αποτέλεσε αυτοσκοπό της θεραπευτικής διαδικασίας. Εντούτοις, οι νεώτερες προσεγγίσεις παρότι αποδέχονται την καθαρτική αξία της βίωσης και έκφρασης συναισθήματος, την θεωρούν ως το πρώτο βήμα, απαραίτητο για την εμφάνιση περαιτέρω θεραπευτικών φαινομένων, αλλά όχι θεραπευτική per se. Το συναίσθημα ως μέσο ενόρασης: Οι ψυχαναλυτικές, ψυχοδυναμικές και πελατοκεντρικές προσεγγίσεις εστιάζουν στην βίωση συναισθήματος ως μέσο για την εμφάνιση της ενόρασης. Εμβαθύνοντας στο συναισθηματικό του βίωμα, ο πελάτης ανακαλεί απωθημένα περιστατικά, γεγονότα, βιώματα, και συναισθήματα και τα αλληλοσυνδέει. Αυτή η εμβάθυνση, διερεύνηση, και εξερεύνηση καταλήγει σε μια βαθύτερη κατανόηση καταστάσεων και προσωπικών βιωμάτων, οδηγεί σε ενόραση, η οποία με την σειρά της προκαλεί μια διαφορετική αφήγηση της προσωπικής του ιστορίας και συντελεί στην θεραπευτική αλλαγή. Το συναίσθημα ως μέσο μάθησης: Η βίωση συναισθημάτων λειτουργεί και ως μέσο μάθησης. Στη συμπεριφορική παράδοση ο πελάτης εκτίθεται στα φοβογόνα ή αγχογόνα ερεθίσματα, προκειμένου να βιώσει στο μέγιστο βαθμό τα αντίστοιχα αρνητικά συναισθήματα, τα οποία τελικά είτε εκφορτίζονται μέσω της διαδικασίας απευαισθητοποίησης, είτε αντικαθίστανται από άλλα. Δηλαδή, ο πελάτης είτε αποβάλλει την αρνητική συναισθηματική αντίδραση μέσω της εξοικείωσης του οργανισμού, είτε μαθαίνει εναλλακτικές και περισσότερο λειτουργικές συμπεριφορές και αντιδράσεις. Το συναίσθημα ως μέσο γνωστικής αναδόμησης: Η γνωστική αναδόμηση είναι η διαδικασία κατά την οποία ο θεραπευτής αντικρούει τις γνωστικές διαστρεβλώσεις ή τις παράλογες πεποιθήσεις του θεραπευόμενου προκειμένου να τις αντικαταστήσει με άλλες πιο ρεαλιστικές και λειτουργικές (Ellis & Cramer, 1988). Για τις γνωστικές θεραπευτικές προσεγγίσεις, η βίωση συναισθήματος θεωρείται ως το πλέον αποτελεσματικό και άμεσο μέσο για την κατανόηση και τροποποίηση βαθύτερων γνωστικών δομών, συναισθηματικών - γνωστικών σχημάτων ή δυσλειτουργικών πρότυπων συμπεριφοράς. Η τροποποίηση αυτή αποτελεί μια γνωστική αναδόμηση που προκαλείται μέσω των έκδηλων αντιφάσεων που εγείρονται με την ανάδυση σχημάτων που οδηγούν σε γνωστική σύγκρουση. Η επίλυση αυτών των γνωστικών συγκρούσεων οδηγεί σε αρμονικότερη και γνωστικά αρτιότερη αντίληψη και κατανόηση του εαυτού και του περιβάλλοντος. Το συναίσθημα ως προσαρμοστικός οδηγός: Για τις υπαρξιακές προσεγγίσεις τα βαθύτερα και αυθεντικά συναισθηματικά βιώματα αποτελούν οδηγούς για δράση, καθώς εμπεριέχουν προσαρμοστικό χαρακτήρα. Για αυτές τις προσεγγίσεις, τα συναισθήματα περιγράφονται να διατρέχουν μια κυκλική πορεία, η οποία ολοκληρώνεται όταν συνδεθούν με τις ανάγκες του ατόμου και βρουν έκφραση μέσω της δράσης. Οι πελάτες παροτρύ- Ο ρόλος της βίωσης θετικών συναισθημάτων στην ψυχοθεραπευτική διαδικασία ◆ 73 νονται να αφεθούν στα πραγματικά αισθητηριακά και συναισθηματικά τους βιώματα, εκπαιδεύονται στον εντοπισμό τους και μαθαίνουν να τα εμπιστεύονται και να καθοδηγούνται από αυτά. Τα συναισθήματα στην ψυχοθεραπευτική έρευνα Ο ρόλος της βίωσης συναισθημάτων στην θεραπευτική συνεδρία αποτελεί αντικείμενο συστηματικής ερευνητικής μελέτης τα τελευταία πενήντα χρόνια (Greenberg & Safran, 1987). Έναυσμα των σχετικών ερευνών αποτέλεσε η πρόταση της ψυχαναλυτικής προσέγγισης αναφορικά με την έννοια της κάθαρσης, η θεραπευτική αξία της οποίας παρέμεινε για αρκετό καιρό στο ερευνητικό επίκεντρο. Παράλληλα, καθώς η έρευνα για τα συναισθήματα εξελίσσεται και η χρησιμότητα της διεξαγωγής ερευνών στην ψυχοθεραπευτική διαδικασία αναγνωρίζεται, το ενδιαφέρον για τη μελέτη των συναισθηματικών διεργασιών εξαπλώνεται σε όλες τις θεραπευτικές προσεγγίσεις. Πλέον, η διαμόρφωση θεωριών και θεραπευτικών μοντέλων ενημερώνεται από, και στηρίζεται στα, ερευνητικά δεδομένα, με αποτέλεσμα η συναισθηματική διέγερση, αναγνώριση και έκφραση να αναδεικνύεται σταδιακά ως θεμελιώδες συστατικό της ψυχοθεραπείας. Ο κύριος όγκος των ερευνών που μελετούν το συναίσθημα στην ψυχοθεραπεία επικεντρώνεται στα αρνητικά συναισθήματα (Greenberg & Paivio, 1998). Ιδιαίτερο ερευνητικό ενδιαφέρον έχουν προσελκύσει το συναίσθημα του θυμού, του άγχους, της θλίψης και του μετατραυματικού στρες. Τα ερευνητικά ερωτήματα αφορούν τον έλεγχο, τη μείωση ή την εξάλειψη αυτών των αρνητικών συναισθημάτων. Σε γενικές γραμμές, η επαφή με το συναίσθημα και η αποδοχή των συναισθηματικών εμπειριών, όσο επίπονη ή δυσάρεστη και εάν είναι, θεωρείται επωφελής για την ψυχική και σωματική υγεία του ατόμου. Αντίθετα, η αποφυγή των δύσκολων συναισθημάτων και ο αποκλεισμός τους από το άμεσο βίωμα θεωρείται επιβλαβής. Τα ερευνητικά ευρήματα στηρίζουν την αναγκαιότητα της συναισθηματικής εμπλοκής του πελάτη στην ψυχοθεραπευτική διαδικασία για την επίτευξη της θεραπευτικής αλλαγής. Έρευνες συνδέουν την έκφραση και κατανόηση των συναισθημάτων με την τελική θετική θεραπευτική έκβαση, την επίλυση ανοιχτών υποθέσεων, την διευκόλυνση της αλλαγής της συμπεριφοράς, την εκτόνωση συσσωρευμένης εσωτερικής έντασης, την ρύθμιση του συναισθήματος, καθώς και την άμεση συναισθηματική ανακούφιση και αλλαγή διάθεσης (Bohart, 1980; Greenberg, 2008; Greenberg & Foerster, 1996; Greenberg & Safran, 1989; Lewis & Butcher, 1992; Mackay, Barkham & Stiles, 1998; Mahrer, White, Howard, & Lee, 1991; Paulson, Truscott & Stuart, 1999; Prochaska, 1999; Stalikas & Fitzpatrick, 2008; Watson, 1996; Young & Bemak, 1998). Έρευνες που μελετούν τη σχέση της έκφρασης δυνατού συναισθήματος και συγκεκριμένων ψυχοθεραπευτικών εννοιών συμπεραίνουν ότι η έκφραση δυνατού συναισθήματος κατά την ψυχοθεραπευτική διαδικασία διευκολύνει την εμφάνιση θεραπευτικών φαινομένων, όπως είναι η ενόραση, η δημιουργία νοήματος, οι καλές ενδό-συνεδριακές στιγμές και η αποδοχή των ερμηνειών του θεραπευτή (Clarke, 1989; Clarke, 1996; Fitzpatrick, Peternelli, Stalikas, & Iwakabe, 1999; Fitzpatrick & Stalikas,1995, 2008; Mergenthaler, 1996; Sexton, 1996; Young & Bemak, 1998). Αντίστοιχα, εμφανίζεται αυξημένο ενδιαφέρον για τον εντοπισμό εκείνων των θεραπευτικών παρεμβάσεων που προωθούν τις συναισθηματικές διεργασίες (Iwakabe, Rogan, & Stalikas, 2000; Mahrer, 1989), και για την κατασκευή κατάλληλων ψυχομετρικών εργαλείων καταγραφής συναισθηματικών φαινομένων και λειτουργιών (Klein, Mathieu-Coughlan, & Keller, 1970; Mahrer, Stalikas, Boissoneault, Trainor, & Pilloud, 1990). Τα θετικά συναισθήματα στην ψυχοθεραπεία Στο επίκεντρο της ενασχόλησης θεωρητικών και ερευνητών αναφορικά με τις συναισθηματικές διεργασίες του θεραπευόμενου βρίσκεται η προσπάθεια κατανόησης και ψυχοθεραπευτικής επεξεργασίας των αρνητικών συναισθημάτων (Greenberg & Safran, 1987). Μέσα σε αυτό το πλαίσιο, η βίωση θετικών συναισθημάτων συνή- 74 ◆ Αναστάσιος Σταλίκας και Αλκμήνη Μπούτρη θως γίνεται αντιληπτή ως δείκτης θεραπευτικής αλλαγής και όχι ως γενεσιουργός παράγοντας αυτής (Korman & Greenberg,1996; Fitzpatrick & Stalikas, 2008). Τόσο οι θεωρητικές διατυπώσεις όσο και τα ερευνητικά ευρήματα αναφορικά με τα θετικά συναισθήματα ως μέσο ψυχοθεραπευτικής αλλαγής παραμένουν περιορισμένα, αποσπασματικά και απομακρυσμένα από το επίκεντρο της θεωρίας και έρευνας. Παρόλα αυτά, υπάρχουν κάποιες προτάσεις σύμφωνα με τις οποίες η βίω ση θετικών συναισθημάτων αποτελεί βασικό συστατικό της θεραπευτικής αλλαγής. Παράλληλα εμφανίζεται ένας σταδιακά αυξανόμενος αριθμός ερευνών, προσφάτων στην πλειοψηφία τους, που αποπειράται να μελετήσει τον ρόλο των θετικών συναισθημάτων στην ψυχοθεραπευτική διαδικασία. Όπως συμβαίνει και με την αξιοποίηση των αρνητικών συναισθημάτων, τόσο η αναγνώριση των επιμέρους θετικών συναισθημάτων όσο και η χρήση τους εξαρτώνται από το θεωρητικό μοντέλο που ακολουθείται στο διαφορετικό πλαίσιο. Στη συνέχεια συνοψίζονται οι θεωρητικές προτάσεις και τα ερευνητικά ευρήματα που αφορούν τη σχέση ανάμεσα στα θετικά συναισθήματα και την ψυχοθεραπευτική αλλαγή. Το χιούμορ και το γέλιο στην ψυχοθεραπεία Η χρήση του χιούμορ από την πλευρά του θεραπευτή και η πρόκληση γέλιου στον πελάτη είναι ίσως η περισσότερο μελετημένη μεταβλητή στον χώρο της ψυχοθεραπείας. Η πλειοψηφία της βιβλιογραφίας, παρότι συχνά αντιφατική, τείνει στο συμπέρασμα ότι το χιούμορ μπορεί να έχει θεραπευτική αξία, αρκεί να χρησιμοποιηθεί κατάλληλα ώστε να μην εκληφθεί από τον πελάτη ως επιθετικότητα ή λειτουργήσει ανασταλτικά και ενισχύσει τις άμυνες του. Ο βασικότερος λόγος αυτής της διχογνωμίας οφείλεται στις διαφορετικές θεωρήσεις αναφορικά με τον ψυχισμό και την πορεία της ψυχοθεραπευτικής διαδικασίας. Ένας από τους πρώτους θεραπευτές που τονίζει την θεραπευτική αξία του χιούμορ στην ψυχοθεραπεία είναι ο Albert Ellis (1977). Σύμφωνα με τον Ellis η ανθρώπινη δυστυχία συχνά οφείλεται στην υπερβολική αξία και σοβαρότητα που προσδίδει το άτομο σε διάφορες καταστάσεις. Έτσι η προσέγγιση αυτών των καταστάσεων μέσα από ένα σαρκαστικό ή αυτοσαρκαστικό πλαίσιο μέσω του χιούμορ και της υπερβολής αποτελεί μια θεραπευτική μέθοδο προκειμένου να έρθουν αντιμέτωποι οι πελάτες με την υπερβολική ανησυχία και υπερβολή τους. Από την δεκαετία του 1960, ο Ellis αναγνωρίζει την θεραπευτική αξία της χρήσης του χιούμορ και εστιάζει σε τρία επίπεδα: στο γνωστικό, στο συναισθηματικό και στο συμπεριφορικό. Στο γνωστικό επίπεδο παρουσιάζει με άμεσο τρόπο σε έναν αρχικά άκαμπτο πελάτη νέες ιδέες και προοπτικές, προσφέροντας μια γνωστική αναπλαισίωση. Στο συναισθηματικό επίπεδο προκαλεί απόλαυση και χαρά, προτείνοντας μια σαρκαστική ή καυστική παρουσίαση της κατάστασης, εισβάλλοντας στην αρνητική διάθεση και τον ψυχικό πόνο του πελάτη, χρησιμοποιώντας το χιούμορ ως αντίδοτο στην θλίψη του. Στο συμπεριφορικό επίπεδο το χιούμορ αποτελεί από μόνο του ένα εργαλείο χαλάρωσης, ενώ προωθεί και την υιοθέτηση νέων και ανατρεπτικών –σε σχέση με τις υπάρχουσες– συμπεριφορών. Παράλληλα, την ίδια χρονική περίοδο μια σειρά από θεραπευτές εισάγουν τεχνικές επικεντρωμένες στο χιούμορ, στην υπερβολή ή τον αυτοσαρκασμό. Ανάμεσα τους ο Frankl (1960) δημιουργεί την τεχνική της «παράδοξης πρόθεσης» κατά την οποία οι πελάτες καλούνταν να προσεγγίσουν τα συμπτώματα τους με υπερβολή και να τα περιγράψουν με τα μελανότερα χρώματα. Ο στόχος αυτής της τεχνικής είναι οι πελάτες –μέσα από την υπερβολή και την ακραία περιγραφή των συμπτωμάτων τους- να αναπτύξουν την ικανότητα αποστασιοποίησης από αυτά, να γελούν με τις νευρωσικές τους αντιδράσεις, και να απομυθοποιήσουν με αυτόν τον τρόπο τα νευρωσικά συμπτώματά τους. Αντίστοιχα, ο Greenwald (1975) χρησιμοποιεί την αντανάκλαση και την καυστική υπερβολή ως μέσο για την αναγνώριση από την μεριά του πελάτη του παράλογου ή γελοίου χαρακτήρα της περιγραφής των προβλημάτων τους στοχεύοντας στην διερεύνηση νέων και διαφορετικών προσεγγίσεων και επιλογών. Ο Grotjahn (1970) χρησιμοποιεί αστεία κατά την διάρκεια της θεραπείας αποτελώντας Ο ρόλος της βίωσης θετικών συναισθημάτων στην ψυχοθεραπευτική διαδικασία ◆ 75 έτσι πρότυπο για τους πελάτες, οι οποίοι μέσω της ταύτισης με τον θεραπευτή μπορούν να αποκτήσουν μια αντίστοιχη συναισθηματική ελευθερία, απόσταση και ευελιξία στην σκέψη τους, και στην κατάρριψη των αμυνών τους. Στη δεκαετία του 1980, οι Farrelly και Matthews (1981) διαμορφώνουν το θεραπευτικό μοντέλο της Προκλητικής Ψυχοθεραπείας (Provocative Therapy). Σύμφωνα με τις αρχές του μοντέλου η θεραπεία θεωρείται αποτελεσματικότερη όταν επικεντρώνεται στο εδώ-και-τώρα, οι πελάτες –ανεξάρτητα από την διάρκεια και την ένταση της ψυχοπαθολογίας τους– θεωρούνται λιγότερο εύθραυστοι και με μεγαλύτερες δυνατότητες αλλαγής από ό,τι πιστεύουν οι θεραπευτές. Η άμεση πρόκληση των δυσλειτουργικών πεποιθήσεων, συναισθημάτων και σκέψεων του πελάτη οδηγεί στην υιοθέτηση των αντίθετων –δηλαδή λειτουργικών- σκέψεων, συναισθημάτων και συμπεριφορών. Μέσα σε αυτό το θεωρητικό πλαίσιο η χρήση του χιούμορ από τον θεραπευτή αποτελεί βασική τεχνική πρόκλησης του πελάτη. Ο στόχος είναι η διακωμώδηση των δυσκολιών του πελάτη μέσα από την υπερβολή, τον σαρκασμό, τη μίμηση και το μαύρο χιούμορ. Ο θεραπευτής παρουσιάζει μια καρικατούρα των συμπτωμάτων του πελάτη και αυτός βλέποντας μπροστά του τους χειρότερους του φόβους να ζωντανεύουν υποχρεώνεται να εγκαταλείψει τις άμυνές του. Οι Mahrer και Gervaize (1984), μελετώντας τη σχετική βιβλιογραφία καταλήγουν στο συμπέρασμα ότι το έντονο γέλιο κατά την διάρκεια της συνεδρίας σχετίζεται με την αποτελεσματική θεραπευτική διαδικασία τουλάχιστον με τέσσερις τρόπους: (α) προκαλεί μια σημαντική βελτίωση της αυτοεικόνας του πελάτη, (β) διευκολύνει την βίωση μιας βελτιωμένης ψυχολογικής κατάστασης που χαρακτηρίζεται από ενέργεια, επίγνωση και ανοιχτή διάθεση, (γ) αποτελεί ένδειξη καλής θεραπευτικής σχέσης καθώς οδηγεί σε ζεστασιά, αποδοχή, οικειότητα και μείωση της συναισθηματικής απόστασης και, (δ) αποτελεί δείκτη θεραπευτικής αλλαγής καθώς φανερώνει έντονη συναισθηματική εμπλοκή, βιωματική διεργασία, και καθαρκτική λειτουργία. Αναφορικά με το είδος του χιούμορ που προκαλεί το «θεραπευτικό» γέλιο, αναγνω- ρίζουν την χρήση του χιούμορ που σχετίζεται με την υιοθέτηση μιας θετικότερης περιγραφής της ζωής, με τον σαρκασμό και την υπερβολή των προβλημάτων και των συμπεριφορών των πελατών, και με μια αυθόρμητη περιπαιχτική ή σατυρική διάθεση που υποδηλώνει τον παραλογισμό της κατάστασης του πελάτη. Επιπλέον, οι Mahrer και Gervaize (1984) στηριζόμενοι στα αποτελέσματα της βιβλιογραφικής τους έρευνας προτείνουν οχτώ θεραπευτικές παρεμβάσεις με τις οποίες ο θεραπευτής μπορεί να προκαλέσει έντονο γέλιο: την κατευθυνόμενη αναγνώριση κάποιας συμπεριφοράς ρίσκου, τον ορισμό της συμπεριφοράς ρίσκου από τον πελάτη ή άλλους, την «γελοιοποιημένη» περιγραφή της συμπεριφοράς ρίσκου από τον πελάτη, την καθοδήγηση στην πραγματοποίηση έντονων συναισθηματικών συμπεριφορών, την πραγματοποίηση συμπεριφορών ρίσκου για τον πελάτη, την λήψη ρίσκου στην εκδήλωση μιας νέας συμπεριφοράς, την έντονη απόλαυση μέσα από την εκδήλωση της νέας συμπεριφοράς, και την εκδήλωση μιας νέας συμπεριφοράς προς τον θεραπευτή. Σε μεταγενέστερη έρευνα τους οι Gervaize, Mahrer, & Markow, (1985) μελετούν τις θεραπευτικές παρεμβάσεις που είχαν προτείνει οι Mahrer και Gervaise (1984). Τα αποτελέσματα τους δείχνουν ότι το έντονο γέλιο προκύπτει: (α) όταν ο πελάτης προσπαθεί να εκφράσει μια νέα συμπεριφορά ρίσκου, η οποία όμως είναι μπλοκαρισμένη, (β) όταν ο θεραπευτής αναγνωρίζει και αποδέχεται την εκδήλωση αυτής της συμπεριφοράς, και (γ) όταν ο θεραπευτής ενθαρρύνει την εκδήλωση αυτής της συμπεριφοράς μέσα από την υπερβολή και την έντονη συναισθηματική διέγερση στο πλαίσιο της θεραπευτικής σχέσης. Αργότερα, οι Falk και Hill (1992) αναπαράγουν την παραπάνω έρευνα, αλλά στα αποτελέσματα τους δεν βρίσκουν συσχετισμό του γέλιου με την εκδήλωση συμπεριφοράς ρίσκου. Μια ερμηνεία που δίνουν οι ερευνητές είναι ότι τα δεδομένα τους προέρχονταν από ποικίλες θεραπευτικές προσεγγίσεις, ελάχιστες από τις οποίες προτρέπουν τον πελάτη στην εκδήλωση συμπεριφοράς ρίσκου στο εδώ-και-τώρα. Τα δικά τους δεδομένα αναδεικνύουν το δυνατό γέλιο από τον πελάτη κυρίως ως αποτέλεσμα τεχνικών 76 ◆ Αναστάσιος Σταλίκας και Αλκμήνη Μπούτρη που σχετίζονται με την εκτόνωση της έντασης. Οι ερευνήτριες προτείνουν την χρήση του χιούμορ από τους θεραπευτές υπό προϋποθέσεις, όπως είναι η ύπαρξη μιας καλής θεραπευτικής σχέσης, η κατάλληλη ταυτόχρονη μη-λεκτική επικοινωνία, αλλά και προσαρμογή του χιούμορ κατά περίπτωση καθώς δεν θεωρούν ότι το χιούμορ είναι εξίσου θεραπευτικό για όλους τους πελάτες. Αρκετοί θεωρητικοί έχουν αναφερθεί στην θεραπευτική δύναμη του χιούμορ σε διαφορετικές ψυχοθεραπευτικές προσεγγίσεις, όπως είναι η συμπεριφορική (Lamb, 1980; Smith, 1973; Ventis, 1973) η Αντλεριανή (Corey, 1986), η Ψυχαναλυτική (Christie, 1994; Mann, 1991), και η Δομική θεραπεία (Furman & Ahola, 1988). Για τον O’Connell (1981) το χιούμορ αποτελεί «την βασιλική οδό για την αυτοπραγμάτωση». Ο ίδιος προτείνει την χρήση του σε μια σειρά από θεραπευτικές τεχνικές, όπως η άδεια καρέκλα, η κατευθυνόμενη φαντασίωση, οι ασκήσεις χαλάρωσης και ελέγχου αναπνοής, το παιχνίδι ρόλων, κ.ο.κ. Επιπλέον, δημιουργεί μια νέα τεχνική ψυχοδράματος, το «χιούμορ-δράμα», όπου κατά την διάρκεια του ψυχοδράματος τα μέλη της ομάδας καλούνται να χρησιμοποιήσουν τεχνικές διακωμώδησης της εκάστοτε συμπεριφοράς ή σκέψης. Στόχος της τεχνικής αυτής είναι η εκμάθηση της χρήσης του χιούμορ για την αντιμετώπιση στρεσσογόνων καταστάσεων (Nezu, Nezu & Blissett, 1988). Ο Kuhlman (1980) υποστηρίζει την θεραπευτική αξία του χιούμορ τόσο βραχυπρόθεσμα –στο εδώ-και-τώρα της συνεδρίας-, όσο και μακροπρόθεσμα στην τελική θεραπευτική αλλαγή. Παράλληλα, τονίζει την ανάγκη για προσεκτική εφαρμογή του χιούμορ ανάλογα με την περίσταση, προκειμένου να μην αποτελεί συγκαλυμμένη επιθετικότητα από την πλευρά του θεραπευτή, αλλά και να μην λειτουργεί ενισχυτικά στις άμυνες του πελάτη, οδηγώντας τον σε αποστασιοποίηση από το πρόβλημά του. Στον αντίποδα των υποστηρικτών της χρήσης χιούμορ βρίσκεται ο Kubie (1971), ο οποίος υπογραμμίζει τις καταστρεπτικές επιπτώσεις που αυτό μπορεί να έχει στην ψυχοθεραπευτική διαδικασία. Υποστηρίζει ότι το χιούμορ μπορεί να αποτελεί καλυμμένη επιθετικότητα, να εκληφθεί από τον πελάτη ως ψυχρότητα και αδιαφορία και να τον αποπροσανατολίσει από την θεραπευτική διαδικασία, ειδικά σε ένα ψυχαναλυτικό μοντέλο ελεύθερου συνειρμού. Ο θεραπευτής μπορεί με την χρήση του χιούμορ να επιδεικνύεται και να χάνει την αντικειμενικότητά του ως προς την διαδικασία, ενώ ιδιαίτερη προσοχή χρήζουν οι περιπτώσεις όπου ο πελάτης έχει υποστεί σκληρούς αστεϊσμούς στην πρώιμη παιδική ηλικία. Τη μειωμένη θεραπευτική αξία του χιούμορ υποστηρίζουν και οι Golan, Rosenhein, & Jaffe (1988), οι οποίοι ερεύνησαν την αντίδραση 60 καταθλιπτικών, ιδεοψυχαναγκαστικών και υστερικών πελατών στις χιουμοριστικές παρεμβάσεις των θεραπευτών τους. Τα αποτελέσματα έδειξαν ότι η πλειοψηφία των πελατών προτιμούσε τις μη-χιουμοριστικές παρεμβάσεις, με την προτίμηση αυτή να μεσολαβείται από την αλληλεπίδραση των χαρακτηριστικών της προσωπικότητας και του είδους του χιούμορ. Τέλος, ο Saper (1987), κάνοντας μια ανασκόπηση των ερευνών της χρήσης του χιούμορ στην ψυχοθεραπεία την δεκαπενταετία 1972-1987, αναφέρει μια σειρά από διδακτορικές διατριβές που εστιάζουν στο θέμα αυτό (Buckman, 1980; Burbridge, 1978; Golub, 1979; Kaneko, 1971; Labrentz, 1973; Peterson, 1980; Schienberg, 1979). Σύμφωνα με τον συγγραφέα τα αποτελέσματα των περίπου είκοσι διατριβών δεν παρέχουν επαρκείς ενδείξεις ως προς την θεραπευτική αξία του χιούμορ, ενώ υπογραμμίζει και τις μεθοδολογικές αδυναμίες των ερευνητικών σχεδιασμών. Η χαρά στην ψυχοθεραπεία Ελάχιστα έχουν ειπωθεί στην βιβλιογραφία της ψυχοθεραπευτικής διαδικασίας για τον ρόλο της χαράς στην θεραπευτική αλλαγή. Οι περισσότερες αναφορές εστιάζουν στο συναίσθημα της χαράς ως συναίσθημα που συνοδεύει το θετικό αποτέλεσμα της θεραπευτικής αλλαγής και ως επιθυμητό στόχο της ψυχοθεραπείας (Howard DiGangi, & Johnson,1988; Misrahi, 1986; Sachs, 1989; Stones, Ivany, & Kosma,1994; Vianu, 1986). Οι Greenberg και Paivio (1998), αναφέρονται στα συναισθήματα της χαράς και του ενδιαφέρο- Ο ρόλος της βίωσης θετικών συναισθημάτων στην ψυχοθεραπευτική διαδικασία ◆ 77 ντος, δίνοντας μια σύντομη περιγραφή των ψυχολογικών δυσλειτουργιών και των θεραπευτικών παρεμβάσεων που σχετίζονται με αυτά. Αναφέρουν ότι σε περιπτώσεις κατάθλιψης, αγχώδους διαταραχής ή μετατραυματικού στρες παρατηρείται μειωμένη παρουσία ή απόλυτη απουσία αυτών των συναισθημάτων. Επιπλέον, σε πελάτες με εξαρτήσεις σε ουσίες, το συναίσθημα της χαράς που βιώνουν τα άτομα με την παράλληλη μείωση των αρνητικών συναισθημάτων, όπως είναι ο φόβος, η ντροπή ή το άγχος μέσω της εξαρτητικής ουσίας, λειτουργεί ενισχυτικά για την εξάρτηση. Σε ό,τι αφορά την θεραπευτική διαδικασία, οι συγγραφείς τονίζουν το ρόλο τον συναισθημάτων της χαράς και του ενδιαφέροντος στην διαμόρφωση θεραπευτικών στόχων. Οι πελάτες ενημερώνονται από αυτά τα συναισθήματα προκειμένου να θέσουν στόχους και να προβούν σε αντίστοιχες πράξεις. Ο θεραπευτής μπορεί να βοηθήσει τον πελάτη να έρθει σε επαφή με αναμνήσεις που σχετίζονται με αυτά τα συναισθήματα προκειμένου να ενθαρρυνθεί και να κινητοποιηθεί. Αναφέρουν τρεις περιπτώσεις, όπου οι θεραπευτές εστιάζουν συνήθως σε αυτά τα συναισθήματα: (α) στις αρχικές συνεδρίες, όπου ο πελάτης νιώθει την ανάγκη να έρθει σε επαφή με τα κομμάτια του εαυτού που βίωναν τα θετικά συναισθήματα, (β) κατά την διάρκεια της θεραπείας, όταν αναδύεται στιγμιαία ένα θετικό συναίσθημα και ο θεραπευτής το αναγνωρίζει και του δίνει χώρο να εκφραστεί στο παρόν, και (γ) ως αποτέλεσμα της θεραπευτικής αλλαγής. Οι Shura και Sidney (1990) προτείνουν ένα θεραπευτικό μοντέλο για την ομαδική θεραπεία με άτομα της τρίτης ηλικίας, το οποίο εστιάζει στην διαμόρφωση χαρούμενων ομαδικών εμπειριών. Για το σκοπό αυτό περιγράφουν δέκα πτυχές του συναισθήματος της χαράς που μπορούν να συντελέσουν στην διαμόρφωση θεραπευτικής ατμόσφαιρας. Οι δέκα πτυχές είναι: (1) η εύρεση του εαυτού, (2) οι σχέσεις μεταξύ των μελών μέσω του μοιράσματος, (3) η αγάπη μεταξύ των μελών, (4) η αυτοπραγμάτωση μέσα από την ενδυνάμωση και την αίσθηση ελέγχου του εαυτού, (5) η παροχή και αποδοχή βοήθειας, (6) το άγγιγμα, (7) η ανακάλυψη μέσω της ενόρασης, (8) η δημιουργικό- τητα, (9) το γέλιο, και (10) το ευ-ζην. Οι συγγραφείς τονίζουν ότι παράλληλα με την προσπάθεια δημιουργίας χαρούμενου ομαδικού κλίματος, οι θεραπευτές πρέπει να δίνουν επαρκή χώρο και στα αρνητικά συναισθήματα που αναδύονται στην ομάδα. Αντίστοιχα, ο Boorstein (1980) προτείνει μια εύθυμη και ανάλαφρη προσέγγιση στην ψυχοθεραπευτική διαδικασία και τονίζει την θεραπευτική αξία ενός θεραπευτή και ενός θεραπευτικού περιβάλλοντος, που είναι χαλαρό και πρόσχαρο. Τεχνικές που προτείνει είναι: (1) η αποδραματοποίηση της σημαντικότητας μιας κατάστασης κρίσης στη ζωή του πελάτη θέτοντας το ευρύτερο πλαίσιο του κοσμικού δράματος, (2) η χρήση διδακτικών ιστοριών από πνευματικές παραδόσεις, (3) η έμφαση στην ιδέα ότι οι άνθρωποι δεν είναι θύματα του παρελθόντος αλλά έχουν επιλογή στις εμπειρίες τις οποίες θα επιλέξουν να κρατήσουν στη ζωή τους, (4) η χρήση του διαλογισμού και η μη-κριτική παρακολούθηση των γεγονότων της ζωής τους, και (5) η προτροπή αντιμετώπισης των αγχωδών καταστάσεων ως προκλήσεων και όχι ως τραυματικών καταστάσεων κρίσης. Οι Beskow, Beskow, και Miro (2004), περιγράφουν τον κεντρικό ρόλο που κατέχει η ευθυμία στην διαμόρφωση σχέσεων, στην κινητοποίηση για αλλαγή, και στην δημιουργία συνείδησης. Οι συγγραφείς αναφέρονται στην ευθυμία ως την πληθώρα των πράξεων μέσω των οποίων ένα άλλο άτομο αναδύεται ως ικανό για δημιουργία σχέσης, με αποτέλεσμα την εμφάνιση τoυ συναισθήματος της χαράς. Η χαρά με τη σειρά της ασκεί σημαντικότατη επίδραση στην θέληση του ατόμου για αλλαγή. Κατά συνέπεια, η ψυχοθεραπευτική συνεργασία, η οποία στηρίζεται στην ισότητα, στην αμοιβαία αποδοχή και εμπιστοσύνη, μέσω του συναισθήματος χαράς που προκαλεί, δημιουργεί ένα κλίμα που ευνοεί την ποικιλομορφία, την δημιουργικότητα και τη δυνατότητα προσωπικής ανάπτυξης. Οι Stones, Ivany, και Kozma (1994), διερευνούν τη σχέση ανάμεσα στο βασικό επίπεδο βιώματος του συναισθήματος της χαράς και την διάρκεια παραμονής σε θεραπευτική παρέμβαση επικεντρωμένη στις αναμνήσεις. Οι συμμετέ- 78 ◆ Αναστάσιος Σταλίκας και Αλκμήνη Μπούτρη χοντες είναι δεκαεννέα εθελοντές που διαμένουν σε διαμερίσματα για άτομα της τρίτης ηλικίας, και χωρίζονται σε ομάδες ανάλογα με το χρονικό διάστημα παραμονής τους στην θεραπευτική παρέμβαση. Τα αποτελέσματα δείχνουν στατιστικά σημαντική σχέση ανάμεσα στο βασικό επίπεδο χαράς και στην παραμονή των συμμετεχόντων στην θεραπευτική παρέμβαση. Συνοψίζοντας, το συναίσθημα της χαράς ως θεραπευτικός παράγοντας συσχετίζεται κυρίως με την κινητοποίηση για στοχοθεσία και θεραπευτική αλλαγή, την διαμόρφωση ενός ευνοϊκού θεραπευτικού κλίματος, την αναγνώριση και ενίσχυση του συναισθήματος προκειμένου να ενθαρρυνθεί ο πελάτης, και με το τελικό θεραπευτικό αποτέλεσμα. Η αγάπη στην ψυχοθεραπεία Ο ρόλος του συναισθήματος της αγάπης στην ψυχοθεραπεία αποτελεί κεντρικό θέμα θεωρητικού διαλόγου στις ψυχαναλυτικές προσεγγίσεις. Το συναίσθημα της αγάπης συχνά συνδέεται στην ψυχαναλυτική βιβλιογραφία με την επιθυμία, τον έρωτα και την σεξουαλική επαφή, πραγματική ή φαντασιωσική (Mitchell & Black, 1995). Άλλες σχολές που πραγματεύονται ζητήματα αγάπης στην ψυχοθεραπεία είναι η συστημική θεραπεία, η θεραπεία ομάδας και η θεραπεία Gestalt, όπου η αγάπη συνδέεται με κεντρικές έννοιες των ανθρωπιστικών προσεγγίσεων, όπως είναι η άνευ όρων αποδοχή και η ενσυναίσθηση (Jarosewitsch, 1997. Shamasundar, 2001. Snyder, 2000). Στην σχετική βιβλιογραφία διακρίνονται τρεις βασικές θεματικές: (α) τα συναισθήματα αγάπης από τον πελάτη προς τον θεραπευτή στα πλαίσια της μεταβίβασης, (β) τα συναισθήματα αγάπης από τον θεραπευτή προς τον πελάτη, στα πλαίσια της αντιμεταβίβασης, και (γ) η ανάπτυξη της ικανότητας του πελάτη για αγάπη μέσα από μια ψυχοθεραπευτική σχέση αμοιβαίας αγάπης. Η έκφραση αγάπης από την πλευρά του θεραπευόμενου προς τον θεραπευτή σχετίζεται κατά κύριο λόγο με τα βιώματα του, τόσο στην πρώιμη παιδική ηλικία όσο και μεταγενέστερα. Ιδιαίτερη έμφαση δίνεται στην ερμηνεία αυτών των συναισθημάτων, τη σύνδεσή τους με τους σημαντικούς άλλους στη ζωή του θεραπευόμενου, και την θεραπευτική αξία της σωστής διαχείρισης αυτών των συναισθημάτων. Τόσο η κλασσική ψυχανάλυση, όσο και η σχολή των αντικειμενοτρόπων σχέσεων, ασχολούνται εκτενώς με το θέμα στο πλαίσιο της μεταβίβασης και της προβολής στο πρόσωπο του θεραπευτή συναισθημάτων που απευθύνονται σε σημαντικούς άλλους. Μέσα σε αυτό το πλαίσιο το συναίσθημα αγάπης που μπορεί να καλλιεργηθεί προς τον θεραπευτή αποτελεί κεντρικό θεραπευτικό άξονα. Ο Ogden (1999) παρουσιάζει την ανάλυση ενός περιστατικού, όπου η θεραπευόμενη ζητάει επίμονα από τον θεραπευτή να εκφράσει το ενδιαφέρον του για αυτήν μέσα από δώρα και σεξουαλική εμπλοκή. Ο θεραπευτής ερμηνεύει την ανάγκη της βάσει του ιστορικού της σε απεγνωσμένους έρωτες και επικεντρώνει στο χιούμορ και την παιχνιδιάρικη διάθεση ως δίοδο αποτελεσματικότερης επικοινωνίας. Παρ’ ό,τι τα συναισθήματα αγάπης από τον θεραπευόμενο προς τον θεραπευτή θεωρούνται κοινός τόπος, οι ψυχαναλυτικοί συγγραφείς φαίνονται διστακτικοί σε ό,τι αφορά την αντιμεταβιβαστική αγάπη. Ο Schamess (1999), καταγράφει το κενό που υπάρχει στην βιβλιογραφία σε ό,τι αφορά τα ερωτικά αντιμεταβιβαστικά συναισθήματα. Αποδίδει την δυσκολία των θεραπευτών να αναφερθούν στις δικές τους αντιμεταβιβαστικές εμπειρίες συναισθημάτων έρωτα και αγάπης σε τρεις βασικούς λόγους: (α) η κλασσική ψυχαναλυτική εκπαίδευση αντιμετωπίζει την αντιμεταβίβαση ως ένδειξη ανεπάρκειας του θεραπευτή, (β) οι αναλυτές πιστεύουν ότι η άμεση επεξεργασία αυτών των συναισθημάτων με τον αναλυόμενο μπορεί να έχει αρνητικές επιπτώσεις στην οργάνωση του ψυχισμού τους, και (γ) η πίστη ότι η επικέντρωση στις αμοιβαίες διεργασίες αλληλεπίδρασης αλλοιώνει την ικανότητα του αναλυτή να αξιολογεί και να ανταποκρίνεται αντικειμενικά στα θέματα του αναλυόμενου. Ο συγγραφέας τονίζει την σημασία που έχει για την θεραπευτική διαδικασία η ικανότητα και προθυμία του αναλυτή να αφουγκράζεται και να αναγνωρίζει τα συναισθήματα έρωτα και αγάπης που αναδύονται προκει- Ο ρόλος της βίωσης θετικών συναισθημάτων στην ψυχοθεραπευτική διαδικασία ◆ 79 μένου να έχει μια πληρέστερη και πιο ανθρώπινη εικόνα της θεραπείας και να αυξάνει την αποτελεσματικότητα των θεραπευτικών παρεμβάσεων. Ο Rabin (2003) διαπραγματεύεται τα συναισθήματα αγάπης και έρωτα που αναπτύσσονται από την πλευρά του θεραπευτή προς τον πελάτη στην ψυχαναλυτική διαδικασία. Θέτει ερωτήματα και παραθέτει τις αντιθετικές απόψεις που έχουν διατυπωθεί σε ό,τι αφορά: (α) την σχέση ανάμεσα το συναίσθημα της αγάπης στην θεραπευτική δια δικασία και στην πραγματική ζωή, (β) την θεραπευτική χρησιμότητα της αυτο-αποκάλυψης του θεραπευτή ως προς αυτά τα συναισθήματα, και (γ) τις περιπτώσεις όπου ο θεραπευτής ερωτεύεται τον πελάτη. Η μελέτη του Rabin (2003) φανερώνει την ποικιλία των θεωρητικών διατυπώσεων και προτάσεων για τον θεραπευτικό χειρισμό ανάλογων συναισθημάτων. O Spurgeon (1987) πραγματεύεται τα συναισθήματα αγάπης που αναπτύσσονται από τον θεραπευτή προς τον πελάτη. Αναγνωρίζει ότι η «διδασκαλία» της αγάπης αποτελεί ένα σημαντικό μέρος της ψυχοθεραπευτικής διεργασίας, επισημαίνοντας παράλληλα τις καταστροφικές συνέπειες στον ψυχισμό του πελάτη που έχει η πιθανή ερωτική εκμετάλλευσή του από τον θεραπευτή. Αντίστοιχα, η Bridges (1998), αναγνωρίζοντας τα ερωτικά και σεξουαλικά συναισθήματα που εγείρονται σε μια ψυχοδυναμική θεραπευτική σχέση, επικαλείται την ανάγκη για ένα μοντέλο εποπτείας το οποίο να εκπαιδεύει τους μελλοντικούς αναλυτές στην αναγνώριση και τον χειρισμό αυτών των συναισθημάτων προς όφελος του θεραπευόμενου. Αρκετοί σύγχρονοι συγγραφείς διαφορετικών προσεγγίσεων αποσυνδέουν την αγάπη από την σεξουαλικότητα και αναφέρονται στην αμοιβαία αγάπη θεραπευτή – θεραπευόμενου ως προϋπόθεση για την επιτυχή θεραπεία. Ο Natterson (2003), σε άρθρο του που επαναδιαπραγματεύεται το ρόλο της αμοιβαίας αγάπης στην ψυχοθεραπεία, υποστηρίζει ότι η θεραπευτική σχέση είναι μια σχέση αγάπης και ότι ο θεραπευτικός διάλογος έχει στην ουσία του ως επίκεντρο την αγάπη και την επέκταση της σε μεγαλύτερο μέρος της ζωής του θεραπευόμενου. Θεωρώντας ως βασικό επίκεντρο της ψυχοθεραπείας την ανάπτυξη του εαυτού, ο συγγραφέας υποστηρίζει ότι η ενδυνάμωση και βελτίωση του εαυτού σχετίζεται άμεσα με την επίτευξη μιας σχέσης αμοιβαίας αγάπης. Επιπλέον, ο συγγραφέας προτείνει ότι οι θεραπευτές οφείλουν να αξιολογούν συστηματικά την ικανότητα των θεραπευομένων για αγάπη, καθώς το βίωμα αγάπης –ή η απουσία τέτοιου βιώματος– αποτελεί ένα σημαντικό κριτήριο της ανάγκης του θεραπευόμενου για θεραπεία, και την επιτυχή έκβαση της θεραπείας. Για το σκοπό αυτό προτείνει τρεις ενδείξεις της ικανότητας του ατόμου να βιώσει αγάπη: την επιθυμία, την πίστη, και την ελπίδα. Η επιθυμία αναφέρεται στην βαθύτερη ανάγκη του ατόμου για αγάπη, η πίστη αφορά την πεποίθηση του ατόμου ότι αξίζει την αγάπη, και η ελπίδα σχετίζεται με την αισιοδοξία ότι η αγάπη θα προκύψει στη ζωή του. Ο θεραπευτής προσφέρει τις κατάλληλες θεραπευτικές συνθήκες επιδεικνύοντας την φροντίδα, το ενδιαφέρον και την γονεϊκή ποιότητα αγάπης που είναι απαραίτητα στην θεραπευτική διαδικασία. H Gerrard (1996) υποστηρίζει ότι οι πελάτες που λαμβάνουν συναισθήματα αγάπης από τον θεραπευτή τους έρχονται σε επαφή με την δική τους δυνατότητα να αγαπηθούν και να αγαπήσουν. O Shamasundar (2001), στηριζόμενος στην φιλοσοφία των αυτοχθόνων της Β. Αμερικής, αναφέρεται στην ικανότητα για αγάπη ως ένα σημαντικό χαρακτηριστικό ενός καλού θεραπευτή, χαρακτηριστικό που σχετίζεται άμεσα με την ικανότητά του θεραπευτή για ενσυναίσθηση και την πρόθεση να βοηθήσει. H Snyder (2000) αναφέρει την αμοιβαία αγάπη στην ψυχοθεραπευτική σχέση στη Συστημική θεραπεία ως μια ουσιαστική παράμετρο για τη συναισθηματική ανάπτυξη του πελάτη. Προτείνει την συνειδητή ανάπτυξη αμοιβαίας αγάπης με την ενεργή συμμετοχή του θεραπευτή. Η Porter (2000), παρουσιάζει συνδυαστικά τρεις περιπτώσεις σε ομάδα δραματοθεραπείας, όπου η επιθυμία για συνένωση με έναν σημαντικό άλλο και η αναζήτηση νοήματος ζωής συγχέονται και στη συνέχεια διαχωρίζονται μέσα στα πλαίσια της θεραπευτικής διαδικασίας. Η συγγραφέας συμπεραίνει ότι τα άγχη και οι αγωνίες που ενυπάρχουν στο βίωμα της αγάπης κατά την θεραπευτική δια δικασία μπορούν να οδηγήσουν σε ψυχολογική 80 ◆ Αναστάσιος Σταλίκας και Αλκμήνη Μπούτρη ενημερότητα και ανάπτυξη, εφόσον ο θεραπευτής τα χειριστεί με ακεραιότητα, κουράγιο, και φροντίδα. O Jarosewitsch (1997), δανείζεται από τον πολιτισμό των Μαόρι της Νέας Ζηλανδίας την εννοιολογική κατασκευή «Aroha», που αναφέρεται στην άνευ όρων αγάπη. Για τον συγγραφέα η «Aroha» αποτελεί την πεμπτουσία της θεραπείας Gestalt. Δεν υπάρχει κάποια τεχνική που οδηγεί σε αυτήν, ούτε μέθοδος να καταγραφεί και να μετρηθεί, αλλά προκύπτει με φυσικό τρόπο μέσα από την θεραπευτική συνάντηση θεραπευτή – θεραπευόμενου. Αναφορές στην θεραπευτική αξία της αγάπης γίνονται και στον χώρο της ομαδικής θεραπείας. Οι συγγραφείς εστιάζουν κατά κύριο λόγο στα δυναμικά που αναπτύσσονται μεταξύ των μελών και την επεξεργασία αυτών σε σχέση με την αναβίωση μέσα στην ομάδα, μέσω της προβολής παλαιότερων σχέσεων και συναισθημάτων ή συστατικών του εαυτού (Ancona, Mangiarotti, & Coope, 1999. Bemak & Epp, 1996. Moeller, & Homolka, 2002). Το συναίσθημα της αγάπης, λοιπόν, στην ψυχοθεραπευτική διεργασία έχει απασχολήσει ιδιαίτερα θεωρητικούς και ερευνητές. Η ερμηνευτική και θεραπευτική αξία της μεταβιβαστικής αγάπης είναι καταγεγραμμένη ήδη από τα πρώτα χρόνια της ψυχανάλυσης. Νεώτερες αναφορές εστιάζουν στην συνείδηση των θεραπευτών για τα δικά τους αντιμεταβιβαστικά συναισθήματα, και τον χειρισμό αυτών στην θεραπευτική διαδικασία. Η αμοιβαία αγάπη τίθεται από πολλούς συγγραφείς στο επίκεντρο της θεραπείας, αποτελώντας πρότυπο αγάπης για τους πελάτες και μέσο για την ανάπτυξη του εαυτού τους. Τα θετικά συναισθήματα στην ψυχοθεραπεία Πέρα από την μελέτη συγκεκριμένων συναισθημάτων, όπως είναι η χαρά ή η αγάπη, κάποιες έρευνες καταγράφουν την εκδήλωση θετικών συναισθημάτων συνολικά χωρίς να διαχωρίζουν το είδος του θετικού συναισθήματος. Κάποιες περιορίζονται στην απλή καταγραφή της ύπαρξης ή όχι θετικών συναισθημάτων στην ψυχοθεραπεία, ενώ αρκετές προχωρούν σε ερμηνείες για έναν πιθανόν ουσιαστικότερο ρόλο που μπορεί τα θετικά συναισθήματα να κατέχουν στην θεραπευτική αλλαγή. Μια από τις πρώτες έρευνες που ασχολούνται με την καταγραφή των θετικών και των αρνητικών συναισθημάτων κατά την διάρκεια των συνεδριών είναι αυτή των Korman & Greenberg (1996). Στόχος της έρευνας είναι η μελέτη των συναισθηματικών επεισοδίων όπως αυτά εξελίσσονται κατά την διάρκεια της θεραπείας 20 πελατών με διάγνωση κατάθλιψης και οι οποίοι ακολούθησαν 15 με 20 συνεδρίες βιωματικής ψυχοθεραπείας. Τα αποτελέσματα της έρευνας δείχνουν ότι στους πελάτες με καλό θεραπευτικό αποτέλεσμα η αναλογία θετικών και αρνητικών συναισθημάτων που βιώνουν κατά την διάρκεια της συνεδρίας διαφοροποιείται, καθώς παρατηρείται σημαντική μείωση του βιώματος αρνητικών συναισθημάτων και αύξηση βιώματος των θετικών συναισθημάτων. Αντίθετα στις θεραπείες με αρνητικό θεραπευτικό αποτέλεσμα δεν παρατηρείται αυτή η διαφοροποίηση. Τα ψυχικά οφέλη της έκφρασης θετικών συναισθημάτων έχουν απασχολήσει τον χώρο της ομαδικής θεραπείας με πελάτες που πενθούν την απώλεια ενός σημαντικού άλλου. Παραδοσιακά το επίκεντρο των παρεμβάσεων στο πένθος είναι η επαφή με τα αρνητικά συναισθήματα (πόνος, θυμός, θλίψη) που βιώνουν τα άτομα και η κατά το δυνατόν πληρέστερη εκτόνωση και επεξεργασία τους. Μέσα σε αυτό το πλαίσιο, η έκφραση θετικών συναισθημάτων αντιμετωπίζεται ως εμπόδιο στην διαδικασία θρήνου και θεωρείται ένδειξη αποφυγής του πένθους, δυσχεραίνοντας έτσι την προσαρμογή του ατόμου που πενθεί (για παράδειγμα, Freud, 1917; Worden, 1991). Την τελευταία δεκαετία μια σειρά ερευνών φέρουν στοιχεία σχετικά με τα οφέλη της έκφρασης θετικών συναισθημάτων μέσα στα πλαίσια της ομαδικής θεραπείας. Οι Bonanno, Keltner, Holen, και Horowitz (1995), βάσει των ερευνητικών τους ευρημάτων υποστηρίζουν ότι η αποφυγή των έντονων δυσάρεστων συναισθημάτων που βιώνει κάποιος το πρώτο εξάμηνο μετά τον θάνατο του σημαντικού άλλου έχει προσαρμοστική αξία και σχετίζεται μακροπρόθεσμα με ταχύτερη προσαρμογή στην απώλεια. Επιπλέον, η έκφραση Ο ρόλος της βίωσης θετικών συναισθημάτων στην ψυχοθεραπευτική διαδικασία ◆ 81 θετικών συναισθημάτων στο πρώτο εξάμηνο της απώλειας φαίνεται να σχετίζεται θετικά με την καλύτερη προσαρμογή του ατόμου εικοσιπέντε μήνες αργότερα, σε σύγκριση με άτομα που στο πρώτο εξάμηνο εκφράζουν έντονα θλίψη και θυμό (Bonanno & Keltner, 1997). Οι ερευνητές αποδίδουν αυτά τα αποτελέσματα στην προσαρμοστική φύση των θετικών συναισθημάτων και στον ρόλο τους στις κοινωνικές σχέσεις. Υποστηρίζουν ότι το βίωμα θετικών συναισθημάτων επιτρέπει στα άτομα να επικεντρωθούν ενεργά στην επίλυση προβλημάτων που οδηγούν σε μακροπρόθεσμη προσαρμογή, ενώ παράλληλα υποβοηθά στην διαμόρφωση ενός κοινωνικού υποστηρικτικού δικτύου, ενθαρρύνοντας τα άτομα του περιβάλλοντος να προσεγγίσουν τον πενθούντα –σε αντίθεση με την έντονη έκφραση αρνητικών συναισθημάτων που τα απομακρύνει (Keltner & Bonanno, 1997. Bonanno & Kaltman, 1999). Η ερμηνεία των ευρημάτων είναι σύμφωνη με την άποψη των Lazarus, Kanner και Folkman (1980), για τους οποίους τα θετικά συναισθήματα σε καταστάσεις έντονου στρες απελευθερώνουν προσωρινά το άτομο από τα έντονα αρνητικά συναισθήματα επιτρέποντάς του να ασχοληθεί με κάτι που θα το αποσπάσει από το άγχος, ενδυναμώνουν το άτομο στην προσπάθειά του να αντιμετωπίσει την αντιξοότητα, και το βοηθούν να αναπληρώσει χαμένους πόρους ή να δημιουργήσει καινούργιους. Σύμφωνα με τους Piper, Orgodniczuk, Joyce, McCallum και Rosie (2002), το καλό θεραπευτικό αποτέλεσμα σε βραχεία ομαδική ψυχοθεραπεία πένθους σχετίζεται θετικά με το βίωμα και την έκφραση θετικών συναισθημάτων, ενώ παρουσιάζουν και κάποιες αρχικές ενδείξεις ότι η έντονη έκφραση αρνητικού συναισθήματος μπορεί να σχετίζεται με αρνητικό θεραπευτικό αποτέλεσμα. Οι Pennebaker, Μayne και Francis (1997), δείχνουν ότι σε περιπτώσεις επεξεργασίας του πένθους μέσω γραπτού λόγου, η συχνότερη χρήση λέξεων που εκφράζουν θετικά συναισθήματα σε σχέση με αυτές που εκφράζουν αρνητικά σχετίζονται με καλύτερη ψυχική και σωματική υγεία. Οι Kennedy-Moore και Watson (2001), παρουσιάζουν μια ανασκόπηση της βιβλιογραφίας σχετικά με την θεραπευτική αξία της έκφρασης της λύπης στην ψυχοθεραπεία. Μεταξύ άλλων συμπεραίνουν ότι η έκφραση λύπης είναι πιθανόν να φέρει θεραπευτικά οφέλη όταν προκύπτει μέσα στα πλαίσια μιας υποστηρικτικής σχέσης, αφορούν ένα μέτριο επίπεδο έντασης και συνοδεύεται από την έκφραση θετικών συναισθημάτων. Οι Alvarez και Nemeny (2001), στα πλαίσια της ψυχαναλυτικής προσέγγισης, προτείνουν ότι οι θετικές εμπειρίες και οι ευχάριστες συναισθηματικές καταστάσεις προωθούν τη σκέψη, ως διαδικασίες μέσω των οποίων προωθείται η συναισθηματική και γνωστική ανάπτυξη. H Fosha (2004), στα πλαίσια της Accelerated Experiential Dynamic Psychotherapy (AEDP), αναγνωρίζει τα θετικά συναισθήματα ως «δείκτες αλλαγής ή μεταμόρφωσης». Υποστηρίζει ότι η αυθόρμητη ανάδυση θετικών συναισθημάτων τη στιγμή που ο πελάτης επεξεργάζεται θέματα που σχετίζονται με αρνητικά και επώδυνα συναισθήματα, σηματοδοτεί την χαλάρωση των αμυνών. Για την Fosha (2004), αυτό το στιγμιαίο βίωμα θετικών συναισθημάτων υποδηλώνει ότι η διαδικασία βρίσκεται στον «σωστό δρόμο». Θεωρεί ότι για εκείνες μόνο τις στιγμές γίνονται διαθέσιμοι ψυχικοί πόροι και θεραπευτικές δυνατότητες που διαθέτει ο πελάτης. Ο θεραπευτής οφείλει να αναγνωρίσει αυτές τις στιγμές, προκειμένου να αξιοποιήσει αυτούς τους πόρους και να οδηγήσει τον πελάτη στην θεραπευτική αλλαγή. Συνοψίζοντας, οι πρόσφατες θεωρητικές προτάσεις και έρευνες για την έκφραση θετικών συναισθημάτων στον χώρο της ψυχοθεραπείας προσφέρουν κάποιες πρώτες ενδείξεις για την πιθανή θεραπευτική τους αξία (Μερτίκα, Μυτσκίδου, & Σεργιάννη, 2011). Η πλειοψηφία των ερευνών αφορούν την αντιμετώπιση του πένθους και αμφισβητούν την κλασσική θεωρητική άποψη που αντιμετωπίζει τα θετικά συναισθήματα ως άμυνα και εμπόδιο στην καλή προσαρμογή των ατόμων. Η νέα θεώρηση παραθέτει τα οφέλη ενός περισσότερο εξισορροπημένου βιώματος μεταξύ αρνητικών και θετικών συναισθημάτων, και αντλεί την επιχειρηματολογία από τον χώρο συναισθηματικών θεωριών, εστιάζοντας στον προσαρμοστικό ρόλο των θετικών συναισθημάτων στη ζωή του ανθρώπου. 82 ◆ Αναστάσιος Σταλίκας και Αλκμήνη Μπούτρη Το μοντέλο Διεύρυνσης και Δόμησης για τα θετικά συναισθήματα στην ψυχοθεραπεία Το μοντέλο της Διεύρυνσης και Δόμησης της Fredrickson (1998. 2001. 2004) προέρχεται από τον χώρο της εξελικτικής ψυχολογίας και είναι το πρώτο μοντέλο που ασχολείται αποκλειστικά με την φύση και την λειτουργία των θετικών συναισθημάτων στον ανθρώπινο οργανισμό. Το μοντέλο αποτελεί προϊόν της συστηματικής μελέτης, καταγραφής και σύνθεσης ερευνητικών δεδομένων τα οποία παρέχουν έμμεση υποστήριξη της εγκυρότητάς του. Παράλληλα, η Fredrickson με την ομάδα της διεξάγουν ένα σημαντικό αριθμό ερευνών μεταγενέστερων της διατύπωσης του μοντέλου, προκειμένου να υποστηρίξουν ερευνητικά τις βασικές θεωρητικές αρχές του. Τα αποτελέσματα των ερευνών είναι ενθαρρυντικά και σε γενικές γραμμές παρέχουν ερευνητική υποστήριξη της εγκυρότητάς του μοντέλου (Fredrickson, 2003. Fredrickson, & Branigan, 200. Fredrickson, Tucade, Waugh, & Larkin, 2003. Galanakis, Galanopoulou, & Stalikas, 2011). Συνοπτικά, το μοντέλο προτείνει τέσσερις βασικές λειτουργίες των θετικών συναισθημάτων: (α) την διεύρυνση του ρεπερτορίου σκέψης και δράσης: η βίωση θετικών συναισθημάτων οδηγεί σε αυξημένη ευελιξία στη σκέψη και τη δράση, καθιστώντας το άτομο περισσότερο ανοικτό στη διαμόρφωση νέων σκέψεων, ιδεών και δυνατοτήτων, περισσότερο δημιουργικό, ικανό για νέες γνωστικές συνδέσεις, με περισσότερες ιδέες για την επίλυση των προβλημάτων του, και πιο κινητοποιημένο και πρόθυμο να ακολουθήσει νέους τρόπους δράσης, (β) την δόμηση πόρων: μέσα από την αλλεπάλληλη βίωση θετικών συναισθημάτων και διεύρυνσης δομούνται πλούσιοι και ισχυροί νοητικοί, ψυχικοί, κοινωνικοί και συναισθηματικοί πόροι, οι οποίοι είναι διαχρονικοί και διαθέσιμοι στο άτομο όταν βρεθεί αντιμέτωπο με μια δύσκολη ή απειλητική κατάσταση επιτρέποντας του να αντεπεξέλθει πιο αποτελεσματικά, (γ) την ύπαρξη της ατέρμονα ανελισσόμενης αλυσίδας: η βίωση θετικών συναισθημάτων και η βίωση διεύρυνσης παρουσιάζουν μια κυκλική ανοδική αλληλεπίδρα- ση, έτσι ώστε όταν το άτομο βρίσκεται σε διαδικασία διεύρυνσης αυξάνονται οι πιθανότητες να βιώσει κάποιο θετικό συναίσθημα, όποτε και να βρεθεί εκ νέου σε διαδικασία διεύρυνσης, και (δ) την αντισταθμιστική δράση της βίωσης θετικών συναισθημάτων ως προς τις συνέπειες της βίωσης αρνητικών συναισθημάτων στον οργανισμό: η βίωση θετικών συναισθημάτων λειτουργεί ως αντίδοτο στις επιδράσεις που έχουν στον οργανισμό τα αρνητικά συναισθήματα, σε γνωστικό και νευροφυσιολογικό επίπεδο. Μέσα από αυτές τις λειτουργίες αποτυπώνεται ο προσαρμοστικός ρόλος της βίωσης των θετικών συναισθημάτων. Το βίωμα θετικών συναισθημάτων εμπλουτίζει το άτομο με δεξιότητες και πόρους μέσα από την κινητοποίησή του να εξερευνήσει το περιβάλλον με ευελιξία και δημιουργικότητα. Οι πόροι αυτοί είναι απαραίτητοι για την αποτελεσματική ανταπόκριση και επιβίωση του. Με αυτόν τον τρόπο η λειτουργία θετικών και αρνητικών συναισθημάτων φαίνεται να είναι συμπληρωματικές, με τα αρνητικά συναισθήματα να βοηθούν στην επιβίωση μέσω της εστίασης στην δράση, και τα θετικά –μέσω της διεύρυνσης– στη συλλογή πόρων, στο χτίσιμο αμυνών και στην απόκτηση δεξιοτήτων και στρατηγικών. Το μοντέλο Διεύρυνσης και Δόμησης απέσπασε την προσοχή ερευνητών της ψυχοθεραπευτικής διαδικασίας. Πρόσφατες έρευνες το χρησιμοποιούν ως έναυσμα για τη μελέτη του ρόλου της βίωσης των θετικών συναισθημάτων στην ψυχοθεραπεία. Οι έρευνες αυτές, παρ’ ότι περιορισμένες σε αριθμό, παράγουν ενθαρρυντικά αποτελέσματα. Οι Joiner, Petit, Perez, Burns, Gencoz, Gencoz και Rudd (2001), στηριζόμενοι στο μοντέλο της Fredrickson διερευνούν τη σχέση ανάμεσα στην θετική και αρνητική διάθεση ασθενών με τάσεις αυτοκτονίας, και την απόκτηση δεξιοτήτων επίλυσης προβλημάτων μέσω της ψυχοθεραπείας. Τα αποτελέσματά τους δείχνουν ότι οι ασθενείς που βίωναν θετική διάθεση παρουσίασαν μεγαλύτερη μείωση των συμπτωμάτων και πιο αναπτυγμένη ικανότητα επίλυσης προβλημάτων, σε σύγκριση με αυτούς που βίωναν αρνητική διάθεση. Βάσει των αποτελεσμάτων οι ερευνητές υποστηρίζουν ότι οι παρεμβάσεις που στοχεύουν στην απόκτη- Ο ρόλος της βίωσης θετικών συναισθημάτων στην ψυχοθεραπευτική διαδικασία ◆ 83 ση μακροπρόθεσμων δεξιοτήτων επίλυσης προβλημάτων είναι σκόπιμο να λαμβάνουν χώρα τις περιόδους που ο ασθενής βρίσκεται σε θετική διάθεση, προκειμένου να βρίσκουν μεγαλύτερη ανταπόκριση από την πλευρά του. Τις στιγμές της αρνητικής διάθεσης, ο θεραπευτής μπορεί να επικεντρώσει σε άλλες πτυχές της θεραπείας, όπως είναι η διαμόρφωση ενός υποστηρικτικού περιβάλλοντος, ή η παρέμβαση στην κρίση. Επιπλέον, προτείνουν μια σειρά από θεραπευτικές τεχνικές που μπορούν να βοηθήσουν τον ασθενή να έρθει σε επαφή με θετικά συναισθήματα. Για παράδειγμα, ο θεραπευτής μπορεί να ζητήσει από τον πελάτη να ανακαλέσει τις καλύτερες στιγμές της ζωής του, προκειμένου περιγράφοντας αυτές τις στιγμές σταδιακά να βελτιωθεί η διάθεσή του στο παρόν. Άλλη τεχνική που αναφέρεται είναι αυτή που πρότειναν οι Zeiss, Lewinsohn και Munoz (1979), όπου ο πελάτης καλείται να προγραμματίσει μια σειρά από ευχάριστες για τον ίδιο δραστηριότητες. Βασική προϋπόθεση για την υιοθέτηση τέτοιων τεχνικών αποτελεί η συμβατότητα της ψυχοθεραπευτικής προσέγγισης με την τεχνική. Η επιλεγόμενη τεχνική πρέπει να συνάδει με τις αρχές και αξιώματα της ψυχοθεραπευτικής προσέγγισης. Οι Σταλίκας και Μπούτρη (2004), αναθεωρούν και επανερμηνεύουν υπάρχοντα ερευνητικά ευρήματα στο ερευνητικό πεδίο της ψυχοθεραπευτικής αλλαγής βάσει του μοντέλου Διεύρυνσης και Δόμησης. Σύμφωνα με αυτήν την οπτική, η βάση κάθε ψυχοθεραπευτικής προσέγγισης είναι η διεύρυνση της αντίληψης του πελάτη σχετικά με το πρόβλημα ή τον εαυτό του συνολικά, καθώς και η εύρεση νέων τρόπων σκέψης και δράσης για να την επίλυση των προβλημάτων του. Παρά το γεγονός ότι κάθε θεραπευτική προσέγγιση έχει διαφορετικό εννοιολογικό σημείο εκκίνησης αναφορικά με την φύση του ανθρώπου, των προβλημάτων που αντιμετωπίζει και τις θεραπευτικές παρεμβάσεις για την αλλαγή, ολόκληρη η θεραπευτική διαδικασία μπορεί να νοηθεί ως μία διαδικασία διεύρυνσης. Επιπροσθέτως, η δόμηση μπορεί να θεωρηθεί ως το επιθυμητό αποτέλεσμα της ψυχοθεραπευτικής διαδικασίας. Ο μακροπρόθεσμος στόχος των ψυχοθεραπευτικών συνεδριών είναι η δόμηση κοινωνικών, ψυχικών, νοητικών και σωματικών πόρων, ώστε ο πελάτης ολοκληρώνοντας την θεραπεία να είναι σε θέση να αντιμετωπίσει τις καθημερινές προκλήσεις και τις αντιξοότητες της ζωής με έναν τρόπο πιο λειτουργικό από τον υπάρχοντα. Οι Σταλίκας και συνεργάτες (2003. 2004. 2005. 2010. 2015) πραγματοποιούν μια σειρά από έρευνες με στόχο να διερευνήσουν την εφαρμογή των βασικών προτάσεων της θεωρίας της Fredrickson στην ψυχοθεραπεία. Πιο συγκεκριμένα, οι ερευνητές προκειμένου να μελετήσουν την λειτουργία της ατέρμονα ανελισσόμενης αλυσίδας, καταγράφουν τα συναισθήματα του πελάτη κατά την είσοδο και την έξοδο του από τρεις συνεχόμενες συνεδρίες. Επιπλέον, προκειμένου να διερευνήσουν το συσχετισμό ανάμεσα στη εμφάνιση και βίωση θετικών συναισθημάτων και την διεύρυνση μέσα στην συνεδρία αποτύπωσαν την διεύρυνση χρησιμοποιώντας δύο εργαλεία για την αποτελεσματικότητα της συνεδρίας, το Session Impact Scale (SIS, Elliot and Wexler, 1994), και το Session Evaluation Inventory (SEQ, Stiles & Snow, 1984). Η επιλογή αυτή στηρίζεται στην εννοιολογική εγγύτητα συγκεκριμένων ερωτημάτων των δύο ερωτηματολογίων με κάποιες πτυχές της διεύρυνσης. Τα ευρήματα τους (α) παρέχουν μερική υποστήριξη για την συσχέτιση ανάμεσα στο βίωμα θετικών συναισθημάτων του πελάτη πριν από την έναρξη της συνεδρίας και την διεύρυνση (ως επίλυση προβλημάτων) κατά την διάρκεια της συνεδρίας, (β) παρέχουν υποστήριξη για τη σχέση ανάμεσα στο βίωμα της διεύρυνσης κατά την διάρκεια της συνεδρίας και την βίωση θετικών συναισθημάτων στο τέλος της συνεδρίας (Stalikas, Mitskidou, & Gazzola, 2004), (γ) συσχετίζουν τη βίωση θετικών συναισθημάτων κατά την έναρξη της συνεδρίας και την αξιολόγηση της θεραπευτικής συμμαχίας και ποιότητας της συνεδρίας, και (δ) υποδεικνύουν ότι η βίωση θετικών συναισθημάτων κατά την έναρξη μιας συνεδρίας επηρεάζεται από την αξιολόγηση του πελάτη αναφορικά με την θεραπευτική συμμαχία και το βάθος της προηγούμενης συνεδρίας (Mertika, Seryianni, Fitzpatrick, & Stalikas, 2005). Ο Mergenthaler (1996) στηριζόμενος στα ευ- 84 ◆ Αναστάσιος Σταλίκας και Αλκμήνη Μπούτρη ρήματα ερευνών του, διατυπώνει το Μοντέλο των Θεραπευτικών Κύκλων. Στόχος του μοντέλου είναι η περιγραφή της διεργασίας που λαμβάνει χώρα κατά την διάρκεια μιας συνεδρίας και οδηγεί σε ενόραση. Το μοντέλο αυτό στηρίζεται σε δύο βασικούς άξονες: τον συναισθηματικό τόνο, και την αφαιρετική σκέψη. Σύμφωνα με το μοντέλο η ύπαρξη ενόρασης και θεραπευτικής αλλαγής σε μια συνεδρία επέρχεται όταν ο θεραπευόμενος περάσει από μια κυκλική ακολουθία φάσεων, με την εξής μορφή: (α) στην αρχή ο πελάτης βρίσκεται σε κατάσταση ηρεμίας, όπου τα συναισθηματικά επίπεδα είναι χαμηλά, (β) στη συνέχεια τα συναισθηματικά επίπεδα αυξάνουν, ενώ η αφαιρετική σκέψη παραμένει χαμηλή, (γ) στην τρίτη φάση το συναίσθημα παραμένει σε υψηλά επίπεδα ενώ αυξάνεται και η γνωστική επεξεργασία του βιώματος, οδηγώντας στην ενόραση, (δ) στη συνέχεια τα επίπεδα του συναισθηματικού βιώματος μειώνονται σταδιακά, ενώ η γνωστική διεργασία παραμένει υψηλή, έως ότου (ε) ο πελάτης επιστρέφει στην αρχική κατάσταση ηρεμίας. Σε μεταγενέστερη έρευνα, ο Mergenthaler (2003) προσθέτει στο μοντέλο μια λεπτομερέστερη περιγραφή της ποιότητας του συναισθήματος που βιώνει ο πελάτης. Σύμφωνα με τον ερευνητή η θετική ή αρνητική χροιά του συναισθήματος συντελεί καθοριστικά στην εμφάνιση της ενόρασης. Πιο συγκεκριμένα, τα αποτελέσματα των ερευνών του έδειξαν ότι: (α) η βίωση αρνητικών συναισθημάτων από μόνη της συνδέεται με αρνητικό θεραπευτικό αποτέλεσμα, (β) το καλό θεραπευτικό αποτέλεσμα συνδέεται με την ταυτόχρονη παρουσία υψηλών επιπέδων θετικού και αρνητικού συναισθήματος, και (γ) το καλό θεραπευτικό αποτέλεσμα συνδέεται με την ύπαρξη διεύρυνσης, η οποία ακολουθεί την βίωση θετικών συναισθημάτων. Έτσι, στην παραπάνω κυκλική ακολουθία των τεσσάρων φάσεων που βιώνει ο πελάτης προκειμένου να επέλθει ενόραση, ανέλυσε το βίωμα έντονου συναισθήματος σε δύο μέρη. Αρχικά ο πελάτης βιώνει έντονο αρνητικό συναίσθημα βάσει του οποίου εμβαθύνει στο πρόβλημά του και αναδύεται νέο συγκρουσιακό υλικό. Στη συνέχεια, και με τις παρεμβάσεις του θεραπευτή γίνεται ένα «επεισόδιο αλλαγής», το οποίο οδηγεί στο βίωμα θετικών συναισθημάτων, το οποίο είναι και αυτό που τελικά οδηγεί στην ενόραση ή χρησιμοποιώντας την ορολογία της Fredrickson σε διεύρυνση. Σε πρόσφατη έρευνα, η Μπούτρη (2008) μελέτησε το μοντέλο Διεύρυνσης και Δόμησης σε ψυχοθεραπευτικές συνεδρίες διαφορετικών προσεγγίσεων. Για την διεξαγωγή της έρευνας διαμορφώθηκε η Κλίμακα Διεύρυνσης, το πρώτο ερευνητικό εργαλείο που καταγράφει την διαδικασία διεύρυνσης του πελάτη στην θεραπευτική συνεδρία, όπως την αξιολογούν ο πελάτης, ο θεραπευτής και κλινικοί κριτές. Συγκεκριμένα, διερευνήθηκε η σχέση ανάμεσα στα θετικά συναισθήματα που βιώνει ο πελάτης κατά την έναρξη και τη λήξη της συνεδρίας με την βίωση της διαδικασίας διεύρυνσης κατά την διάρκεια της συνεδρίας, καθώς και η θεραπευτική αξία της βίωσης διεύρυνσης. Τα ερευνητικά ευρήματα παρείχαν υποστήριξη στις βασικές αρχές του μοντέλου Διεύρυνσης και Δόμησης και στην εφαρμογή του στην ψυχοθεραπευτική διαδικασία. Πιο συγκεκριμένα, διαφάνηκε μια ακολουθία θεραπευτικών φαινομένων, σύμφωνα με την οποία η βίωση του θετικού συναισθήματος του ενθουσιασμού κατά την έναρξη της συνεδρίας οδήγησε σε αυξημένο βίωμα διεύρυνσης κατά την διάρκεια της συνεδρίας, το οποίο με τη σειρά του οδήγησε σε αυξημένο βίωμα θετικών συναισθημάτων κατά τη λήξη της συνεδρίας. Η βίωση θετικών συναισθημάτων κατά τη λήξη της συνεδρίας οδήγησε σε αυξημένο βίωμα θετικών συναισθημάτων και ενθουσιασμού κατά την έναρξη της επόμενης συνεδρίας, προωθώντας την επανάληψη της κυκλικής διαδικασίας μεταξύ θετικών συναισθημάτων και διεύρυνσης και στην επόμενη συνεδρία. Η διεύρυνση φάνηκε να λειτουργεί ανεξάρτητα από άλλα γνωστά ενδοσυνεδριακά φαινόμενα, όπως είναι η ενόραση, η επίλυση προβλημάτων και η θεραπευτική συμμαχία. Η βίωση της διεύρυνσης είχε ως αποτέλεσμα την αξιολόγηση της συνεδρίας από τον πελάτη ως ομαλή και βαθιά, δηλαδή ως ουσιαστική για την θεραπευτική αλλαγή του πελάτη. Συνοψίζοντας, οι παραπάνω έρευνες αποτελούν τις πρώτες εφαρμογές του μοντέλου Διεύ ρυνσης και Δόμησης στην ψυχοθεραπευτική Ο ρόλος της βίωσης θετικών συναισθημάτων στην ψυχοθεραπευτική διαδικασία ◆ 85 διαδικασία και στοχεύουν στην διερεύνηση της δυνατότητας των θετικών συναισθημάτων να δράσουν ως παράγοντες θεραπευτικής αλλαγής. Τα ερευνητικά αποτελέσματα παρέχουν αρχική στήριξη για την εφαρμογή του μοντέλου στην ψυχοθεραπεία, υποδεικνύοντας την σχέση ανάμεσα στη βίωση θετικών συναισθημάτων, την διεύρυνση και την θεραπευτική αλλαγή. Συμπεράσματα Παρά τον κεντρικό ρόλο που κατέχουν οι συναισθηματικές διαδικασίες για την ανθρώπινη λειτουργία και την ψυχοθεραπευτική πρακτική, η συντριπτική πλειοψηφία των θεωρητικών προτάσεων και των ερευνών εστιάζεται στα αρνητικά συναισθήματα. Η έκφραση θετικών συναισθημάτων μέχρι πρόσφατα θεωρούταν κατά βάση προϊόν θεραπευτικής αλλαγής, και αντιμετωπιζόταν ως φαινόμενο μειωμένης θεραπευτικής αξίας. Ανεκδοτολογικές αναφορές από την θεραπευτική πρακτική, αλλά και οι πρώτες έρευνες στον χώρο των θετικών συναισθημάτων στην ψυχοθεραπεία, παρέχουν κάποιες αρχικές ενδείξεις για έναν ενεργό ρόλο των θετικών συναισθημάτων ως γενεσιουργό παράγοντα θεραπευτικής αλλαγής. Το βίωμα χαράς, το γέλιο και η χρήση χιούμορ μέσα στον ψυχοθεραπευτικό χώρο βοηθάει τον πελάτη στην αντιμετώπιση των προβλημάτων του με έναν διαφορετικό τρόπο και την αλλαγή προοπτικής. Η βίωση ενός είδους αγάπης φαίνεται να βρίσκεται στην βάση της θεραπευτικής σχέσης, η οποία με τη σειρά της αποτελεί τον κινητήριο μοχλό της συνολικής θεραπευτικής διαδικασίας. Σε περιπτώσεις πένθους, η ικανότητα βίωσης του ατόμου θετικών συναισθημάτων αποτελεί προγνωστικό παράγοντα ταχύτερης ολοκλήρωσης αυτής της διαδικασίας. Κάποιες έρευνες προτείνουν ότι η ίδια η θεραπευτική αξία των αρνητικών συναισθημάτων εξαρτάται από την συνύπαρξη, ταυτόχρονη ή με χρονική αλληλουχία, των θετικών συναισθημάτων. Η παράλληλη παρουσία αυτών των θετικών συναισθημάτων φαίνεται να οδηγεί τελικά στην γνωστική αναδόμηση και την θεραπευτική αλλαγή. Η διαδικασία της διεύρυνσης, που απορρέει από τη βίωση θετικών συναισθημάτων, φαίνεται να συνδέεται με την θεραπευτική αλλαγή του πελάτη και την αποτελεσματικότητα της συνεδρίας. Οι παραπάνω προτάσεις συνολικά παρέχουν κάποια αρχική θεωρητική και ερευνητική υποστήριξη για την υπόθεση ότι η βίωση θετικών συναισθημάτων στην θεραπευτική διαδικασία αποτελεί γενεσιουργό παράγοντα ψυχοθεραπευτικής αλλαγής και συμβάλλουν στην ψυχική υγεία και ευεξία. 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Psychiatrie Francaise, 17 (6), 28-36. Young, M.E. & Bemak, F. (1998). The role of emotional arousal and expression in mental health counseling. Journal of Mental Health Counseling, 18 (4), 316-332. Ο ρόλος της βίωσης θετικών συναισθημάτων στην ψυχοθεραπευτική διαδικασία ◆ 91 The role of experiencing positive emotions in the psychotherapeutic process: a review of the theory and research literature Anastassios Stalikas1 Alkmini Boutri2 En this article we critically present the main theories, models and research findings regarding the role of positive emotions in psychotherapy. In the first part we present an overview of the role of the emotional presence, expression, and experience in psychotherapy and the role of emotions in different psychotherapeutic models. In the second part we focus on the role of humor, joy and love in psychotherapy, while in the last part we present and discuss the Broadenand-build model and its application to psychotherapy theory and practice. Abstract Keywords: positive emotions, psychotherapy 1. Panteion University of Political and Social Sciences. * Correspondance: A. Stalikas, Panteion Univeristy of Social and Political Sciences, Department of Psychology, 17671, Athens. Email: anstal@panteion.gr Powered by TCPDF (www.tcpdf.org)
https://openalex.org/W4298282612	https://zenodo.org/records/2678104/files/ma-thesis-patel.pdf	English	null	How the Abstract Becomes Concrete: Irrational Numbers are Understood Relative to Natural Numbers and Perfect Squares	Zenodo (CERN European Organization for Nuclear Research)	2,017	cc-by	13,084	How the Abstract Becomes Concrete: Irrational Numbers Are Anchored on Natural Numbers and Perfect Squares Running head: UNDERSTANDING IRRATIONAL NUMBERS Running head: UNDERSTANDING IRRATIONAL NUMBERS 1 Sashank Varma Keywords: irrational, perfect square, magnitude, number line, strategy, arithmetic, anchor UNDERSTANDING IRRATIONAL NUMBERS 2 2 UNDERSTANDING IRRATIONAL NUMBERS 3 How the Abstract Becomes Concrete: Irrational Numbers Are Anchored on Natural Numbers and Perfect Squares q The historical development of number systems can be characterized as a gradual progression from the concrete to the abstract. Natural numbers, which denote the cardinalities of sets, were understood directly and formalized first. According to the mathematician Leopold Kronecker, “God created” this relatively concrete number system, whereas more abstract number systems like the integers, rationals, irrationals, and reals are “the work of man.” In contrast, The historical development of number systems can be characterized as a gradual progression from the concrete to the abstract. Natural numbers, which denote the cardinalities of sets, were understood directly and formalized first. According to the mathematician Leopold Kronecker, “God created” this relatively concrete number system, whereas more abstract number systems like the integers, rationals, irrationals, and reals are “the work of man.” In contrast, cognitive science research emphasizes how number systems are grounded in analog magnitude representations (Moyer & Landauer, 1967). The natural numbers are thought to be grounded in directly perceptible objects and are internally represented as continuous magnitudes on a mental number line. For more abstract number systems, the necessity of mastering the associated symbol systems has been acknowledged. However, the focus is often on how these symbol systems recruit and restructure magnitude representations. Examples include incorporating the inverse relationship between positive and negative integers into the mental number line and capturing the ratio relationship between the numerators and denominators of fractions more precisely (Matthews & Chesney, 2015; Varma & Schwartz, 2011). cognitive science research emphasizes how number systems are grounded in analog magnitude representations (Moyer & Landauer, 1967). The natural numbers are thought to be grounded in directly perceptible objects and are internally represented as continuous magnitudes on a mental number line. For more abstract number systems, the necessity of mastering the associated symbol systems has been acknowledged. However, the focus is often on how these symbol systems recruit and restructure magnitude representations. Examples include incorporating the inverse relationship between positive and negative integers into the mental number line and capturing the ratio relationship between the numerators and denominators of fractions more precisely (Matthews & Chesney, 2015; Varma & Schwartz, 2011). Comparatively little attention has been directed towards abstract mathematical concepts that are both imperceptible and incapable of being fully grounded in visuospatial referents. Abstract Mathematical cognition research has largely emphasized concepts that can be directly perceived or grounded in visuospatial referents. These include concrete number systems like natural numbers, integers, and rational numbers. Here, we investigate how a more abstract number system, the irrationals denoted by radical expressions like , is understood across three tasks. Performance on a magnitude comparison task suggests that people interpret irrational numbers – specifically, the radicands of radical expressions – as natural numbers. Response times and strategy self-reports during number line estimation reveal that the spatial locations of irrationals are determined by anchoring to neighboring perfect squares. Perfect squares also facilitate the evaluation of complex arithmetic expressions. These converging results align with a constellation of related anchoring phenomena spanning tasks and number systems of varying complexity. Accordingly, we propose that the task-specific recruitment of more concrete representations, formal anchoring, is an important mechanism for understanding and teaching mathematics. UNDERSTANDING IRRATIONAL NUMBERS 4 numerical cognition and for instructional design in mathematics education. It builds on literature concerned with how people understand natural numbers, integers, and rational numbers. numerical cognition and for instructional design in mathematics education. It builds on literature concerned with how people understand natural numbers, integers, and rational numbers. UNDERSTANDING IRRATIONAL NUMBERS For such concepts, do magnitude representations continue to be heavily recruited or does notation- specific strategic processing become more essential? In this paper, we use irrational numbers as a test case to address this question. To our knowledge, this is the first study to explore how familiar irrational numbers denoted by symbolic radical expressions like are mentally represented and processed by typical adults. Our study has implications for extending theories of UNDERSTANDING IRRATIONAL NUMBERS Some definitions include zero within the set of natural numbers, though others do UNDERSTANDING IRRATIONAL NUMBERS 5 5 marking on paper or clicking on a virtual number line with a computer mouse. Performance is typically measured by average absolute error – the absolute difference between the correct position of the number and the position selected by the participant. Higher error values indicate worse performance. Over development, children’s accuracy on NLE tasks improves gradually (Fazio, Bailey, Thompson, & Siegler, 2014). More specifically, their estimation patterns suggest that natural numbers are initially represented in a compressed, logarithmically spaced fashion. marking on paper or clicking on a virtual number line with a computer mouse. Performance is typically measured by average absolute error – the absolute difference between the correct position of the number and the position selected by the participant. Higher error values indicate worse performance. Over development, children’s accuracy on NLE tasks improves gradually (Fazio, Bailey, Thompson, & Siegler, 2014). More specifically, their estimation patterns suggest that natural numbers are initially represented in a compressed, logarithmically spaced fashion. This incorrect representation is revised later by spacing the same numerical magnitudes more linearly. Performance gains on these tasks are driven in part by anchoring estimates to number lines’ midpoints and endpoints (Ashcraft & Moore, 2012; Barth & Paladino, 2011; Opfer et al., 2016; Siegler & Thompson, 2014). This incorrect representation is revised later by spacing the same numerical magnitudes more linearly. Performance gains on these tasks are driven in part by anchoring estimates to number lines’ midpoints and endpoints (Ashcraft & Moore, 2012; Barth & Paladino, 2011; Opfer et al., 2016; Siegler & Thompson, 2014). Individual differences in the magnitude representations of natural numbers, as indexed by the slope of the distance effect in the MC task, predict variation in mathematical achievement for elementary (De Smedt, Verschaffel, & Ghesquière, 2009) and middle school students (Halberda, Mazzocco, & Feigenson, 2008). Likewise, NLE performance predicts mathematical achievement in elementary school students, although not older students learning more advanced topics Individual differences in the magnitude representations of natural numbers, as indexed by the slope of the distance effect in the MC task, predict variation in mathematical achievement for elementary (De Smedt, Verschaffel, & Ghesquière, 2009) and middle school students (Halberda, Mazzocco, & Feigenson, 2008). Likewise, NLE performance predicts mathematical achievement in elementary school students, although not older students learning more advanced topics (Sasanguie, De Smedt, Defever, & Reynvoet, 2012). Instructional studies support a causal link between numerical magnitude representations and arithmetic skills. Natural Numbers The natural numbers are the set of countable numbers .1 Their mental representation has been studied extensively using the magnitude comparison (MC) task. When comparing which number in a pair is greater or lesser, response time decreases as the difference between the numbers increases (Moyer & Landauer, 1967). For instance, people can judge the greater or lesser number in the pair (2, 9) more quickly than in the pair (5, 6). This well-replicated performance pattern is known as the distance effect. It manifests whether participants compare symbolic numbers or visuospatial numerosities like sets of dots (Buckley & Gillman, 1974). The distance effect is commonly interpreted as evidence that natural numbers are represented in part as psychophysically scaled magnitudes. It has been observed as early as infancy with numerosities (Xu & Spelke, 2000) and kindergarten with symbolic numbers (Sekuler & Mierkiewicz, 1977). Over development, children’s representation of numerical magnitude changes such that response times decrease overall and the slope of the distance effect decreases (Sekuler & Mierkiewicz, 1977). These changes indicate improved precision of magnitude representations. The magnitude representation of natural numbers and its development have also been probed by the number line estimation (NLE) task. In the bounded version of this task, participants are presented with a number line labeled zero at the left endpoint and ten at the right endpoint (Cohen & Blanc-Goldhammer, 2012). The middle segment is left blank. Numbers are presented sequentially and participants are prompted to map their positions to the line by UNDERSTANDING IRRATIONAL NUMBERS Young children who were tutored on the magnitudes of small natural numbers via a board game performed better on simple arithmetic problems than their peers who received a control lesson (Siegler & Ramani, 2009). Integers Integers The natural numbers combined with their additive inverses, the negative numbers, form the integers {…, -2, -1, 0, 1, 2, …}. Comprehending the meaning of this number system requires engaging in additional symbolic processing. For instance, when judging the greater number in UNDERSTANDING IRRATIONAL NUMBERS UNDERSTANDING IRRATIONAL NUMBERS 6 the pair (-3, -9), it is necessary to inhibit the whole number interpretations of “-3” as “3” and “- 9” as “9”. Children initially compare such integer pairs by using a symbolic strategy. First, they convert the negative numbers to natural numbers by ignoring the minus signs. Next, they compare the numerical magnitudes and then reverse this judgment (Varma & Schwartz 2011) the pair (-3, -9), it is necessary to inhibit the whole number interpretations of “-3” as “3” and “- 9” as “9”. Children initially compare such integer pairs by using a symbolic strategy. First, they convert the negative numbers to natural numbers by ignoring the minus signs. Next, they compare the numerical magnitudes and then reverse this judgment (Varma & Schwartz, 2011). This strategy results in a distance effect when comparing pairs of negatives numbers. But when comparing mixed integer pairs like (-4, 9), judgments can be made by noticing that positives are greater than negatives. The result is the absence of a distance effect in elementary school children. Over development, this strategic processing of integers is substituted by accessing a restructured magnitude representation that incorporates the symmetry of positives and negatives around the anchor zero. Adults represent the negative number line as a reflection of the positive number line, incorporating the additive inverse property . This shift manifests as an inverse distance effect when comparing mixed integer pairs – as the distance between the integers increases, so does the responses time. the pair (-3, -9), it is necessary to inhibit the whole number interpretations of “-3” as “3” and “- 9” as “9”. Children initially compare such integer pairs by using a symbolic strategy. First, they convert the negative numbers to natural numbers by ignoring the minus signs. Next, they compare the numerical magnitudes and then reverse this judgment (Varma & Schwartz, 2011). This strategy results in a distance effect when comparing pairs of negatives numbers. But when comparing mixed integer pairs like (-4, 9), judgments can be made by noticing that positives are greater than negatives. The result is the absence of a distance effect in elementary school children. Over development, this strategic processing of integers is substituted by accessing a restructured magnitude representation that incorporates the symmetry of positives and negatives around the anchor zero. UNDERSTANDING IRRATIONAL NUMBERS 7 7 studies inspired the design of a manipulative that encourages learners to incorporate the zero anchor in their magnitude representation of the integers. Doing so improves arithmetic problem- solving on difficult items like (Tsang et al., 2015). UNDERSTANDING IRRATIONAL NUMBERS Adults represent the negative number line as a reflection of the positive number line, incorporating the additive inverse property . This shift manifests as an inverse distance effect when comparing mixed integer pairs – as the distance between the integers increases, so does the responses time. This strategy results in a distance effect when comparing pairs of negatives numbers. But when comparing mixed integer pairs like (-4, 9), judgments can be made by noticing that positives are greater than negatives. The result is the absence of a distance effect in elementary school children. Over development, this strategic processing of integers is substituted by accessing a restructured magnitude representation that incorporates the symmetry of positives and negatives around the anchor zero. Adults represent the negative number line as a reflection of the positive number line, incorporating the additive inverse property . This shift manifests as an inverse distance effect when comparing mixed integer pairs – as the distance between the integers increases, so does the responses time. As with natural numbers, NLE tasks have also uncovered a logarithmic-to-linear shift in the mental representation of integers. When estimating the positions of positive numbers in the range 0 – 1,000 and negative numbers in the range -1,000 – 0, second graders exhibit logarithmic magnitude representations. By contrast, fourth and sixth graders exhibit linear representations for both ranges (Brez, Miller, & Ramirez, 2015). This logarithmic-to-linear shift extends to larger number ranges and older people. Middle school students’ estimates of integers in the negative range -10,000 – 0 and the combined range -1,000 – 1,000 are linear, though with lower accuracy for negative numbers (Young & Booth, 2015). Performance on NLE tasks like these depends partly on recognizing the symmetry of the integers around the zero anchor (Saxe, Earnest, Sitabkhan, Haldar, Lewis, & Zheng, 2010; Tsang, Blair, Bofferding, & Schwartz, 2015). These UNDERSTANDING IRRATIONAL NUMBERS UNDERSTANDING IRRATIONAL NUMBERS 8 8 The precision of rational number magnitude representations is also associated with higher-level mathematical skills. Symbolic fraction magnitude representations predict achievement across a range of domains and measures – fraction arithmetic, algebra, grade school standardized exams, and high school mathematical achievement (Siegler et al., 2012). Similarly, accuracy on NLE tasks using decimals predicts mathematics achievement in elementary school students (Schneider, Grabner, Zurich, & Paetsch, 2009). Even nonsymbolic ratio precision predicts college students’ knowledge of fractions and algebra (Matthews, Lewis, & Hubbard, 2016). Causal support for the link between magnitude knowledge and arithmetic skills comes from interventions that devote more instructional time to mastering the magnitudes of fractions. Results show that emphasizing magnitude boosts arithmetic skills for high and low achievers (Fuchs et al., 2013). Rational Numbers Rational numbers are the set of numbers that can be written as a ratio of two integers such that the denominator is non-zero. Magnitude comparison with symbolic fractions and decimals, as well as with non-symbolic ratios, produces the distance effect (DeWolf, Grounds, Bassok, & Holyoak, 2014; Matthews & Chesney, 2015; Matthews & Lewis, 2016; Siegler, Thompson, & Schneider, 2011; Varma & Karl, 2013). Thus, rational numbers are also thought to be integrated on the mental number line. As learners begin to understand fraction magnitudes, the slope of the distance effect is nearly zero. With time, fraction magnitude information becomes more accessible and the slope increases gradually. By adulthood, the slope stabilizes (Gabriel, Szucs, & Content, 2013). These behavioral changes are paralleled by the discovery and use of strategies to facilitate performance. For instance, children and young adults often report using unit fractions like ½ as anchors to perform fraction magnitude comparison (Fazio, Dewolf, & Siegler, 2016; Siegler & Thompson, 2014). In contrast to natural numbers and integers, NLE tasks suggest that the representation of rational numbers does not undergo a logarithmic-to-linear developmental shift. When children and adults perform NLE with decimal proportions, they both exhibit highly linear estimation patterns (Iuculano & Butterworth, 2011). However, estimates do become more accurate in the 0 – 1 and 0 – 5 ranges. These performance gains may result partly from using unit fractions like ½ as anchors (Siegler & Thompson, 2014; Spinillo & Bryant, 1991). Irrational Numbers Irrational numbers are incommensurable. Unlike rational numbers, they cannot be expressed as ratios of integers such that the denominator is non-zero. Their decimal expansions are infinitely long, non-repeating, and ostensibly random. When they were first proposed, the notion seemed outlandish. According to a famous anecdote that originates with Vitruvius, the Greek mathematician who first proved their existence was drowned at sea for challenging the ratio doctrine of numbers. It was not until the late 1800s that irrationals were formalized and properly integrated onto the real number line (Dedekind, 1963). Two subclasses can be distinguished – algebraic irrationals and transcendental numbers. Algebraic irrationals are the solutions to polynomial equations and can be denoted by expressions of the form . Transcendental numbers, which are not the solutions to any polynomial equations, include and . UNDERSTANDING IRRATIONAL NUMBERS 9 9 Just one other study has investigated the cognitive bases of algebraic irrationals. It explored whether the same human “number sense” that allows us to compare the magnitudes of natural numbers, integers, and rational numbers extends to irrationals (Obersteiner & Hofreiter, 2017). Participants performed magnitude comparisons with irrationals of the form . Both the root y and the radicand x could vary. The authors tested whether participants compared the magnitudes of irrationals by accessing their holistic magnitudes or by focusing on the root and radicand components. Comparisons were faster when both numbers contained a common root component, as in the pairs (. Likewise, comparisons were faster when both numbers contained a common radicand component, as in the pair (. When both the roots and radicands differed, as in the pair (, response times were slow. Unexpectedly, response times for the common component pairs were not predicted by the distances between the root components or radicand components. These mixed results require explanation. One problem is the use of unfamiliar irrational numbers. Another is the highly expert sample of mathematics graduate students and professors. Therefore, it is unclear how typical adults understand familiar irrational numbers like . In our study, we focus on algebraic irrationals. Our stimuli are familiar square root expressions of the form . These expressions denote irrational numbers when the radicand x is not a perfect square (e.g., ) and natural numbers when the radicand is a perfect square (e.g., ). In the following sections, we will collectively refer to these as radical expressions because both contain the radical sign. Research Questions and Hypotheses First, we asked whether radical expressions are represented like natural numbers, integers, and rational numbers – as continuous magnitudes integrated on the mental number line. We refer to this proposal as the mental number line hypothesis. An alternative proposal is that UNDERSTANDING IRRATIONAL NUMBERS 11 Third, we investigated whether individual differences in the mental representation and processing of irrationals explain variation in conceptual and procedural knowledge of this number system. Some researchers have proposed that magnitude knowledge is at the core of numerical and arithmetic learning (Link, Nuerk, & Moeller, 2014; Siegler, 2016). Contra this view, we hypothesize that the influence of magnitude representations on arithmetic problem- solving attenuates as the abstraction of number systems increases. Arithmetic expertise with irrationals may not depend as much on magnitude knowledge. UNDERSTANDING IRRATIONAL NUMBERS 10 people use processes that capitalize on the structure of radical expressions. For instance, when judging the greater or lesser number in the pair (, ), people may ignore the radical signs and only compare the radicand components (3, 8) using their magnitude representations of natural numbers. This is possible because when x and y are non-negative, judgments of (, ) and (x, y) are equivalent. We refer to this as the equivalence strategy hypothesis. Second, we investigated whether people process irrational numbers by strategically anchoring them on more concrete concepts like natural numbers and perfect squares. anchoring them on more concrete concepts like natural numbers and perfect squares. Specifically, we hypothesized the possible use of a multiplication strategy on the MC task whereby perfect square pairs like (, ) are transformed to computationally analogous “tie” multiplication problems like Such a transformation might facilitate comparison because tie problems are processed more quickly than non-tie problems of comparable size (Ashcraft, 1992; Parkman, 1972). On the NLE task, irrational numbers might be estimated in relation to perfect squares – the landmark strategy. For example, people may estimate the positions of irrational numbers like by referencing the positions of neighboring perfect squares like and . Finally, people may leverage their knowledge of perfect squares during arithmetic problem-solving. When simplifying , a naïve strategy would be to decompose the radicand into its prime factors and then shift pairs of common factors outside the radical sign –the prime factorization strategy. Consider the following solution procedure: A more efficient approach is to decompose the radicand into its perfect square factors – the perfect squares factorization strategy: Participants Overall, 81 undergraduate students (24 males, 57 females) from a large Midwestern university were recruited via flyers distributed across campus. Their ages ranged from 18 to 24 (M = 20.5 years, SD = 1.7). On a questionnaire, 30.3% of participants self-reported that they were enrolled in a major emphasizing quantitative and analytic skills such as science, h l i i h i fi i E i l i d Overall, 81 undergraduate students (24 males, 57 females) from a large Midwestern university were recruited via flyers distributed across campus. Their ages ranged from 18 to 24 (M = 20.5 years, SD = 1.7). On a questionnaire, 30.3% of participants self-reported that they were enrolled in a major emphasizing quantitative and analytic skills such as science, technology, engineering, mathematics, finance, or economics. Experimental sessions spanned about 60 minutes and all participants received $12 in compensation. The protocol was approved by the local Institutional Review Board. Design We employed a within-subjects experimental design, with each participant completing all levels of the four experimental tasks. See Table 1 for an overview of the design and materials. Table 1. Overview of the design and materials used in this study. Table 1. Overview of the design and materials used in this study. Task Condition Stimuli Dependent Variables Magnitude natural numbers 0, 1, 2…, 9 accuracy UNDERSTANDING IRRATIONAL NUMBERS UNDERSTANDING IRRATIONAL NUMBERS UNDERSTANDING IRRATIONAL NUMBERS 12 comparison (MC) response time one-digit radicals , , , , Number line estimation (NLE) natural numbers 1, 2, 3, …, 9 absolute error response time R2 value one-digit radicals , ,, , perfect squares , , , two-digit radicals , , , Strategy questionnaire natural numbers How did you place 7? strategy type one-digit radicals How did you place ? perfect squares How did you place ? two-digit radicals How did you place ? Irrationals knowledge test number classification Is rational or irrational? accuracy strategy type density knowledge How many irrationals are between 0 – 1? operation concepts The sum of a rational and an irrational is ...? simple arithmetic Simplify . complex arithmetic Simplify . Note. The stimuli for the strategy questionnaire and the irrationals knowledge test are examples from the larger set of items. response time Magnitude comparison task. The first task was magnitude comparison and its stimuli were defined by four variables – type, distance, size, and perfect square status. The type variable refers to whether a number pair consisted of natural numbers or radical expressions. For the distance and size variables, we used two definitions to evaluate the equivalence strategy and mental number line hypotheses (research question 1). The distance variable was the absolute difference between the numbers. The first definition of distance focused on the radicand Magnitude comparison task. The first task was magnitude comparison and its stimuli were defined by four variables – type, distance, size, and perfect square status. The type variable refers to whether a number pair consisted of natural numbers or radical expressions. For the distance and size variables, we used two definitions to evaluate the equivalence strategy and mental number line hypotheses (research question 1). The distance variable was the absolute difference between the numbers. The first definition of distance focused on the radicand components. For example, the distance of the pair (, ) was . The second definition focused on the actual magnitude values of the radical expressions. For example, the distance of the pair (, ) was . The size variable was defined as the average of the two numbers. The first definition again used the radicand components. For example, the size of the pair (, ) was . The second definition again components. For example, the distance of the pair (, ) was . UNDERSTANDING IRRATIONAL NUMBERS The second definition focused on the actual magnitude values of the radical expressions. For example, the distance of the pair (, ) was . The size variable was defined as the average of the two numbers. The first definition again used the radicand components. For example, the size of the pair (, ) was . The second definition again UNDERSTANDING IRRATIONAL NUMBERS UNDERSTANDING IRRATIONAL NUMBERS 13 used the actual magnitude values of the radical expressions. For example, the size of the pair (, ) was . The perfect square variable was defined by whether both natural numbers or radicands were perfect squares like (4, 9) or (, ). Two dependent variables, accuracy and response time (RT) in milliseconds, were collected. Number line estimation task. The second task was number line estimation (NLE) and it included one within-subjects variable (type) with four levels – natural numbers, one-digit radicals, perfect square radicals, and two-digit radicals. The natural numbers were 0, 1, 2, 3, …, 10; the one-digit radicals were ; the perfect square radicals were ; and the two-digit radicals were In each level, the first and last values served as manipulation checks. Data associated with these values were not included in the main analyses. Three dependent variables were formed – average absolute error, response times, and linearity of estimates. Average absolute error was the absolute difference between a participant’s selected position and the target position. Response times were measured in milliseconds. Linearity of estimates was the relationship between participants’ selected positions and target positions as estimated by linear regression (R2). Strategy questionnaire. The third task was the strategy questionnaire for the NLE task. The independent variable was number type and the dependent variable was self-reported estimation strategies. Irrationals knowledge test. The fourth task was the irrationals knowledge test. It consisted of five subsections that were collapsed to form two independent variables – conceptual knowledge and procedural knowledge. Two dependent variables were computed – accuracy on each section and problem-solving strategies on the complex arithmetic subsection. UNDERSTANDING IRRATIONAL NUMBERS 14 All experimental materials are available via the Open Science Framework (https://osf.io/6s9pc/#). UNDERSTANDING IRRATIONAL NUMBERS 15 decide where to place x?”, where x was a value from one of the four blocks. See Table 1 for examples. Irrationals knowledge test. We constructed a novel paper-and-pencil test to measure conceptual and procedural reasoning about irrational numbers. Many items were adapted from literature documenting students’ misconceptions (Van Hoof et al., 2015). Correct items earned one point each; partial credit was not awarded. The conceptual items were distributed across three subsections. They required participants to (1) classify numbers like as either rational or irrational, (2) answer questions about the density of rational and irrational numbers, and (3) reason about the results of arithmetic operations on rational and irrational numbers. Most of these items were in the selected response format. The procedural items were distributed over two subsections. Subsection (4) required simplifying radical expressions and subsection (5) required performing arithmetic operations on multiple radical expressions. These were all fill-in-the-blank items. Example items from all five subsections are in Table 1. (https://osf.io/6s9pc/#). Magnitude comparison task. To investigate the magnitude representations of radical expressions, we created two sets of number pairs. The first set included natural number pairs formed from the combinations of 0, 1, 2, …, 9, with same-number pairs excluded (90 pairs). We were particularly interested in pairs where both numbers were perfect squares, like (4, 9) or (). Hence, we included an additional copy of each of the 12 perfect square pairs in the stimulus set. In total, 102 natural number pairs were used in blocks 1 and 2. Participants judged the greater of two numbers in the first block and the lesser in the second block. Natural number pairs were mirrored by 102 pairs of radical expressions formed in the same way from , , , …, . These pairs were used in blocks 3 and 4. Block 3 required greater judgments and block 4 required lesser judgments. Within each block, number pairs were presented in random order. Overall, there were 408 trials. Number line estimation task. Each of the four levels of the type variable was associated with one NLE block. The first block consisted of the natural number stimuli 0, 1, 2, …, 10. The second block consisted of the one-digit radical stimuli , , , …, . This block contained perfect squares and irrationals. The third block included perfect squares like , , , …, and the fourth block included two-digit radicals from , , , …, . These were all irrational numbers. Within each block, the 11 stimuli were presented in random order. Overall, there were 44 trials. Strategy questionnaire. Strategy prompts were developed to examine reasoning during the NLE task. Participants completed an eight-item paper-and-pencil strategy questionnaire composed of two prompts for each of the four blocks – natural numbers, one-digit radicals, perfect square radicals, and two-digit radicals. All prompts were in the form “How did you UNDERSTANDING IRRATIONAL NUMBERS Procedure After obtaining informed written consent, participants were seated alone in a quiet laboratory room in front of a Windows PC with an extended keyboard, a mouse, and a monitor measuring 55.6 cm diagonally. Magnitude comparison task. Participants first completed the MC task, implemented in the experimental design program OpenSesame 2.9.5 (Mathôt, Schreij, & Theeuwes, 2012). All number pairs were shown in white text against a black background. Each trial began with a blank screen for 250 ms, after which a fixation dot was shown in the center of the screen for 1000 milliseconds, followed by a blank screen for 500 milliseconds. A number pair was presented next, with each number offset about 5 centimeters on either side of the center. All numbers were UNDERSTANDING IRRATIONAL NUMBERS 16 displayed in the font Cambria Math (point 80). Participants indicated the larger or smaller number by pressing the key below the target number – “Z” for the number on the left and “M” for the number on the right. The stimuli were visible until participants made a response. Correct responses were followed by Correct in green text and incorrect responses by Incorrect in red text. This feedback was displayed for 500 milliseconds. Participants required about 6 minutes to complete each of the four blocks. Number line estimation task and strategy questionnaire. Next, participants completed the NLE task written in JavaScript and hosted online (www.psycholab.org/FreeLab). They were instructed to be as accurate as possible and that speed was irrelevant. On each trial, a natural number or radical expression was shown above a number line. The number line was only labeled at the endpoints, zero on the left and ten on the right. The middle was left blank. Participants estimated the number’s position on the line by left-clicking with a computer mouse. To prevent them from rushing through the task, a blank screen was shown for three seconds between trials and clicks during this interval were ignored. Feedback was not provided. Overall, participants needed about six minutes to complete this task. Afterward, they were given the strategy questionnaire to complete at their own pace. Irrationals knowledge test. Finally, participants completed the irrationals knowledge test without feedback or the use of electronic devices. Approximately 25 minutes were allotted to respond. Afterward, demographic information was collected, participants were debriefed, and compensated with $12 in cash. Results Results UNDERSTANDING IRRATIONAL NUMBERS 17 Magnitude Comparison Task The average accuracy across magnitude comparison blocks was high (98.2%). To determine whether there was a speed-accuracy tradeoff, we correlated the average response times and average accuracy of all participants. These variables were not significantly correlated (r = - .079, p = .486). Because of the ceiling effect in accuracy and the lack of a speed-accuracy tradeoff, subsequent analyses concentrated on the response time data only. We trimmed all trials on which participants made an incorrect response. This was 1.8% of the overall data. Following common practice in the literature, we also trimmed response times outside of the interval 200- 2000 ms (Varma & Karl, 2013). This was a further 2.6% of the overall data. Recall that the first research question asks whether the magnitudes of radical expressions are compared by referencing a mental number line or by using the equivalence strategy. The second research question, concerning the use of anchoring strategies, is also relevant here for answering whether perfect square radicals like () are processed more quickly via the multiplication strategy. Addressing these questions demanded sensitivity to the natural confound between size and perfect square status in our stimulus set. Between 0 and 10, there are three “small” perfect squares (0, 1, and 4), but only one “large” perfect square (9). Thus, the average size and distance of perfect squares pairs is smaller than that of non-perfect square pairs. To control for this confound, we used hierarchical linear regression. We explored whether the canonical distance and size effects replicated for natural number comparisons and extended to radical expression comparisons. The size effect is the finding that, controlling for distance, numerical magnitude comparisons require more time as the operands grow larger (Parkman, 1971). For instance, the pair (2, 3) can be compared more quickly than the pair (7, 8). Additionally, we investigated whether the type of a stimulus (natural UNDERSTANDING IRRATIONAL NUMBERS 18 numbers or radical expressions) and perfect square status (yes or no) affected RTs. If there is an advantage for comparing perfect squares, then it may only apply to radical expression pairs like (, ), but not to natural number pairs like (1, 4). Hence, we included the interaction between type and perfect square status in the hierarchical regression analysis. The dependent variable was RT and the five predictor variables were entered sequentially: distance (1 – 9), size (0.5 – 8.5), type (natural numbers or radical expressions), perfect square status (yes or no), and the type by perfect square interaction. We addressed the first research question in two hierarchical regression analyses. The first defined the distance and size variables by the radicand components of the radical expressions. This was consistent with the equivalence hypothesis. The second defined them by the actual magnitude values of the radical expressions. This was consistent with the mental number line hypothesis. The final model from the first analysis, which evaluated the equivalence strategy hypothesis, is in Table 2. Distance was entered during step 1; it was a significant predictor of RT, R2 = .278, F(1, 88) = 33.9, p < .001. Size was entered during step 2 and it accounted for significant additional variance, ∆R2 = .183, R2 = .461, F(1, 87) = 29.6, p < .001. Thus, we replicated the distance and size effects for natural numbers and extended them to radical expressions (Fig. 1). Number type was entered during step 3; it explained the most additional variance in RTs, ∆R2 = .471, R2 = .932, F(1, 86) = 598.5, p < .001. The positive β weight means that radical expressions required more time to compare than natural numbers. Perfect square status was entered during step 4; it did not explain significant additional variance in RTs, ∆R2 = .001, R2 = .993, F(1, 85) = 1.07, p = .303. The interaction between number type and perfect UNDERSTANDING IRRATIONAL NUMBERS 19 UNDERSTANDING IRRATIONAL NUMBERS square value was entered during step 5; it was also a nonsignificant predictor, ∆R2 = .000, R2 = .993, F(1, 84) = .01, p = .935. square value was entered during step 5; it was also a nonsignificant predictor, ∆R2 = .000, R2 = .993, F(1, 84) = .01, p = .935. Table 2. Final hierarchical linear regression models predicting RTs during magnitude comparison when distance and size were defined by (a) the radicand components and (b) the actual magnitude values of the radical expressions. Model Predictor Std. β t p R2 Equivalence Strategy Constant 107.49 .000 .933 Distance -.535 -18.40 .000 Size .434 15.06 .000 Type .687 22.68 .000 Perfect Square -.033 -.804 .424 Type X Perfect Square .003 .082 .935 Mental Number Line Constant 43.03 .000 .838 Distance -.481 -8.99 .000 Size .199 -3.74 .000 Type .687 14.56 .000 Perfect Square .024 .368 .714 Type X Perfect Square -.003 -.053 .958 UNDERSTANDING IRRATIONAL NUMBERS Second, although perfect squares may not be privileged on speeded tasks like magnitude comparison, they may benefit from special processing on unspeeded tasks like number line estimation and arithmetic problem-solving. For these reasons, we return to the second question with data from the NLE task. Finally, we estimated RTs using the predictors from the equivalence strategy model separately for each participant. There was considerable variation between participants in the magnitudes of the β weights for the five predictors (Fig. 2). Later, these individual differences will be used to predict performance on the irrationals knowledge test. XXX Fig. 1. Distance and size effects, defined by the radicands and consistent with the equivalence strategy hypothesis, were found for natural number and radical expression magnitude comparisons. Shaded regions are 95% confidence envelopes. To summarize, the first regression analysis defined the distance and size variables by the radicand components of radical expressions. The distance, size, and type effects were observed. In the second regression analysis, the distance and size variables were defined by the actual magnitude values of the radical expressions. The same three effects were observed (Table 2). Critically, the first analysis explained more of the variance in RTs than the second analysis UNDERSTANDING IRRATIONAL NUMBERS UNDERSTANDING IRRATIONAL NUMBERS 20 (93.3% versus 83.8%). Thus, these findings favor the equivalence strategy hypothesis over the mental number line hypothesis. Radical expressions like (, ) appear to be converted to natural numbers like (8, ) and then compared using the mental number line. Recall that the second research question asked whether people strategically anchor abstract irrational numbers on concrete natural numbers and perfect squares. The regression model favoring the equivalence strategy supports the idea that natural numbers are used as anchors. Moreover, both regression models indicate that perfect squares are not used as anchors – perfect square status and the type by perfect square status interaction were nonsignificant. Recall that the second research question asked whether people strategically anchor abstract irrational numbers on concrete natural numbers and perfect squares. The regression model favoring the equivalence strategy supports the idea that natural numbers are used as anchors. Moreover, both regression models indicate that perfect squares are not used as anchors – perfect square status and the type by perfect square status interaction were nonsignificant. People do not appear to speed comparison of natural number or radical expression pairs composed of perfect squares by automatically activating associated “tie” multiplication problems. However, there are two reasons to be cautious when interpreting this failed prediction. First, the distance, size, and type variables entered during the initial steps explained a large percentage of the variance in RTs. Little variability remained to be explained by the time the perfect square variables were entered. Second, although perfect squares may not be privileged on speeded tasks like magnitude comparison, they may benefit from special processing on unspeeded tasks like number line estimation and arithmetic problem-solving. For these reasons, we return to the second question with data from the NLE task. p q yp y p q g People do not appear to speed comparison of natural number or radical expression pairs composed of perfect squares by automatically activating associated “tie” multiplication problems. However, there are two reasons to be cautious when interpreting this failed prediction. First, the distance, size, and type variables entered during the initial steps explained a large percentage of the variance in RTs. Little variability remained to be explained by the time the perfect square variables were entered. UNDERSTANDING IRRATIONAL NUMBERS UNDERSTANDING IRRATIONAL NUMBERS 21 Number Line Estimation Task Data from one participant were lost due to computer failure. Thus, the analyses included responses from 80 participants. Due to a programming error, 5.8% of the natural number trials were lost. These lost trials were random and did not affect the results. We trimmed outlier responses by discarding trials for which the RT was outside of the interval 1.5 – 15 sec (4.6% of the overall data). Our analyses focused on the second research question: Do people use anchoring strategies to facilitate performance? Average absolute error, response time, and linearity. First, we analyzed average absolute error using a one-way ANOVA with type (natural numbers, one-digit radicals, perfect square radicals, and two-digit radicals) as the factor. See Fig. 3a. There was a main effect of type F(3, 316) = 14.13, p < .001, η2 = .118. Post-hoc comparisons using Tukey’s procedure found that average absolute error was comparable for natural numbers and perfect squares (p = .836). Error for these two number types was lower than for the one-digit radicals and two-digit radicals (ps < .001), which were comparable to each other (p = .706). XXX Fig. 2. Distributions of the β weights for the distance, size, type, perfect square, and type by perfect square interaction variables. UNDERSTANDING IRRATIONAL NUMBERS 22 two-digit radicals (ps < .001). Finally, response times were lower for one-digit radicals than for two-digit radicals (p < .001). two-digit radicals (ps < .001). Finally, response times were lower for one-digit radicals than for two-digit radicals (p < .001). How linear were participants’ estimates for natural numbers, perfect squares, one-digit radicals, and two-digit radicals? For each of the four types, best-fitting lines predicting the selected position from the actual position were plotted (Fig. 4). Overall performance was high across all four blocks. For natural numbers and perfect squares, the R2 values were both 1.00. For one-digit and two-digit radicals, the R2 values were .97 and 1.00, respectively. XXX Fig. 3. Average absolute error (a) and response times (b) on the number line estimation task revealed that, compared to natural numbers and perfect squares, one- and two-digit radicals w more difficult to process. Next, we analyzed response time using a one-way ANOVA with type as a factor, finding a main effect F(3, 316) = 27.95, p < .001, η2 = .210). See Fig. 3b. Post-hoc comparisons using Tukey’s procedure revealed that natural numbers and perfect squares were estimated equally quickly (p = .908). Response time for these two number types was lower than for one-digit and UNDERSTANDING IRRATIONAL NUMBERS XXX Fig.4. Number line estimation performance was highly linear on average. Fig.4. Number line estimation performance was highly linear on average. Fig.4. Number line estimation performance was highly linear on average. These analyses concerned the overall accuracy of estimates. To examine whether averaging performance concealed individual differences, we repeated them separately for each participant, estimating the best-fitting lines for each of the four number types (Fig. 5). Estimates for natural numbers and perfect squares were highly linear across participants. By contrast, there was greater variation in the linearity of participants’ estimates for one-digit radicals like and two-digit radicals like . XXX Fig. 5. Individual differences in the linearity of participants’ estimates were greatest for two-digit radicals. Estimation strategies. On the strategy questionnaire, participants self-reported the strategies they used for each block of the NLE task. After dropping illegible and incoherent responses, full data were available for 63 of 81 questionnaires. All 63 questionnaires were coded by the first author and a naïve volunteer. Interrater agreement, measured by Cohen’s kappa, UNDERSTANDING IRRATIONAL NUMBERS UNDERSTANDING IRRATIONAL NUMBERS 23 averaged .74 across the eight prompts. Participants reported using multiple strategies, summarized in Table 3. Table 3. Strategies self-reported on the number line estimation strategy questionnaire and their frequency of use. Number Type Strategy % Use Natural numbers landmark of 0, 5, or 10 93.7 other 6.3 Perfect squares simplify & map 95.2 other 4.8 One-digit radicals single landmark – 1 perfect square 26.2 double landmark – 2 perfect squares 43.7 other – correct 19.0 other – incorrect 7.9 Two-digit radicals single landmark – 1 perfect square 25.4 double landmark – 2 perfect squares 60.3 other – correct 2.4 other – incorrect 8.7 Table 3. Strategies self-reported on the number line estimation strategy questionnaire and their frequency of use. Given natural numbers, the most frequent strategy was to anchor on the nearby landmarks 0, 5, and 10, and to adjust slightly to the left or right (reported by 93.7% of participants). When estimating the position of the number 3, for instance, most participants found the midpoint (5) and then shifted their estimate slightly to the left. All additional strategies were categorized as other (6.3%). Given perfect squares like , most participants chose to simplify the expression to 3 and map it to the natural number line – the simplify & map strategy (95.2%). The use of this strategy, which is related to the equivalence strategy participants used for the MC task, suggests that perfect squares enjoy privileged processing for the NLE task. All other strategies were labeled other (4.8%). UNDERSTANDING IRRATIONAL NUMBERS 24 Given one-digit and two-digit radicals, participants continued to anchor on nearby landmarks during estimation. In this case, however, the landmarks were perfect squares. This finding informs the second research question. Two strategies were observed; we illustrate them using as an example. The single landmark strategy was to reason that and , so estimate slightly to the left of 3. The double landmark strategy was to bound the estimate from both sides. One might reason that , , and . Thus, one should estimate between 2 and 3. Participants were more likely to use the double anchor strategy than the single anchor strategy for unfamiliar two-digit radicals like (60.3%) than for familiar one-digit radicals like (40.7%). Correct, but rarely used strategies were categorized as “other – correct.” Vague, incorrect, or guess responses were categorized as “other – incorrect.” Next, we evaluated whether the use of landmark strategies improved accuracy when estimating the locations of one-digit radicals ( and ) and two-digit radicals ( and ). All four prompts were analyzed via one-way ANOVAs. The dependent measure was average absolute error and the factor was strategy – single landmark, double landmark, other-correct, and other- incorrect. See Table 4 for the results. For each prompt, there was a main effect of strategy (F(3, 58) > 5.28, p < .001, η2 > .215). Post-hoc comparisons using Tukey’s procedure revealed that the single landmark and double landmark strategies were associated with the lowest average absolute errors. Landmark strategies were most beneficial for two-digit radicals. The average absolute errors were lower than the “other – correct” strategies. This is perhaps because the distances between one-digit radicals are lower than between two-digit radicals. For one-digit radicals like and , the difference is about 1.4. Hence, multiple strategies can be used without compromising accuracy. In contrast, the difference between two-digit radicals like and is greater (about 4.5). For this reason, anchoring irrationals like to perfect squares facilitated estimation considerably. UNDERSTANDING IRRATIONAL NUMBERS 25 Table 4. ANOVA results evaluating whether different strategies on the NLE task are associated with average absolute errors. UNDERSTANDING IRRATIONAL NUMBERS Number Type Prompt F(3, 58) p η2 Post-hoc strategy comparisons One-digit radicals 5.28 < .001 .215 single landmark = double landmark = other – correct > other – incorrect 10.02 < .001 .341 single landmark = double landmark = other – correct > other – incorrect Two-digit radicals 14.66 < .001 .431 single landmark = double landmark > other – correct > other – incorrect 9.96 < .001 .340 single landmark = double landmark > other – correct > other – incorrect a Computed by Tukey’s procedure. Table 4. ANOVA results evaluating whether different strategies on the NLE task are associated with average absolute errors. Table 4. ANOVA results evaluating whether different strategies on the NLE task are associated with average absolute errors. a Computed by Tukey’s procedure. a Computed by Tukey’s procedure. Irrationals Knowledge Test All 81 participants completed a paper-and-pencil irrationals knowledge test composed of two sections – conceptual knowledge and procedural knowledge. These were in turn composed of five subsections – number classification, density knowledge, operation concepts, simple arithmetic, and complex arithmetic. Recall that Table 1 lists examples items from each subsection. Table 5 summarizes performance across all sections and subsections. Table 5. Performance on all sections and subsections of the irrationals knowledge Section Subsection Mean (%) SD Conceptual Number classification 75.1 20.4 Density knowledge 49.0 26.4 Operation concepts 66.9 22.6 average 63.6 17.2 Procedural Simple arithmetic 61.0 27.0 Complex arithmetic 35.8 35.2 average 48.4 29.3 5. Performance on all sections and subsections of the irrationals knowledge test. UNDERSTANDING IRRATIONAL NUMBERS 27 = = = = To evaluate whether arithmetic strategy use affected participants’ procedural scores on the irrationals knowledge test, a one-way ANOVA was conducted. The strategy factor had four levels: prime factorization, perfect squares factorization, both strategies, and other. The category “other” refers to participants who used incorrect strategies or guessed. There was a main effect of strategy on procedural scores (F(3, 76) = 51.59, p < .001, η2 = .671). Post-hoc comparisons with Tukey’s procedure revealed that procedural scores were comparable between participants who used the prime factorization strategy and those who used the perfect squares factorization strategy (p = .455). The only significant score differences were between participants who used “other” strategies and those who used using the remaining strategies (ps < .001). Although the use of the perfect squares strategy is not associated with higher accuracy on complex arithmetic problems than the use of the prime factorization strategy, it may reduce processing time and working memory demand. We could not test this possibility because we did not measure these dependent variables. UNDERSTANDING IRRATIONAL NUMBERS 26 To determine the number of constructs being measured across these subsections, we conducted a principal components analysis using varimax rotation. The two procedural subsections loaded onto a single procedural factor (eigenvalue = 2.3, 46.0% of variance explained) and the three conceptual subsections loaded onto a single conceptual factor (eigenvalue = 1.1, 22.8% of variance explained).2 Hence, subsequent analyses focused on average scores on the conceptual section and the procedural section. To address the second research question, which concerns the use of anchoring strategies during arithmetic problem-solving, we coded the strategies used on the complex arithmetic subsection of the irrationals knowledge test. Interrater agreement, measured by Cohen’s kappa, was .95. We focused on whether participants used inefficient or efficient strategies. The inefficient prime factorization strategy was used by only 12% of participants. It involves solving arithmetic problems by decomposing radicands into prime factors and then shifting pairs of common factors outside the radical sign. See Table 6 for an example. By contrast, 40% of participants used the perfect squares factorization strategy. It involves directly identifying the largest perfect squares factors of the radicand. This chunking strategy enabled them to solve problems using fewer operations (Table 6). Table 6. Strategy use on the complex arithmetic subsection of the irrationals knowledge test. Strategy Example Use (%) Prime Factorization = = = = = = = = = 12 Perfect Squares Factorization = = 40 2 See Fig. S1 of the supplementary materials for a plot of the two factors. Table 6. Strategy use on the complex arithmetic subsection of the irrationals knowledge test. Strategy Example Use (%) Prime Factorization = = = = = = = = = 12 Perfect Squares Factorization = = 40 2 See Fig. S1 of the supplementary materials for a plot of the two factors. See Fig. S1 of the supplementary materials for a plot of the two factors. Predicting Individual Differences in Knowledge of Irrationals Recall that the third research question concerned whether individual differences in the magnitude representation and strategic processing of radical expressions predicts conceptual and procedural knowledge of irrationals. To answer this question, we extracted two sets of predictor variables from each participant, as described above. The first set consisted of the β weights for the three significant variables when predicting response times on the magnitude comparison task (distance, size, and type). The β weights for the distance and size variables index the precision of UNDERSTANDING IRRATIONAL NUMBERS 28 magnitude representations and are common predictors of mathematical achievement in children (De Smedt et al., 2009). The β weight for the type variable indicates the additional time required to compare radical expressions versus natural numbers. In other words, it reflects the time needed to apply the equivalence strategy. The second set of predictor variables were the average absolute errors when estimating the positions of one-digit radicals and two-digit radicals on the NLE task. These variables reflect the efficiency of anchoring to neighboring perfect squares. The magnitude comparison variables were entered first and the NLE variables were entered second in hierarchical linear regressions predicting accuracy on the conceptual section and the procedural section. For the conceptual knowledge measure, the magnitude comparison variables added during the first step were nonsignificant (R2 = .074, F(3, 76) = 2.02, p = .119). Adding the NLE variables during the second step accounted for significant additional variance (∆R2 = .115, R2 = .188, F(2, 74) = 5.23, p = .007). In the final model, shown in Table 7, only the magnitude comparison variable type and the NLE variable two-digit radicals were significant predictors (ps < .05). Table 7. Final models predicting conceptual and procedural knowledge of irrationals. Measure Predictor Std. β t p R2 Conceptual Knowledge Constant 11.87 .000 .188 MC – β Distance .039 .271 .788 MC – β Size .053 .370 .712 MC – β Type .235 2.21 .030 NLE 1D Radicals -.022 -.184 .855 NLE 2D Radicals -.355 -2.97 .004 Procedural Knowledge Constant 8.28 .000 .322 MC – β Distance .125 .945 .348 MC – β Size -.045 -.343 .732 MC – β Type .151 1.55 .125 Table 7. Final models predicting conceptual and procedural knowledge of irrationa UNDERSTANDING IRRATIONAL NUMBERS 29 NLE 1D Radicals -.104 -.952 .344 NLE 2D Radicals -.411 -3.76 .000 For the procedural knowledge measure, the magnitude comparison variables accounted for significant variance in step 1 (R2 = .140, F(3, 76) = 4.14, p = .009), and the NLE variables for significant additional variance in step 2 (∆R2 = .182, R2 = .322, F(2, 74) = 9.91, p < .001). In the final model, only the NLE variable two-digit radicals was significant (Table 7). These findings inform the second and third research questions. With respect to the second research question, participants’ ability to estimate the spatial locations of two-digit radicals (all irrational numbers) predicted conceptual and procedural scores. UNDERSTANDING IRRATIONAL NUMBERS This indicates that strategically anchoring irrational numbers to perfect squares is crucial for succeeding on higher level tests of mathematical knowledge. Concerning the third research question, neither the distance nor the size variables from the magnitude comparison task predicted knowledge of irrationals in the final models. This suggests that although magnitude representations are significant predictors of RTs in the speeded magnitude comparison task, they are less important when predicting performance on unspeeded tasks measuring higher-level mathematical knowledge. Further, the significance of the magnitude comparison variable type for predicting conceptual scores supports the role of the equivalence strategy. Understanding when judgments of irrational numbers can and cannot be simplified to judgments of natural numbers depends on conceptual knowledge of number systems. UNDERSTANDING IRRATIONAL NUMBERS 31 classroom mathematics tests. The type variable for the magnitude comparison task, which indicates whether the stimuli are natural numbers or radical expressions, was a significant predictor of conceptual knowledge. We interpret this as reflecting the importance of selectively accessing the natural number components of radical expressions (i.e., radicands and roots) when reasoning about irrationals. Accuracy with two-digit radicals on the NLE task was a significant predictor of both conceptual and procedural knowledge. This reflects the cognitive value of anchoring irrational numbers to perfect squares. Nevertheless, we note that most of the variation in conceptual and procedural knowledge of irrational numbers was left unexplained. More generally, magnitude representations are necessary for arithmetic competence, but not to the same extent as for natural numbers, integers, and rational numbers. The role of magnitude representations seems to attenuate as the abstraction of number systems increases. Hence, alternative mechanisms must be invoked to explain arithmetic expertise with irrationals. UNDERSTANDING IRRATIONAL NUMBERS 30 To study how people understand highly abstract mathematical concepts, we used irrational numbers as a test case. Specifically, we addressed three research questions. The first asked whether radical expressions are compared solely by referencing a mental number line or using the equivalence strategy. We addressed this question by fitting two models to the magnitude comparison RT data. The first model, consistent with the equivalence strategy, defined the distance and size variables by the radicands’ magnitudes. The second model, consistent with the mental number line strategy, defined the distance and size variables by the actual magnitudes of the radical expressions. The equivalence strategy model fit the data best. The type variable, which indicates whether the stimuli being compared are natural numbers or radical expressions, was a significant predictor above and beyond the distance and size variables Collectively, these MC results suggest that people compare radical expressions by anchoring them on natural numbers. To study how people understand highly abstract mathematical concepts, we used irrational numbers as a test case. Specifically, we addressed three research questions. The first asked whether radical expressions are compared solely by referencing a mental number line or using the equivalence strategy. We addressed this question by fitting two models to the The second research question asked whether people anchor their understanding of abstract irrational numbers to concrete natural numbers and perfect squares. Examples of such anchoring, besides the equivalence strategy, are evident on the NLE task. Strategy self-reports suggest that people anchor their estimates of one-digit and two-digit irrationals to nearby perfect squares – the single and double landmark strategies. Anchoring strategies were also observed on the complex arithmetic subsection of the irrationals knowledge test. When simplifying radical expressions, 40% of participants factored radicands into perfect squares, a more efficient strategy than the prime factorization strategy used by 12% of participants. The third research question asked whether individual differences in the speed and accuracy with which irrational numbers are processed on the simple MC and NLE tasks predicts performance on measures of higher-level conceptual and procedural knowledge comparable to Theoretical Implications – Formal Anchoring Hypothesis Dominant accounts of numerical and arithmetic development emphasize the recruitment, repurposing, and refinement of perceptuo-motor and visuospatial systems (Blair, Tsang, & Schwartz, 2013; Landy, Allen, & Zednik, 2014; Siegler, 2016). Based on our results that natural numbers and perfect squares support an understanding of the irrationals on a range of tasks, we hypothesize that abstract mathematical concepts are understood by selectively recruiting mathematical representations that align with task demands, a process we call formal anchoring. Our hypothesis builds on broader evidence of the use of anchors in decision-making, landmarks in spatial navigation, and reference points on a range of tasks (Holyoak & Mah, 1982; Rosch, 1975; Siegler & Thompson, 2014). UNDERSTANDING IRRATIONAL NUMBERS 33 Formal anchoring also results in differential performance gains depending on the task to be performed. For MC and NLE, it is highly beneficial for answering quickly and accurately. As discussed earlier, the use of anchoring strategies on NLE improved participants’ accuracy for irrational numbers. In contrast, anchoring to perfect squares during arithmetic problem-solving was not associated with higher accuracy. The benefits afforded in this context are likely to be more modest and restricted to speed. UNDERSTANDING IRRATIONAL NUMBERS 32 Mathematical tasks like magnitude comparison, number line estimation, and arithmetic problem-solving can impose high computational demands on limited cognitive resources. Formal anchoring efficiently alleviates these demands in task-specific ways. Magnitude comparison with radical expressions, for instance, can be performed using an informal “guess and check” algorithm to determine the greater or lesser value in number pairs like . Instead, people use the more efficient equivalence strategy. Radical expression pairs are interpreted as natural number pairs like (7, 4) because the judgments are the same in both cases. On the NLE task, the computational demands of estimating the positions of one-digit and two-digit radicals are also high. People handle these demands by anchoring their estimates on neighboring perfect squares – the single and double landmark strategies. Arithmetic problem-solving on the irrationals knowledge test also benefitted from formal anchoring. Expressions like can be simplified laboriously via prime factorization: Mathematical tasks like magnitude comparison, number line estimation, and arithmetic problem-solving can impose high computational demands on limited cognitive resources. Formal anchoring efficiently alleviates these demands in task-specific ways. Magnitude comparison with radical expressions, for instance, can be performed using an informal “guess and check” algorithm to determine the greater or lesser value in number pairs like . Instead, people use the more efficient equivalence strategy. Radical expression pairs are interpreted as natural number pairs like (7, 4) because the judgments are the same in both cases. On the NLE task, the computational demands of estimating the positions of one-digit and two-digit radicals are also high. People handle these demands by anchoring their estimates on neighboring perfect squares – the single and double landmark strategies. Arithmetic problem-solving on the irrationals knowledge test also benefitted from formal anchoring. Expressions like can be simplified laboriously via prime factorization: or more efficiently via perfect square factorization or more efficiently via perfect square factorization A key feature of formal anchors is that they are task-specific. During NLE and arithmetic problem solving, perfect squares anchored people’s understanding of irrationals. On the magnitude comparison task, however, the appropriate anchors were natural numbers. To explain why particular concrete mathematical concepts are appropriate on some tasks but not others, it is useful to focus on the alignment between what a task demands and what a representation provides. An appropriate representation simplifies a task without altering its underlying nature. By doing so, the representation minimizes computational demands and optimizes performance. UNDERSTANDING IRRATIONAL NUMBERS 34 Evidence for the use of anchoring strategies extends to more abstract number systems. For integers, zero is often used as a cognitive anchor when performing magnitude comparison, number line estimation, line bisection, and arithmetic computations (Gullick & Wolford, 2013; Tsang et al., 2015; Tsang & Schwartz, 2009; Varma & Schwartz, 2011). Fractions are often processed in relation to unit fractions like ½ when engaging in magnitude comparison and number line estimation judgments (Fazio et al., 2016; Siegler & Thompson, 2014). When rational numbers are expressed as decimal proportions, their magnitudes are anchored to natural numbers (Varma & Karl, 2013). During number line estimation with decimals, quartiles like .25 and tenths like .10 are anchored upon (Houseworth, 2016). Table 8. A sample of formal anchors used across number systems and tasks. Naturals Integers Rationals Irrationals one-digit multi-digit fractions decimals Magnitude Comparison none multiples of 10 0 unit fractions, decimals naturals naturalsa Number Line Estimation 0, 5, 10 quartiles thirds 0 unit fractions, decimals quartiles tenths perfect squaresb Arithmetic Computation 10 multiples of 10 0 ? ? perfect squaresc a Supported by the equivalence strategy on the MC task. b Supported the single and double landmark strategies on the NLE task. c Supported by the perfect squares factorization strategy on the irrational knowledge test. Table 8. A sample of formal anchors used across number systems and tasks. sample of formal anchors used across number systems and tasks. pp y q gy b Supported the single and double landmark strategies on the NLE task. g g ted by the perfect squares factorization strategy on the irrational knowledge test. Formal Anchoring Varies Across Number Systems and Tasks Our formal anchoring hypothesis may unify a constellation of seemingly disparate phenomena in the mathematical cognition literature. Here, we consider examples that apply to natural numbers, integers, rationals, and irrationals (Table 8). For natural numbers, formal anchoring strategies are not needed because the comparison of one-digit numbers is directly supported by magnitude representations (Moyer & Landauer, 1967). In contrast, number line estimation with one-digit numbers is performed by referring to the midpoint 5 and the endpoints 0 and 10 (Barth & Paladino, 2011; Opfer et al., 2016; Siegler & Thompson, 2014). Further, natural number arithmetic can be supported by anchoring to 10, as when the problem 9 + 8 is solved by decomposition: 9 + 8 = 9 + (1 + 7) = (9 + 1) + 7 = 10 + 7 = 17 (Baroody, Purpura, Eiland, Reid, & Paliwal, 2015). Comparisons of multi-digit numbers are anchored to intervening multiples of 10 (Brysbaert, 1995; Franklin, Jonides, & Smith, 2009). When estimating the positions of multi-digit natural numbers, midpoint and endpoint landmarks are also referenced (Dotan & Dehaene, 2013; Peeters, Verschaffel, & Luwel, 2017). For arithmetic with multi-digit numbers, the anchors are often multiples of 10, as when 49 + 38 is transformed to 50 + 37 (Torbeyns, de Smedt, Ghesquiere, & Verschaffel, 2009). For natural numbers, formal anchoring strategies are not needed because the comparison of one-digit numbers is directly supported by magnitude representations (Moyer & Landauer, 1967). In contrast, number line estimation with one-digit numbers is performed by referring to the midpoint 5 and the endpoints 0 and 10 (Barth & Paladino, 2011; Opfer et al., 2016; Siegler & Thompson, 2014). Further, natural number arithmetic can be supported by anchoring to 10, as when the problem 9 + 8 is solved by decomposition: 9 + 8 = 9 + (1 + 7) = (9 + 1) + 7 = 10 + 7 = 17 (Baroody, Purpura, Eiland, Reid, & Paliwal, 2015). Comparisons of multi-digit numbers are anchored to intervening multiples of 10 (Brysbaert, 1995; Franklin, Jonides, & Smith, 2009). When estimating the positions of multi-digit natural numbers, midpoint and endpoint landmarks are also referenced (Dotan & Dehaene, 2013; Peeters, Verschaffel, & Luwel, 2017). For arithmetic with multi-digit numbers, the anchors are often multiples of 10, as when 49 + 38 is transformed to 50 + 37 (Torbeyns, de Smedt, Ghesquiere, & Verschaffel, 2009). pp y q gy b Supported the single and double landmark strategies on the NLE task. Supported by the perfect squares factorization strategy on the irrational knowledge Instructional Implications Our results have three potential implications for instructional design in mathematics. First, performance patterns on the MC and NLE tasks suggest that most people represent the magnitudes of irrationals accurately. Nonetheless, some people placed irrationals on the number line haphazardly. Drawing on the cognitive science literature on learning and instruction UNDERSTANDING IRRATIONAL NUMBERS 35 (Schwartz & Bransford, 1998), we recommend teaching the compression of the irrationals on the number line by contrasting their nonlinear spacing with the linear spacing of other numbers. Students may benefit from explicit strategy instruction to estimate the values of irrational numbers by referencing perfect squares via the single and double landmark strategies. The second pedagogical implication is based on strategy differences during arithmetic problem-solving. Participants who used the prime factorization strategy required more steps and likely spent more time on task. Those who used the perfect squares factorization strategy could skip steps by recognizing the perfect square factors under the radical signs. Not all learners discover such strategies on their own. Hence, the perfect squares factorization strategy should be taught during formal instruction. Doing so may improve the adaptive expertise of students, or their ability to recognize and apply optimal strategies (Hatano & Oura, 2003). Third, it is worth noting that the correlation between magnitude knowledge and arithmetic expertise is weaker for irrationals than other number systems. Although interventions that improve magnitude knowledge result in greater arithmetic expertise for natural numbers, integers, and rational numbers (Fuchs et al., 2013; Siegler & Ramani, 2009; Tsang et al., 2015), similar learning gains may be unlikely for the irrationals. The individual differences analyses did not find a relationship between the precision of magnitude representations as measured by distance and size effects and conceptual and procedural knowledge of irrationals. We tentatively suggest a generalizable guideline for teaching highly abstract mathematical concepts like the irrationals. Instruction should focus on strategies for anchoring abstractions to task-appropriate formal concepts. UNDERSTANDING IRRATIONAL NUMBERS 36 Most research in numerical cognition focuses on relatively concrete concepts like natural numbers, integers, and rational numbers. Our study of irrationals hints at how the mind grasps abstract mathematical concepts incapable of being directly perceived or fully grounded in visuospatial referents. We suggest that the numerical magnitudes and symbolic procedures associated with irrationals are understood by task-specific anchoring to natural numbers and perfect squares. 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Individual differences in the linearity of participants’ estimates were greatest for two-digit radicals.
https://openalex.org/W2020322297	https://www.scielo.br/j/tce/a/5MYTbsYbmysYRp6PKcpqjGD/?lang=pt&format=pdf	Portuguese	null	O alcoolismo na história de vida de adolescentes: uma análise à luz das representações sociais	Texto & contexto enfermagem	2,013	cc-by	7,036	ALCOHOLISM IN ADOLESCENTS’ LIFE HISTORIES: AN ANALYSIS IN THE LIGHT OF SOCIAL REPRESENTATIONS ABSTRACT: The study aimed to describe adolescents’ social representations of alcoholism and to analyze alcoholism’s implications in the adolescents’ life histories. It is qualitative-descriptive research, which used the life history method for data collection from 40 adolescents, concomitantly with the technique of non-participant observation. Thematic content analysis led to two categories: ‘Alcoholic drinks, the good and the bad’, and ‘Alcoholism and its consequences’. Adolescents’ social representations on alcohol couple it to two symbolic meanings: the association of alcoholic drinks with pleasure and fun, and the negative aspect of its use, related to violence and the loss of one’s senses. It is concluded that living with alcoholism in the family influences how the adolescents perceive alcohol throughout their lives. DESCRIPTORS: Alcoholism. Nursing. Adolescents. Life history. Social psychology. g g O ALCOOLISMO NA HISTÓRIA DE VIDA DE ADOLESCENTES: UMA ANÁLISE À LUZ DAS REPRESENTAÇÕES SOCIAIS1 Sílvio Éder Dias da Silva2, Maria Itayra Padilha3 1 Este texto é parte da tese - História de vida e alcoolismo: representações sociais sobre o alcoolismo, apresentada ao Programa de Pós-Graduação em Enfermagem (PEN) da Universidade Federal de Santa Catarina (UFSC), em 2010. p p ç p g de Pós-Graduação em Enfermagem (PEN) da Universidade Federal de Santa Catarina (UFSC), em 2010. 2 Doutor em Enfermagem. Professor Adjunto II da Faculdade de Enfermagem da Universidade Federal do Pará. Belém, Pará, Brasil. E-mail: silvioeder2003@yahoo.com.br ç g ( ) ( ), 2 Doutor em Enfermagem. Professor Adjunto II da Faculdade de Enfermagem da Universidade Federal do Pará. Belém, Pará, Brasil. E-mail: silvioeder2003@yahoo.com.br y 3 Doutora em Enfermagem. Professora Associado do Departamento de Enfermagem e do PEN/UFSC. Pesquisadora do CNPq. Florianópolis, Santa Catarina, Brasil. E-mail: padilha@ccs.ufsc.br RESUMO: O estudo tem como objetivos descrever as representações sociais de adolescentes sobre alcoolismo e analisar as implicações do alcoolismo na história de vida dos adolescentes. Trata-se de uma pesquisa qualitativo-descritiva, que utilizou o método de história de vida para coleta de dados com 40 adolescentes, concomitantemente à técnica de observação livre. A análise de conteúdo temática levou a duas categorias: ‘O bom e o ruim das bebidas alcoólicas’ e ‘Alcoolismo e suas consequências’. As representações sociais dos adolescentes sobre o álcool o atrelaram a dois significados simbólicos: a associação da bebida alcoólica com o prazer e a diversão, e a negatividade do seu uso, relacionada à violência e perda dos sentidos. Conclui-se que o convívio com o alcoolismo na família influencia o modo como os adolescentes percebem o álcool no decorrer de sua vida. DESCRITORES: Alcoolismo. Enfermagem. Adolescente. História de vida. Psicologia social. - 576 - Artigo Original - 576 - Artigo Original OBJETIVOS Descrever as representações sociais de ado- lescentes sobre alcoolismo e o hábito de consumir bebidas alcoólicas e analisar as implicações do alcoolismo nas histórias de vida dos adolescentes. O contato precoce dos adolescentes com as bebidas alcoólicas é relevante para o surgimento do alcoolismo. A Organização Mundial de Saú- de (OMS) considera a doença um problema de saúde pública, uma vez que impõe à sociedade uma carga considerável de agravos indesejáveis. Atualmente, estima-se que a consequência do ál- cool corresponda a 1,5% das mortes entre adultos que são alcoolistas.4 A OMS enfatiza, também, que é uma ação impactante à população, devido a alta mortalidade e incapacidade que ocasiona. Essa realidade se faz presente tanto em países desen- volvidos quanto naqueles em desenvolvimento. Por tal motivo, o consumo excessivo de bebidas alcoólicas é considerado importante fator de risco nas projeções da próxima década – 2010 a 2020.4 EL ALCOHOLISMO EN LAS HISTORIAS DE VIDA DE LOS ADOLESCENTES: UNA ANALISE A LA LUZ DE LAS REPRESENTACIONES SOCIALES RESUMEN: El estudio tiene como objetivo describir las representaciones sociales de los adolescentes acerca del alcoholismo y analizar las consecuencias del alcoholismo en la historia de vida de los adolescentes. Se trata de una investigación descriptiva-cualitativa que empleó el método de la historia de la vida para recolectar los datos con 40 adolescentes concomitantemente con la observación libre. El análisis de contenido dirigido a dos categorías: Lo bueno y lo malo del alcohol y el alcoholismo y sus consecuencias. Las representaciones sociales de los adolescentes sobre el alcohol lo entrelazaron a dos significados simbólicos: la asociación de alcohol con el placer y la diversión, y la negatividad de su uso, relacionados con la violencia y pérdida del conocimiento. Se llegó a la conclusión de que vivir con el alcoholismo en la familia influye en la manera en que los adolescentes perciben el alcohol durante su vida. DESCRIPTORES: Alcoholismo. Enfermería. Adolescentes. Historia de vida. Psicología social. Texto Contexto Enferm, Florianópolis, 2013 Jul-Set; 22(3): 576-84. O alcoolismo na história de vida de adolescentes: uma análise... O alcoolismo na história de vida de adolescentes: uma análise... - 577 - INTRODUÇÃO Além dos problemas de saúde ocasiona- dos por seu uso nos indivíduos de qualquer faixa etária, é na adolescência que esta questão toma vulto diferenciado, em virtude do momento/fase da vida em que se encontram.3 INTRODUÇÃO lidando com fortes pressões socioeconômicas, com padrões educacionais ríspidos e punitivos no relacionamento com os filhos, aturdidas com o embate de valores culturais, tendo muitas vezes como única possibilidade de lazer a ingestão de bebidas alcoólicas. Esses fatores, quando aliados aos hábitos de seu uso abusivo, têm sido associa- dos, frequentemente, à violência intrafamiliar.6 A problemática do álcool é uma questão de saúde pública que atinge a população mun- dial em todas as faixas etárias.1 A adolescência é um período crítico na vida das pessoas, no qual ocorrem novas descobertas significativas que são fundamentais para a construção da personalidade e da individualidade, sob o ponto de vista biopsi- cossocial. Nesse período, o conceito de interação grupal é perceptível e o adolescente busca perten- cer a um grupo com o qual se identifica, que terá a capacidade de influenciar suas ações e fará com que ele adote atitudes que serão a prova de sua aceitação na “tribo”.2 Cabe mencionar que tribo, etimologicamente, diz respeito à comunidade ét- nica, porém, o termo como está sendo empregado, diz respeito à formação de adolescentes que têm em comum os mesmos ideais, compondo assim um grupo de pertença consensual.2 O alcoolismo, ao ser inserido no cotidiano do adolescente, passa a fazer parte do seu cognitivo e de sua comunicação com o seu grupo de pertença, passando a doença para uma dimensão psicos- social, que será fundamental para adoção de um comportamento diante de uma droga tão presente na sua rotina familiar. Por tal motivo, compreen- der a relação entre a história de vida dos jovens ao conviverem com um membro da família que é alcoolista irá propiciar um melhor entendimento de sua representação sobre a doença e, a partir dela, a sua atitude frente às bebidas alcoólicas. É flagrante que as questões relevantes à saúde dos adolescentes são primordiais para com- preender as razões que os levam ao uso e abuso de drogas. Além dos problemas de saúde ocasiona- dos por seu uso nos indivíduos de qualquer faixa etária, é na adolescência que esta questão toma vulto diferenciado, em virtude do momento/fase da vida em que se encontram.3 É flagrante que as questões relevantes à saúde dos adolescentes são primordiais para com- preender as razões que os levam ao uso e abuso de drogas. Texto Contexto Enferm, Florianópolis, 2013 Jul-Set; 22(3): 576-84. METODOLOGIA A iniciativa visa retirá-los das ruas e envolvê-los em atividades socioeducativas.9 Os sujeitos do estudo foram 40 adolescentes de ambos os sexos, sendo 30 do sexo masculino e 10 do feminino, que participam do Projeto Tribos Urbanas. Os critérios de inclusão foram: estar na faixa etária entre 12 e 20 anos; fazer parte do pro- grama da instituição mencionada; ter a permissão dos adolescentes e de seus responsáveis legais para participação no estudo; conviver com um familiar alcoolista; e fazer uso de bebidas alcoólicas. O número de sujeitos da pesquisa deve ser represen- tativo de um grupo, e neste sentido empregamos a técnica da saturação de dados, que diz respeito à repetição dos discursos, como forma de delimitar a amostragem deste estudo.10 Destaca-se que a satu- ração dos dados obteve-se na trigésima entrevista, visto já se repetirem as respostas. Mesmo assim, foram feitas mais 10 entrevistas para reforçar o cri- tério de saturação. Outro aspecto relevante é que a pesquisa do tipo levantamento de dados preconiza trabalhar com mais de 30 depoentes, o que justifica o total de 40 depoentes. Ressalta-se, também, estes terem familiares alcoolistas que é um critério para se captar RSs, pois elas somente existem a partir do fenômeno vivenciado. Por conta disso era ne- cessário que os jovens entrevistados convivessem com o fenômeno alcoolismo, por meio da interação cotidiana com o familiar alcoolista. Outra técnica empregada para validar os da- dos foi a observação. Esta técnica foi utilizada pelo fato de permitir a captura, por parte do entrevis- tador, do comportamento global do entrevistado, durante todo o percurso da entrevista, que servi- ram como dados importantes para a conclusão do estudo. Na observação devem ser valorizados, além dos componentes físicos da palavra, também os múltiplos elementos da apresentação pessoal, aspectos do comportamento global e em particular a comunicação (linguagem) não-verbal do infor- mante.12 Esta técnica possibilita um contato pessoal e estreito do pesquisador com o fenômeno pesqui- sado. A observação permitiu captar o gestual e a emergência de sentimentos que surgiam a cada aplicação das técnicas de coletas já mencionadas. Estes dados foram fundamentais para uma melhor compreensão do enfoque afetivo que circundava as representações sociais do grupo em questão.12 A observação, semelhante à entrevista, possui várias modalidades, dentre as quais op- tou-se pela observação livre. Nesta modalidade, o investigador fica fora da comunidade que deseja observar, anotando espontaneamente tudo que lhe parecer conveniente. METODOLOGIA Este estudo é do tipo descritivo-exploratório, com o emprego de abordagem qualitativa, utili- zando o método de história de vida para captar as Representações Sociais (RSs) dos sujeitos do estudo acerca do tema em questão. Tal método é tido como uma vertente da história oral, que se compõe como um autêntico e eficiente instrumento de investigação quando o pesquisador atribui um aspecto científico a seu estudo.7 A história de vida consiste na narrativa contada pelo sujeito, servindo como ponte de interação entre o indivíduo e o meio social, tendo como principal característica a rememoração so- bre algum aspecto da vida desse indivíduo que influencia no seu viver. A abordagem qualitativa foi selecionada, pois permite investigar o objeto de estudo por meio da apreensão do universo sub- jetivo de um determinado grupo de indivíduos. Ressalta-se que essa modalidade de estudo tem como fundamento uma relação dinâmica, uma interdependência viva entre o indivíduo e o objeto, um vínculo indissociável entre o mundo objetivo e a subjetividade do sujeito.8 A história de vida pos- sibilita a compreensão do universo do adolescente O fato do adolescente conviver na família com um ou mais alcoolistas pode influenciar positiva ou negativamente na formação deste indivíduo. Filhos de dependentes químicos do álcool apresentam risco elevado para o consumo de bebidas alcoólicas, quando comparados com filhos de não-dependentes, numa proporção de risco aumentado em quatro vezes para o desen- volvimento do alcoolismo.5 Evidencia-se que a imagem da família atual é de grupos humanos Silva SED, Padilha MI - 578 - fundamental para captação de dados de uma pesquisa, pois a fala que emerge a partir de sua realização é reveladora de categorias estruturais, de princípios de valores, normas e símbolos e, ao mesmo tempo, tem a magia de transmitir, por meio de um porta-voz, as representações de grupos determinados, em condições históricas, socioeconômicas e culturais específicas.11 Como forma de respeitar o anonimato dos adolescentes foi empregado o sistema alfanumérico, utilizando- se E de entrevista, seguida pelo número de ordem do depoimento. a partir de seu passado com a construção de suas representações sociais sobre o alcoolismo, que se- rão primordiais para sua tomada de atitude frente à prática social de consumo de bebidas alcoólicas. O campo de pesquisa foi o Projeto Tribos Urbanas, que é um programa da Prefeitura de Belém-PA, criado em 2006, com o objetivo de atender jovens e adolescentes que se envolvem com gangues. Grupo de adolescentes organizados que infligem a lei. Texto Contexto Enferm, Florianópolis, 2013 Jul-Set; 22(3): 576-84. Caracterização dos sujeitos O caminho teórico que se optou ao produzir este estudo considera relevante a relação entre sujeito e objeto, favorecendo a compreensão da construção simbólica dos adolescentes sobre o al- coolismo. É necessário, ainda, conhecer o contexto sociocultural do indivíduo, por propiciar o vínculo entre o seu universo e suas representações sociais. Outro ponto a considerar são as experiências an- teriores e pontos comuns, que favorecem tanto a adequação dessas construções simbólicas quanto sua aceitação pelo grupo do qual faz parte. No que se refere ao estado civil, destacou- se que entre os homens 29 são solteiros e um mantém união estável. Já entre as mulheres, são oito solteiras, uma mantém união estável e uma é casada. Considerando-se a idade dos sujeitos do estudo, este dado vai ao encontro da realidade brasileira, na qual os adolescentes, na sua maioria, são solteiros. Entre os casados, 100% são mulheres que, muitas vezes, assumem o casamento como forma de sair de casa ou por terem engravidado neste período. Cabe enunciar que os sujeitos não são somen- te receptores pessoais de ideologias dominantes produzidas e veiculadas por classes sociais, por meio das instituições sociais, tais como igreja, estado, escola, entre outras. Existe a opção autô- noma, de modo que eles constantemente estão produzindo e comunicando representações que compartilham com seus grupos, que têm influên- cia decisiva sobre suas relações, suas escolhas e suas vidas. Assim, os indivíduos sempre estão trocando conhecimentos que encontram em seu cotidiano, por meio de discussões que se realizam no trabalho e em outros locais. Quanto ao grau de escolaridade, entre os ho- mens observou-se que 16 têm o nível fundamental incompleto, 13 têm o segundo grau incompleto e um tem o ensino médio completo. Já entre as mulheres, oito apresentam o ensino fundamental incompleto e duas apresentam o ensino médio incompleto. No Projeto Tribos Urbanas, os adolescentes fazem cursos profissionalizantes para capacitá-los ao primeiro emprego. As profissões nas quais os homens se formaram foram: três como operadores de caixa, dois como torneiros mecânicos, quatro em informática básica, três em segurança eletrôni- ca, um pintor, um em mecânica automotiva, três como eletricista predial e industrial, sendo que 13 adolescentes ainda estavam fazendo os cursos. En- tre as mulheres, três ainda estavam fazendo cursos profissionalizantes, três se formaram garçonetes, uma cabeleireira, duas operadoras de caixa e uma em informática básica. METODOLOGIA Para realização desta etapa utilizou-se um diário de campo, no qual foram anotadas todas as impressões percebidas no transcorrer do trabalho de campo, evidencia-se que todas foram realizadas logo após o término das entrevistas.12 A pesquisa foi orientada pela Portaria 196/96 do Conselho Nacional de Saúde e foi aprovada pelo Comitê de Ética da Universidade Federal do Pará, recebendo o número de protocolo 004/08 CEP-ICS/UFPA. O período da coleta dos dados foi de março a julho de 2009. A técnica de coleta das narrativas dos adolescentes para produção de fontes orais foi a entrevista semiestruturada, orientada por roteiro composto de 15 perguntas, contextualizando da infância até a adolescência dos jovens, o que possibilitou compreender a in- fluência dos seus familiares alcoolistas na adesão do adolescente ao uso de álcool. Cabe esclarecer que a entrevista semiestruturada é uma técnica Para trabalhar as informações, optou-se pela análise temática, a qual consiste na significação que se depreende do texto, permitindo sua interpreta- ção sob o enfoque da teoria que guia o estudo. Esta técnica de análise propicia conhecer uma realidade por meio das comunicações de indivíduos que tenham vínculos com ela.13 Buscou-se desdobrar a análise temática em três etapas: 1ª) a pré-análise, que consistiu na seleção e organização do material, * Grupo de adolescentes organizados que infligem a lei. Texto Contexto Enferm, Florianópolis, 2013 Jul-Set; 22(3): 576-84. - 579 - O alcoolismo na história de vida de adolescentes: uma análise... mínimo e meio, uma tem dois salários mínimos e duas têm três salários mínimos. quando realizou-se a leitura flutuante e a consti- tuição do corpus; 2ª) a exploração do material; e 3ª) o tratamento dos dados.13 Ao final da análise, chegou-se às seguintes categorias temáticas: O bom e o ruim das bebidas alcoólicas e Alcoolismo e suas consequências. Percebe-se nesses dados que a maioria dos adolescentes pertence a famílias que têm renda de, no máximo, um salário mínimo e meio, o que significa fazerem parte da classe econômica e social considerada baixa. Este fato dificulta o acesso à ali- mentação adequada, escolas adequadas e cuidado à saúde orientado. Texto Contexto Enferm, Florianópolis, 2013 Jul-Set; 22(3): 576-84. O bom e o ruim das bebidas alcoólicas Nesta unidade temática, fica notório como os adolescentes vislumbram em suas RSs da bebida alcoólica algo benéfico, visto esta propiciar espon- taneidade e descontração. Porém, eles também destacam que o consumo em excesso, que leva à embriaguez, é um fato danoso para sua saúde física e mental. Outro ponto identificado foi de que, apesar de o consumo do álcool fazer parte das atividades festivas de determinados grupos sociais e de ser considerado normal se ingerido sem provocar em- briaguez, os adolescentes conseguem identificar os seus efeitos negativos quando ele é consumido de forma excessiva, o que pode ser identificado nas seguintes falas: Por ser consumida por vários povos e cultu- ras diferentes, a bebida alcoólica adquiriu alguns significados positivos na população mundial, pois, por meio de sensações como as de relaxar e de se divertir, o indivíduo abstrai a ideia de “bom” no consumo do álcool. [...] o álcool é ruim porque deixa a gente de ressaca no outro dia. A gente gasta dinheiro para beber e tudo. A pessoa acorda no outro dia liso com dor de cabeça, bafo escroto na boca, não sabe o que fez, se brincou ou arrumou confusão. A gente não sabe o que faz (E10). O álcool é bom porque faz a gente viajar. Quando a gente bebe, ficamos mais empolgados, se acha o tal. Eu bebia muito, porque achava que o meu problema era em casa, porque a vovó me criticava muito, porque eu fumava cigarro e bebia, aí eu achava que fazendo aquilo eu ia esquecer os problemas de casa. Outra questão para gente beber é a influência dos amigos, pois para ficar perto dos moleques lá tinha que beber, porque senão era careta, era mocinha, essas frescuras de macho (E2 ). [...] o álcool, quando é bebido de forma excessiva, ocasiona um grande malestar físico e psicológico. Além do que, a pessoa faz às vezes um monte de besteira sobre o efeito do álcool, que, depois, se arrepende (E1). Diante das falas expostas, apreendeu-se que o significado “ruim” em relação ao consumo do álcool foi relacionado ao não-controle da quantida- de ingerida. Em outro momento, o sentido mudou seu enfoque para a perda financeira resultante do uso excessivo de bebidas alcoólicas. Caracterização dos sujeitos Nessa perspectiva, buscou-se caracterizar o universo dos sujeitos do estudo com dados re- ferentes à identificação (nome, endereço, idade, sexo, estado civil, número de filhos, procedência, naturalidade, escolaridade, profissão e religião). Os sujeitos do estudo foram 40 adolescentes, sendo 30 do sexo masculino e 10 do feminino. A faixa etária entre os homens foi de 15 a 20 anos, com predominância da faixa etária de 17 a 20 anos. Já entre as mulheres, a idade variou entre 15 e 19 anos, sendo predominante a faixa etária de 16 e 19 anos. Quanto à atividade de lazer no Programa Tribus Urbanas, ocorreu a seguinte distribuição entre os depoentes do sexo masculino: três não possuem atividade de lazer, 14 jogam futebol, nove têm como forma de lazer as festas, um rea- liza passeios e três frequentam igrejas. Entre os depoentes do sexo feminino duas não possuem atividade de lazer, duas jogam futebol, quatro possuem as festas como lazer, uma realiza passeios e uma joga capoeira. No que se refere à renda mensal da família dos sujeitos da pesquisa, notou-se, que entre os homens, 11 têm como renda familiar um salário mínimo, sete têm um salário mínimo e meio, seis têm dois salários mínimos, quatro têm meio salário mínimo e dois têm três salários mínimos. Entre as mulheres, observou-se que cinco têm renda fami- liar de um salário mínimo, duas têm um salário No que se refere à religião, observou-se que entre os homens dois referiram não ter religião, 12 são católicos e 16 são evangélicos. Entre as Silva SED, Padilha MI - 580 - sujeitos deste estudo está relacionada ao consumo ocasional, ao entretenimento, embora muitas vezes o consumo não se restrinja a estas variáveis. O consumo de qualquer bebida alcoólica representa para as pessoas algo “normal”. Este é considerado excessivo quando culmina com pessoas bêbadas e origina condutas agressivas que ultrapassam as regras do convívio social.14 mulheres, uma referiu não ter religião, quatro são católicas e cinco são evangélicas. RESULTADOS E DISCUSSÃO Na primeira unidade temática, O bom e o ruim das bebidas alcoólicas, evidenciam-se os pontos positivos e negativos do consumo de álcool pelos adolescentes. Na segunda unidade, Alcoo- lismo e suas consequências, estão presentes os fenômenos representacionais dos adolescentes as consequências geradas pelo alcoolismo, presente em um familiar alcoolista que, na maioria das vezes, era o pai ou a mãe, no seu cotidiano. Uma das características dos adolescentes é a percepção de que nada acontecerá e de que podem controlar todas as situações. Esse aspecto leva-os a ter uma menor percepção do risco, e pode aumentar o uso de drogas. Dessa forma, as sensações obtidas durante o efeito do álcool no organismo tornam-se prazerosas, porém a busca de novas e intensas sensações pode tornar o jovem um dependente químico.15 Texto Contexto Enferm, Florianópolis, 2013 Jul-Set; 22(3): 576-84. O bom e o ruim das bebidas alcoólicas A bebida alcoólica deixa a gente mais solto, alegre para aproveitarmos as festas, ela acaba com a minha ini- bidez, mas também ela não pode ser bebida em excesso, porque a gente só faz besteira, e no outro dia o mal-estar é muito grande (E7). O meu pai bebia muito, ele gastava tudo que ganhava. O álcool fez ele perder o emprego e ele passou a ser autônomo, mas mesmo assim todo dinheiro que ele ganhava com os trabalhos, ele gastava com bebidas alcoólicas. Ele bebia até cair (E13). Durante a fala dos adolescentes, identificou- se a relação da bebida alcoólica com a sensação de calmaria, com os sentimentos de empolgação e diversão, com fortalecimento de vínculos afetivos, como a amizade, e também a correlação do uso do álcool com o esquecimento dos problemas. Também foram percebidas RSs que retratam a bebida alcoólica como algo “ruim”, acarretado pelo exagerado e também pela “ressaca”, a qual representa um conjunto de sintomas, tais como náuseas, vômitos, cefaléia, desidratação e amnésia alcoólica. Ressalta-se que, apesar de amplamente aceito e até estimulado socialmente, o consumo de Vários subgrupos sociais veem as bebidas alcoólicas como forma de proporcionar alegria. A avaliação positiva para o consumo do álcool pelos O alcoolismo na história de vida de adolescentes: uma análise... - 581 - ção por parte dos profissionais de saúde e solicitam respostas e políticas públicas apropriadas, que se proponham a resolver ou ao menos minimizar esses problemas, nas distintas sociedades. O co- nhecimento sobre as questões relacionadas ao uso de substâncias e às dependências químicas possui ainda muitas lacunas a serem preenchidas, daí a importância de se somarem esforços provenientes de representantes de todos os segmentos sociais: políticos, legisladores, pesquisadores, profissio- nais de saúde e outros grupos da sociedade civil.14 álcool, quando excessivo, torna-se um problema importante e acarreta altos custos para a socieda- de. As intercorrências causadas pelo álcool extra- polam as amplamente divulgadas na literatura, caracterizando-o como um problema que ocasiona graves consequências sociais e colocando-o entre os principais problemas de saúde pública da atualidade.15 Cabe considerar que as pessoas dependentes de álcool apresentam uma série de episódios rela- cionados ao abuso antes de serem diagnosticadas como alcoolistas e encaminhadas para tratamento específico. O bom e o ruim das bebidas alcoólicas Considerando que a porta de entrada para os serviços de saúde são as unidades de atenção primária é possível inferir que muitos indivíduos com padrão de consumo abusivo são atendidos nesses serviços, em decorrência das complicações precoces do alcoolismo, tais como traumas, mal-estar, dentre outras. Em diversos momentos, quando indagados sobre o motivo que leva um ser humano a apro- ximar-se da bebida alcoólica, os entrevistados, em sua maioria, relatam os problemas de caráter fami- liar ou social. Sabe-se que os eventos estressantes da vida são subjetivos para cada indivíduo, e que a capacidade de superação depende de fatores que implicam, a princípio, a maturidade da pessoa no contexto sociocultural no qual está inserida. As falas a seguir reforçam tais afirmações: O meu pai, quando bebia, ele enchia a cara. Uma vez ele bebeu tanto que ficou desmaiado, aí nos tivemos medo dele morrer e o levamos para a Unidade Básica da Pedreira. Chegando lá, o doutor passou um soro e disse que o remédio era não beber mais (E12). [...] eu acho que às vezes é pra esquecer os pro- blemas, e começa a beber pra curtir mesmo, beber pra fazer briga com os outros, criarem coragem, esse é meu ponto de vista. Eu bebo hoje com a minha namorada, mas não bebo com os moleques da rua, porque ali não tem futuro não. Através de amizades, através de ver todo mundo bebendo, aí vai querer experimentar e vê que é gostoso (E12). [...] eu acho que às vezes é pra esquecer os pro- blemas, e começa a beber pra curtir mesmo, beber pra fazer briga com os outros, criarem coragem, esse é meu ponto de vista. Eu bebo hoje com a minha namorada, mas não bebo com os moleques da rua, porque ali não tem futuro não. Através de amizades, através de ver todo mundo bebendo, aí vai querer experimentar e vê que é gostoso (E12). A minha irmã bebia que caía. Uma vez levamos no Pronto Socorro Municipal, mas o médico disse que o remédio dela era café e água fria (E25). A relação direta e mais comum entre o ál- cool e a agressão é por meio da intoxicação, que ocorre pela falta de inibição do medo, em função da ação ansiolítica, podendo também aumentar a percepção da dor, que pode ser uma das causas de maior agressão defensiva. Texto Contexto Enferm, Florianópolis, 2013 Jul-Set; 22(3): 576-84. O bom e o ruim das bebidas alcoólicas No entanto, pode servir como um mecanismo de gatilho para desencadear reações violentas independentemente de motivos. Exemplo disso são as alterações nas funções cog- nitivas que diminuem, assim como a capacidade do indivíduo em planejar ações em resposta às situações de ameaça, ou seja, ele age sem pensar.16 eu tomo somente cerveja, eu tomo sempre quando eu saio para ir para festa todo final de semana. Eu bebo para não ficar porre. Um dia desse eu fui para uma festa e tinha um menino que pediu que secasse um copo, eu disse que não era tão alcoólatra assim para secar um copo. Na festa eu só bebo para brincar e me divertir, se divertir mesmo! Até porque tem gente que bebe para fazer confusão. Pois é, eu não, eu fico quieta e não mexo com ninguém, e entro na festa meia-noite e saio por volta das três horas da madrugada, eu só tomo duas latinhas, assim eu não fico porre. Depois que eu bebo, a festa melhora (E9). […] até de mim mesmo, pois ainda bebo. Eu acho que a pessoa está só entregando a sua vida. A bebida alcoólica não faz bem pra pessoa, mas elas vivem be- bendo. É só a gente ver o jornal, ele sempre traz notícia de problemas ocasionados por pessoas alcoolizadas, tipo acidente de trânsito, que o filho bateu na mãe ou o marido na mulher. Coisas desse tipo fazem as pessoas que usam o álcool. A bebida muda muito a pessoa. Uma vez um amigo de porre tentou dar um tiro na namora- da, aí eu o segurei e a bala pegou na minha perna, ela atravessou a minha perna (E2). Constatou-se nas falas que um dos princi- pais problemas que levam os sujeitos do estudo a se aproximarem da bebida alcoólica é para tan- genciar-se de circunstâncias incômodas da vida. Apesar de a bebida alcoólica obter tal simbologia na percepção dos adolescentes entrevistados, sabe- se que o álcool pode ocasionar comportamentos agressivos como já mencionados neste estudo. A adolescência é também um período de cri- se para os pais, pois eles terão de conviver com o adolescente em processo de desenvolvimento para a vida adulta. Alcoolismo e suas consequências na família Nesta unidade temática se percebe como as bebidas alcoólicas trazem transtornos nas histórias de vida dos adolescentes, impregnando, assim, RSs que destacam como a vida familiar é conflituosa, principalmente levando em consideração que muitos adolescentes conviviam com familiares alcoolistas. O alcoolismo é uma doença endêmica, porém percebe-se nas RSs dos adolescentes que ele traz consequências para quem é dependente químico, como se observa nas falas a seguir: A bebida alcoólica, se consumida de for- ma abusiva, pode acarretar também problemas psiquiátricos e psicológicos, como os quadros de delirium tremens, ilusões e paranoias. Os comporta- mentos agressivos têm muito a ver com o consumo exagerado de álcool. Vários estudos demonstram que mais de 50% dos casos de espancamento de es- posas têm relação direta com o consumo de álcool pelo agressor. Abusos sexuais e atos incestuosos contra crianças também foram comprovadamente cometidos sob a influência do álcool. Vítimas de um estilo de vida baseado na violência doméstica podem apresentar problemas de longo prazo ou até incuráveis, como doenças de natureza afetiva, neurótica e de desenvolvimento.14 Esse momento é percebível nos seguintes relatos: [...] eu vou dar como exemplo do meu pai, que é dependente, que nunca bebeu pra brigar conosco. Quan- do ele bebe e quer falar as coisas, ele fala pra mamãe, pra ela nos chamar a atenção. Quando é no outro dia que ele não está mais porre aí ele senta e fala o que tem que falar. Tem pessoas que bebem e esperam ficar porre para falar as coisas, espancam mulher e filhos (E36). [...] no meu caso, a minha mãe é alcoolista, pois eu falo com ela e ela faz que nem é com ela, mas eu in- sisto assim mesmo, porque eu não vou deixar ela ficar bebendo, porque ela serve de palhaço lá, pois ela fala um bocado de besteira, e faz muita confusão. A minha mãe sempre que bebe serve de palhaço para o pessoal lá em casa, até porque ninguém lá em casa, ninguém gosta dela, nem a família da minha avó gosta dela. Só quem gosta é a minha avó, porque é mãe dela, mas os irmãos e irmãs dela, não gostam da minha mãe (E1). [...] o meu pai, quando bebia, ficava muito bravo, ele chegava em casa batia na minha mãe e mandava a gente dormir para continuar se embebedando (E30). O bom e o ruim das bebidas alcoólicas Essas mudanças fazem o adolescente Os vários problemas de saúde associados ao consumo e à dependência do álcool e de outras substâncias lícitas e ilícitas demandam maior aten- Silva SED, Padilha MI - 582 - (últimos 30 dias) ainda se encontrava no patamar de 43% nas cidades pesquisadas e o consumo semanal ou diário chegava a 22% no último ano pesquisado.17 sentir a necessidade de estar menos tempo com seus pais, permitindo a possibilidade de estabelecer novas relações, principalmente com outros adoles- centes. O processo de transformação que ocorre é, naturalmente, mais complexo. De certo modo, podemos dizer que a perturbação do equilíbrio traz sentimentos agradáveis e desagradáveis.16 Por meio de um acordo com o Ministério da Saúde, essa Secretaria recebeu, desde 1988, informa- ções sobre hospitais em todo o país, que admitem pacientes com transtornos relacionados ao consumo de substâncias psicotrópicas. Embora aproximada- mente 450 hospitais devessem enviar informações sobre seus pacientes – incluindo gênero, idade e diagnóstico –, apenas cerca da metade em média (variação: 35,5% – 79,6%) o fizeram. No último ano analisado, 1999, foram relatadas 44.680 admissões, das quais 84,5% referentes a bebidas alcoólicas.18 Texto Contexto Enferm, Florianópolis, 2013 Jul-Set; 22(3): 576-84. Alcoolismo e suas consequências na família A pesquisa evidenciou que antecedentes de violência e uso de álcool ou outras drogas em algum membro da família são fatores observados consistentemente nas dimen- sões exploradas.19 de diferentes classes sociais, em uma cidade do México, encontrou uma prevalência de 42% de violência sexual, 40% de violência física e 38% de violência emocional. A pesquisa evidenciou que antecedentes de violência e uso de álcool ou outras drogas em algum membro da família são fatores observados consistentemente nas dimen- sões exploradas.19 Cabe mencionar, também, que essa convi- vência com um familiar alcoolista propiciou aos adolescentes a aprendizagem de ingerir bebidas alcoólicas quando se deparavam com problemas que levaram os seus pais a se tornarem depen- dentes químicos. O adolescente assume o papel de aprendiz seguindo os passos do perito, que são seus pais. O consumo abusivo de álcool também pro- voca, direta ou indiretamente, custos altos para o sistema de saúde, pois as morbidades desenca- deadas por ele são caras e de difícil manejo. Além disso, a dependência do álcool aumenta o risco para transtornos familiares.19 Sabe-se, hoje, que a inten- sidade e as complicações do consumo de drogas psicotrópicas variam ao longo de um continuum de gravidade. Desse modo, não existe apenas para o dependente de álcool que ingere duas garrafas por dia, tem tremores matinais e cirrose hepática, mas também aquele que bebe dentro dos padrões considerados normais, e se acidenta ao dirigir. Portanto, não basta olhar para o consumo em si, é necessário considerar os danos que ele acarreta aos indivíduos e seus grupos de convívio.20 A adolescência é uma fase de mudança na qual se busca romper com os laços familiares, em busca de um grupo que aceite a sua atitude mais independente pelo novo. Este é o momento que mais oportunizou ao jovem a aproximação das bebidas alcoólicas, conforme foi visível nas suas RSs. Por tal motivo, desvelar o universo do alcoo- lismo dos adolescentes favorece a implementação de estratégias que devem ser aplicadas para im- pedir que esta clientela, tão carente de cuidados, faça uso de álcool, seja por experimentação ou de forma abusiva. Esta pesquisa destaca o fenômeno das RSs como uma forma de conhecimento adequado para desvelar o universo de interação entre os adolescentes e as bebidas alcoólicas. Alcoolismo e suas consequências na família Os estudos que trazem a Teoria das Representações Sociais como fenômeno e não como constructo teórico, fa- vorecem conhecer a realidade que é reapresentada pelo sujeito social, por tal motivo são relevantes para o universo acadêmico, visto propiciar ao pesquisador contemplar a realidade na forma de conhecimento prático. É essencial conhecer as re- presentações dos adolescentes aqui pesquisados, pelo fato de se poder, assim, compreender como o conhecimento consensual influencia nas atitudes e comportamentos dos jovens frente ao objeto psicossocial que é o alcoolismo. Alcoolismo e suas consequências na família [...] o meu pai e minha mãe, no início que come- çavam a beber, era uma maravilha, mais depois, quando estavam porres, sempre surgia discussão. Teve uma vez que meu pai bateu na minha mãe que tivemos de separá-lo, senão ele ia matar ela (E33). Analisando-se as falas dos adolescentes, inferiu-se que eles são conscientes dos efeitos do álcool em seu organismo, e também de seus efeitos sociais. Isso foi expresso pelas palavras vício, vio- lência, laços familiares abalados e autodegradação. Porém, é relevante ressaltar que muitos afirmam não suspender o consumo do álcool porque pos- suem controle, uma vez que não o ingerem em grandes quantidades. [...] a minha mãe é alcoolista, pois ela não sabe beber. Quando ela começa, ela se joga mesmo, só pára quando cai. Eu também tenho uma irmã que é viciada na bebida alcoólica, pois todo final de semana ela tá na festa bebendo, ela não sabe beber, se tiver festa todo dia, ela vai para festa, tem vez que ela passa três dias na rua. Eu tenho dificuldade de lidar com ela, porque quando ela está porre quer fazer onda com todo mundo (E3). Segundo levantamento realizado pela Se- cretaria Nacional Antidrogas, os cinco estudos realizados até o momento (1987/1989/1993/1997 e 2003) apontam que o consumo de álcool é bas- tante alto entre crianças e adolescentes de nove a 18 anos. Para esses jovens, o álcool não apareceu como a droga favorita, mas seu consumo recente A incidência de violência doméstica tem sido considerada maior em abusadores de substâncias psicoativas na maioria das sociedades e culturas, estando presente nos diferentes grupos econômi- cos. Um estudo transversal de violência doméstica no qual foram entrevistadas 384 mulheres casadas, - 583 - O alcoolismo na história de vida de adolescentes: uma análise... alcoolista que, no caso da maioria dos adolescen- tes do estudo, era retratado pelo pai ou pela mãe. Essa realidade favoreceu um ambiente familiar conflituoso para os adolescentes. de diferentes classes sociais, em uma cidade do México, encontrou uma prevalência de 42% de violência sexual, 40% de violência física e 38% de violência emocional. Texto Contexto Enferm, Florianópolis, 2013 Jul-Set; 22(3): 576-84. REFERÊNCIAS 12. Lüdke M, André MEDA. Pesquisa em educação: abordagens qualitativas. São Paulo (SP): EPU; 1986. 1. Braga ABB, Bastos AFB. Formação do acadêmico de enfermagem e seu contato com as drogas psicoativas. Texto Contexto Enferm. 2004 Abr-Jun; 13(2):241-9. 13. Bardin L. Análise de conteúdo. Lisboa (PT): Edições 70; 2008. 14. Castillo CO, Costa MCS. Significados do consumo de álcool em famílias de uma comunidade pobre venezuelana. Rev Latino-Am Enfermagem. 2008; 16(esp):535-42. 2. Cavalcante MBPT, Alves MDS, Barroso MGT. Adolescência, álcool e drogas: uma revisão na perspectiva da promoção de saúde. Esc Anna Nery Rev Enferm. 2008 Set; 12(3):555-9. 15. Vargas D, Luis MAV. Alcohol, alcoholism and alcohol addicts: conceptions and attitudes of nurses from district basic health centers. Rev Latino-Am Enfermagem [online]. 2008 [acesso 16 Abr 2010]; 16(esp):543-50 Disponível em: http://www.scielo. br/scielo.php?script=sci_arttext&pid=S0104- 11692008000700007&lng=pt&nrm=iso 3. Rocha RM. O enfermeiro na equipe interdisciplinar do Centro de Atenção Psicossocial e as possibilidades de cuidar. Texto Contexto Enferm [online]. 2005 [acesso 10 fev 2010];14(3):350-7. Disponível em: http://www.scielo.br/pdf/tce/v14n3/v14n3a05. pdf 16. Pillon SC, O’brien B, Chavez KAP. The relationship between drugs use and risk behaviors in Brazilian university students. Rev Latino-Am Enfermagem [online]. 2005 [acesso 20 Abr 2010]; 13(esp2):1169-76. Disponível em: http://www. scielo.br/scielo.php?script=sci_arttext&pid=S0104- 11692005000800011&lng=pt&nrm=iso 4. World Health Organization. Global status report on alcohol 2004. Geneva (CH): Department of Mental Health and Substance Abuse; 2004. 5. Moscovici S. Representações sociais: investigações em psicologia social. 5ª ed. Petrópolis (RJ): Vozes; 2007. 17. Sena ELS, Boery RNSO, Carvalho PAL, Reis HFT, Marques AMN. Alcoolismo no contexto familiar: um olhar fenomenológico. Texto Contexto Enferm [online]. 2011 Jun [acesso 27 Ago 2011]; 20(2):310-8. Disponível em: http://www.scielo.br/pdf/tce/ v20n2/a13v20n2.pdf 6. Bertaux D. Los relatos de vida. Barcelona (ES): Edicions Bellaterra; 2005. 7. Chizzotti A. Pesquisa em ciências humanas e sociais. 6ª ed. São Paulo (SP): Cortez; 2003. 8. Fundação Papa João XXIII. Projeto Tribos Urbanas [online]. Belém (PA): Prefeitura de Belém; 2008. [acesso 10 Fev 2010] Disponível em: http:// www.belem.pa.gov.br/portal/new/index2. php?option=com_events&task=view_detail&agid= 506&year=&month=&day=&Itemid=280&pop=1 18. Castillo BAA, Marziale MHP, Castillo MMA, Facundo FRG, Meza MVG. Stressful situations in life, use and abuse of alcohol and drugs by elderly in Monterrey, Mexico. Rev Latino-Am Enfermagem [online]. 2008 [acesso 20 Abr 2010]; 6(esp):509-15. Disponível em: http://www.scielo. br/scielo.php?script=sci_arttext&pid=S0104- 11692008000700002&lng=pt&nrm=iso 9. Padilha MICS, Guerreiro DMVS, Coelho MS. Aspectos teórico-metodológicos das representações sociais e seu uso na enfermagem. Online Braz J Nurs [online]. 2007 [acesso 25 Mar 2010];6(2). Disponível em: http://www.uff.br/objnursing/index.php/ nursing/article/view/j.1676-4285.2007.601/199 19. Chalub M, Telles LEB. CONSIDERAÇÕES FINAIS As RSs reveladas pelo método de história de vida propiciaram a compreensão do universo consensual dos adolescentes que tiveram as be- bidas alcoólicas inseridas no seu cotidiano, o que favoreceu compreender a verdadeira extensão deste problema. Na unidade sobre O bom e o ruim das bebi- das alcoólicas, ficou notório como os adolescentes, em suas representações sociais, visualizavam os benefícios e os malefícios do álcool. A parte positiva dessa droga lícita era evidenciada pela sensação de bem-estar e espontaneidade que o efeito do álcool ocasionava nos adolescentes. Já a parte negativa era retratada pelos problemas que a embriaguez lhes provocava. Existe a necessidade de se instituir estra- tégias de prevenção ao consumo de bebidas alcoólicas de forma abusiva pelos adolescentes que não se centrem unicamente em transmitir os conhecimentos científicos, mas em fornecer uma relação de influência mútua com o conhecimento, que venha servir aos interesses e necessidades do jovem para interagir na sociedade, a fim de que o adolescente se sinta capaz de utilizar seus saberes para participar da sociedade como cida- dão, enfatizando a importância da aplicabilidade prática desses conhecimentos para sua relação com o mundo. As bebidas alcoólicas são amplamente difun- didas, principalmente porque seus apreciadores gostam da sensação de prazer que a droga ocasio- na. Porém, passam despercebidos pela maioria dos consumidores, os malefícios do álcool, tais como a dependência química, os acidentes automobilísti- cos e outros problemas de vínculo social. Já na segunda unidade temática, foi per- cebível como o uso das bebidas alcoólicas causa transtornos na vida familiar dos adolescentes, sendo o principal gerador de problemas o familiar Texto Contexto Enferm, Florianópolis, 2013 Jul-Set; 22(3): 576-84. Silva SED, Padilha MI - 584 - Correspondência: Sílvio Éder Dias da Silva Avenida 25 de setembro, 1965, ap. 901 66093-005 – Bairro do Marco, Belém, PA, Brasil E-mail: silvioeder2003@yahoo.com.br Recebido: 10 de Outubro de 2011 Aprovação: 20 de Julho de 2012 REFERÊNCIAS Álcool, drogas e crime. Rev Bras Psiquiatr [online]. 2006 [acesso 20 Abr 2010]; 28(supl2):69-73. Disponível em: http://www. scielo.br/scielo.php?script=sci_arttext&pid=S1516- 44462006000600004&lng=pt&nrm=iso 10. Minayo MCS. O desafio do conhecimento: pesquisa qualitativa em saúde. 10ª ed. São Paulo (SP): HUCITEC-ABRASCO; 2007. 20. Mendes IAC, Luis MAV. Uso de substâncias psicoativas, um novo velho desafio. Rev Latino- am Enfermagem. 2004 [acesso 21 Abr 2010]; 12(esp) Disponível em: http://www.scielo. br/scielo.php?script=sci_arttext&pid=S0104- 11692004000700001&lng=pt&nrm=iso 11. Turato ER. Tratado da metodologia da pesquisa clínico-qualitativa: construção teórica- epistemológica, discussão comparada e aplicada nas áreas de saúde e humanas. Petrópolis (RJ): Vozes; 2003. Texto Contexto Enferm, Florianópolis, 2013 Jul-Set; 22(3): 576-84.
https://openalex.org/W2753004326	https://europepmc.org/articles/pmc5580291?pdf=render	English	null	Young adult non-smokers’ exposure to real-world tobacco marketing: results of an ecological momentary assessment pilot study	BMC research notes	2,017	cc-by	4,672	© The Author(s) 2017. 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: The aims of this pilot study were to assess and characterize non-current smoking young adults’ expo‑ sure to tobacco marketing through an ecological momentary assessment protocol. Methods: Ecological momentary assessment (EMA) consists of repeated measurement of momentary phenomena and is well-suited to capture sporadic experiences in the real-world, such as exposure to tobacco marketing. EMA has the potential to capture detailed information about real-world marketing exposures in ways that reduce recall bias and increase ecological validity. In this study, young adults (n = 31; ages 18–25) responded to random prompts regarding their momentary exposure to tobacco marketing via text messages on their smartphones for 14 days (n = 1798 observations). Unadjusted and adjusted analyses were conducted using multilevel logistic regression to assess the odds of exposure accounting for correlation of multiple repeated measures within individuals while con‑ trolling for variability between individuals. Results: Respondents reported, on average, two momentary exposures to tobacco advertising in the 14-day study period. In adjusted analyses, African–American (aOR 3.36; 95% CI 1.07, 10.54) and Hispanic respondents (aOR 5.08; 95% CI 1.28, 20.13) were more likely to report exposure to tobacco advertising. Respondents were also more likely to report exposure when also exposed to others using tobacco products and when they were at stores compared with at home (aOR 14.82; 95% CI 3.61, 60.88). Conclusion: Non-smoking young adults report exposure to tobacco marketing particularly at the point-of-sale, with the highest likelihood of exposure among African-American and Hispanic young people. EMA protocols can be effec‑ tive in assessing the potential impact of point-of-sale tobacco marketing on young adults. Keywords: District of Columbia, Marketing, Pilot projects, Tobacco, Young adult, Health equity rds: District of Columbia, Marketing, Pilot projects, Tobacco, Young adult, Health equity *Correspondence: srose@truthinitiative.org 1 Schroeder Institute at Truth Initiative, Washington, DC, USA Full list of author information is available at the end of the article © The Author(s) 2017. 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. Rose et al. BMC Res Notes (2017) 10:435 DOI 10.1186/s13104-017-2758-7 Rose et al. BMC Res Notes (2017) 10:435 DOI 10.1186/s13104-017-2758-7 BMC Research Notes Open Access Recruitment As part of an EMA pilot study for young adult non- smokers that also examined store visiting behavior (not reported in this study), participants were recruited in summer 2015 via online and print advertisements (e.g., Craigslist, local newspaper, and flyers). In total, 258 individuals responded to the screener within a 1-month period. Eligible respondents were (1) between the ages of 18–25, (2) did not smoke cigarettes in the past 30 days, (3) lived and worked/attended school in Washington, DC, (4) owned their own android or iPhone smartphone with an unlimited text message plan, and (5) agreed to participate in the study (n = 31). This pilot protocol was developed to determine the feasibility of engaging young adult non-smokers in reporting on their exposure to tobacco marketing and store visiting building, in part, on Ethical considerationsh The study was approved by the Chesapeake institutional review board (Study Number #Pro00012367). Respond- ents were provided with informed consent to partici- pate and identifiable information was kept separate from respondent survey and EMA text message responses. The EMA data was transmitted via standard text mes- sages (SMS) via a respondents’ smartphone to a secure database. In the EMA, respondents were simply asked to provide numeric responses to questions without provid- ing other identifiable information. In transit, SMS mes- sages are encrypted. Once received, the data were stored in a secure firewall-protected and password-protected database via an encrypted representational state transfer (REST) application programing interface with access only to authorized users. Measures Participant baseline measures included gender, race/eth- nicity (White, African–American, Hispanic, other), past 30-day cigarette smoking status (y/n), age, self-reported personal financial status (live comfortably, meet needs with a little left, just meet basic needs, don’t meet basic needs), and smoking status as ever (even one puff) or never. They were also asked about their susceptibility to future cigarette use using the validated three-item sus- ceptibility to smoke index measured on a four point scale of definitely yes, probably yes, probably no, and definitely no (“Do you think in the future you might experiment with cigarettes?,” “At any time in the next year do you think you will smoke a cigarette,” and “If one of your best friends were to offer you a cigarette, would you smoke it?”) [20]. Respondents who answered definitely no to all three questions were deemed non-susceptible, all oth- ers were deemed susceptible. EMA prompts measured tobacco marketing exposure as the outcome [“In your current location, do you see any advertisements or signs promoting tobacco?” (y/n)], current location (home, work, other’s home, bar/restaurant, vehicle/in transit, outside, store, other), and exposure to tobacco use (“Is anyone using tobacco products in your current location?” Yes, in view, yes, in my group, no) adapted from a prior study [17]. Participants self-defined what constituted tobacco marketing. Data collection All 31 participants completed a baseline survey of par- ticipant demographics, tobacco use history, and sus- ceptibility to future cigarette use (see Additional file 1). Respondents downloaded the study application to their phone which enrolled them in a text message protocol. Over the next 14 days they were randomly prompted 6 times a day about their exposure to tobacco advertising. As a result, each respondent was assessed over 80 times. They had 1 h to respond to each prompt via text message before it expired. All 31 respondents completed the EMA period. Background to tobacco marketing may also be more prevalent among minority populations. For instance, there are significantly more menthol advertisements in areas with higher pro- portions of African–American residents [8, 9] and near schools with more African American students [10]. Exposure to tobacco marketing and brand recognition is higher among non-smoking African–American [11, 12] and Hispanic [12] youth and young adults and tends to be associated with greater risk of smoking initiation. Identifying how young adult non-smokers are exposed to tobacco marketing can help to understand who is at risk of exposure and potentially future tobacco use. Exposure to tobacco marketing has been associated with increased risk of smoking in youth [1, 2] and young adults [3, 4]. Individual exposures throughout time and places are cumulative and can change positive expectancies and normative beliefs about tobacco products [5]. Exposure to tobacco marketing in real world settings affects many facets of tobacco use, but its role in tobacco use initiation among non-users is not well understood [6, 7]. Exposure Rose et al. BMC Res Notes (2017) 10:435 Page 2 of 6 an approach used in a prior study of smoker’s relapse and store visiting used by members of this study team [18]. an approach used in a prior study of smoker’s relapse and store visiting used by members of this study team [18]. This study utilized a real-time and ecologically valid measure of exposure to tobacco advertising through ecological momentary assessment (EMA) [13, 14]. EMA can provide real-time tracking of exposure to and atti- tudes towards tobacco advertising closer to the moment of exposure, rather than through retrospective recall [1, 15, 16]. Prior EMA studies have examined self-reported overall tobacco advertising exposure [3, 17] or exposure to POS tobacco outlets [18] However, few studies have focused on exposure of non-current smokers to tobacco marketing. One study of college students found that almost 70% of self-reported exposure to tobacco market- ing measured through an EMA protocol was at point of sale [19], and that exposure was related to future risk of smoking among both smokers and non-smokers [3]. The current study tested a protocol to assess real time expo- sure to tobacco advertising through EMA and identified correlates of exposure. Analysis Descriptive statistics were used to characterize partici- pants in terms of sociodemographic factors, smoking status and EMA responses. The analysis utilized multi- level logistic regression modeling using Stata version 13 in a two-level model, to account for clustering of mul- tiple EMA responses within individual respondents. We anticipate that responses by the same individual are correlated and thus a multilevel modelling approach Rose et al. BMC Res Notes (2017) 10:435 Page 3 of 6 Table 1 Descriptive statistics of participant characteristics and ecological momentary assessments a Smoking status defined as having ever tried at least one puff of a cigarette Mean/N SD/% Sociodemographics Mean age 22.5 1.9 Female 23 74.0 Race/ethnicity Non-Hispanic African American 18 58.1 Non-Hispanic White 9 29.0 Non-Hispanic Asian 2 6.5 Hispanic 2 6.5 Education Some college, no degree 11 35.5 Associate’s degree 1 3.2 Bachelor’s degree 18 58.1 Master’s degree 1 3.2 Financial situation Live comfortably 5 16.1 Meet needs with a little left 15 48.4 Just meet basic expenses 10 32.3 Don’t meet basic expenses 1 3.2 Smoking statusa Never 11 35.5 Ever 20 65.5 EMA responses Completed 1798 70.2 Abandoned 83 3.2 Expired 674 26.3 properly adjusts standard errors and associated confi- dence intervals. Additionally, multilevel models using repeated measure data allows for including all subjects’ observations despite the potential for missing data points for incomplete response to the EMA by participants. We modeled the likelihood of respondent momentary exposure to tobacco advertising when responding to the random prompts. Multilevel modeling extends to binary outcomes by using a logit link function, in which the log odds of momentary exposure to tobacco advertising was modeled as a linear combination of correlates. We first estimated the null model (a model with no predic- tors) finding that the logistic multilevel model was pref- erable to an ordinary logistic regression model based on a likelihood ratio test provided by STATA showing a highly significant result [χ2 (1) 28.99 p = 0.00]. We report 95% confidence intervals with significance levels set at the p < 0.05 level for all statistical analyses. Correlates of exposure included gender, race/ethnicity, financial situation, baseline cigarette susceptibility, location when responding to EMA prompts, and momentary exposure to smoking. We report both bivariate crude odds ratios and adjusted odds ratios in the multilevel logistic regres- sion models along with 95% confidence intervals report- ing significance at the p < 0.05 level. Analysis We retained all correlates in the adjusted model regardless of significance in the bivariate analyses to examine the potential joint effects of these variables on advertising exposure. Table 1 Descriptive statistics of participant characteristics and ecological momentary assessments Resultsh The respondents (Table 1) were predominantly female (74%) and African–American (58%). Respondents were highly educated with 61% having a Bachelor’s degree or higher and had a comfortable personal financial situation with 65% meeting needs with a little left or living com- fortably. The majority (66%) had ever had even a puff of a cigarette but only one respondent had smoked 100 life- time cigarettes. The remaining participants were never cigarette smokers. At baseline, 48% were susceptible to future cigarette use. needs, those exposed to tobacco use in their group or in their view, and respondents who were outside, at work or in a store had higher odds of being exposed to tobacco advertising. g Though based on small sample sizes, when adding covariates to the model (Table 2), African–Americans (aOR 3.36, 95% CI 1.07, 10.54) and Hispanics (aOR 5.08, 95% CI 1.28, 20.13) had higher odds of reporting momen- tary exposure to tobacco advertising than Whites. Asians were no more likely to report exposure than Whites. Respondents who did not meet financial needs com- pared with those who lived comfortably were more likely to report exposure to advertising (aOR 6.12, 95% CI 1.16, 32.37). Females also reported greater odds of exposure in adjusted analyses (aOR 3.92, 95% CI 1.10, 13.96). Respond- ents were more likely to report exposure to tobacco adver- tising if someone in their group was using tobacco (aOR 17.04, 95% CI 7.47, 38.87) or someone in their view was using tobacco (aOR 14.43, 95% CI 6.38, 32.65). There were no significant differences in odds of reporting expo- sure based on baseline susceptibility to future cigarette During the EMA, the respondents were asked a total of 2560 random prompts, responding to 73% and com- pleting 70%. The average number of complete random prompts was 58 per subject (median 59, range 25–75). On average, over the 14 day EMA period, respondents reported 1.9 exposures to tobacco advertising (range 0–10). For the null multilevel model of exposure to tobacco advertising, the intraclass correlation was 0.24 indicating that 24% of the variance in tobacco advertising exposure was accounted for by clustering of observations within participants. In unadjusted analyses (Table 2), Hispanic respondents, participants not meeting basic financial Rose et al. Resultsh BMC Res Notes (2017) 10:435 Page 4 of 6 Table 2 Unadjusted and adjusted multilevel logistic regression models of reported momentary exposure to tobacco advertising (N = 1794a) Italic responses have 95% confidence intervals that do not include 1 and are significant at the p < 0.05 level of significance a Number of observations in final fully adjusted model Variable %b Unadjusted model Adjusted model OR 95% CI aOR 95% CI Female 72.58 1.67 0.51 5.42 3.92 1.10 13.96 Race/ethnicity Hispanic 6.79 8.01 1.45 44.21 5.08 1.28 20.13 NH AA 58.51 2.94 0.94 9.17 3.36 1.07 10.54 NH Asian 6.90 0.63 0.05 8.13 1.60 0.14 18.29 White (ref) 27.81 – – – – – – Personal financial situation Live comfortably (ref) 16.52 – – – – – – Meet needs with a little left 46.55 0.95 0.27 3.35 0.47 0.15 1.46 Just meet basic needs 33.65 1.43 0.39 5.23 1.06 0.34 3.35 Don’t meet basic needs 3.28 10.41 1.31 82.63 6.12 1.16 32.37 Baseline susceptibility to future tobacco use Yes 47.89 1.32 0.51 3.45 1.73 0.74 4.06 No (ref) 52.11 – – – – – – Using tobacco Yes, in my group 2.89 17.05 7.60 38.24 17.04 7.47 38.87 Yes, in my view 4.62 13.42 6.56 27.45 14.43 6.38 32.65 No (ref) 92.27 – – – – – – Location Bar/restaurant 4.89 1.29 0.29 5.88 0.99 0.20 4.95 Other 4.45 1.22 0.27 5.56 0.80 0.16 4.01 Others home 7.17 1.58 0.41 6.07 0.95 0.23 4.01 Outside 4.73 4.14 1.50 11.41 1.46 0.45 4.77 Store 1.00 10.66 2.50 45.53 14.82 3.61 60.88 Vehicle/transit 7.17 2.18 0.88 5.42 1.17 0.42 3.21 Work 14.52 2.46 1.06 5.67 2.39 0.96 5.94 Home (ref) 56.06 – – – – – – Table 2 Unadjusted and adjusted multilevel logistic regression models of reported momentary exposure to tobacco advertising (N = 1794a) smoking. Finally, respondents had 15 times the odds of reporting exposure to tobacco advertising if they were at a store (aOR 14.82, 95% CI 3.61, 60.88) compared to when they were at home. In adjusted analyses, no other locations were significantly different than home. in the environment. Point-of-sale tobacco marketing exposure may be particularly problematic, as exposure in stores, but not through other media, has been associ- ated with increased future smoking risk [23]. Resultsh Addition- ally, store type where respondents shop may play a role in these results, with convenience stores compared with other store types like grocery stores, more likely to have tobacco marketing [24]. We did not ask respondents who were at stores at EMA response to report the type of store visited, though this or automated geolocation mon- itoring through the smartphone could be added to future studies. However, though significant, these results should be interpreted cautiously and replicated in larger studies due to low sample sizes and wide confidence intervals. Discussion When controlling for other factors, African American and Hispanic participants are more likely to report expo- sure to tobacco advertising. Respondents were also signif- icantly more likely to report exposure to advertisements in stores. A large body of research has found increased tobacco advertising in stores in minority neighborhoods [8–10, 21, 22] suggesting that greater exposure among minority respondents may be due to more advertising Rose et al. BMC Res Notes (2017) 10:435 Page 5 of 6 tobacco use disparities in vulnerable groups, and greater likelihood of tobacco use initiation over time. Respondents were also more likely to report advertis- ing exposure when in a group or in view of others using tobacco products. This finding is concerning as a prior EMA study of young adults found that being with a friend when exposed to pro-tobacco marketing was asso- ciated with greater intention to smoke in the future com- pared with exposure when alone, which was unrelated to intention [25]. Additionally, experiencing tobacco adver- tising in places where tobacco use is present may both serve as cues for use [26, 27]. Multiple consistent ‘social exposures’ (such as smoking and tobacco marketing) may interact to make tobacco use more normative which can in turn influence smoking risk [28]. Author details 1 1 Schroeder Institute at Truth Initiative, Washington, DC, USA. 2 College of Global Public Health, New York University, New York, NY, USA. Additional file Additional file 1: Appendix A.Baseline study instrument. Funding This pilot study finds that this EMA approach is feasi- ble to engage young adult non-smokers in reporting on their momentary exposure to tobacco marketing. We intend to develop a larger study which examines differ- ences in exposure between vulnerable population young adults and non-vulnerable young adults. This future study will assess whether differential neighborhood and individual exposure to tobacco marketing is associ- ated with greater susceptibility to future use of tobacco, g This study was funded by Truth Initiative and the funder did not have a role in the design of the study and collection, analysis, and interpretation of data or in writing the manuscript. Authors’ contributions SWR designed the study and was in charge of all aspects of data collection and analysis and served as lead author of the manuscript. HE conducted statis‑ tical data analyses. AAR and RN provided input into study design and analytic plan. All authors provided substantive contributions to the development and editing of the manuscript and approved the final manuscript. All authors read and approved the final manuscript. Abbreviations OR d d d aOR: adjusted odds ratio; CI: confidence interval; EMA: ecological momentary assessment. Ethics, approval, and consent to participate , pp , p p The study was approved by the Chesapeake Institutional Review Board (Study Number #Pro00012367). All respondents provided written and signed consent to participate consistent with this approved study protocol. Acknowledgements h h ld l k Strengths of the study are the wide applicability of the text messaging approach due to 85% smartphone own- ership among young adults and 100% of text messaging use among smartphone owners in that age group [32]. Additionally, the response to the random prompts was relatively high, comparable with other studies of youth or young adults and generated nearly 60 observations on average per respondent [33, 34]. Momentary reporting of tobacco marketing exposure reduces recall bias and was more strongly correlated with future susceptibility than recall measures [19]. Additionally, the self-reported measure was broad-based in assessing marketing expo- sure through multiple channels and not just at the point-of-sale.hi The authors would like to thank Ashley Mayo for assisting with participant recruitment and monitoring, Morgane Bennett for assistance with the EMA system, and Thomas Kirchner for consultation with the pilot study protocol. Strengths and limitationsh This study has several limitations. The small sample size for the pilot study precludes broad generalizations and the study sample was predominately female; however, it did include a relatively diverse sample and is in line with the sample size in other pilot EMA studies of tobacco- related behaviors [25, 29, 30]. This study also focused on responses to random prompts, rather than also incor- porating user-initiated responses as in other studies of marketing exposure [19, 25]. This approach may, there- fore, have missed some advertising exposures, however, it reduces potential bias introduced by study-induced heightened participant awareness of tobacco marketing, which has been associated with increased susceptibility [31] and future use [16]. Availability of data materials D id tifi d d t il bl Availability of data materials D id tifi d d t il bl -identified data are available upon request from the study authors. Consent for publication P i i id d i p Participants provided written informed consent to publish results. p Participants provided written informed consent to publish results. Conclusionsh This pilot study shows that both susceptible and non-sus- ceptible young adult nonsmokers are routinely exposed to tobacco marketing in their daily life, particularly at the point-of-sale. EMA protocols can be effective in assessing the potential impact of point-of-sale tobacco marketing on young adults. 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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. Received: 4 January 2017 Accepted: 23 August 2017 Received: 4 January 2017 Accepted: 23 August 2017 Rose et al. BMC Res Notes (2017) 10:435 Page 6 of 6 References 19. Martino SC, Scharf DM, Setodji CM, Shadel WG. Measuring exposure to protobacco marketing and media: a field study using ecological momen‑ tary assessment. Nicotine Tob Res. 2012;14(4):398–406. 1. Fulmer EB, Neilands TB, Dube SR, Kuiper NM, Arrazola RA, Glantz SA. Pro‑ tobacco media exposure and youth susceptibility to smoking cigarettes, cigarette experimentation, and current tobacco use among US youth. PLoS ONE. 2015;10(8):e0134734. 1. Fulmer EB, Neilands TB, Dube SR, Kuiper NM, Arrazola RA, Glantz SA. 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https://openalex.org/W4206621338	https://escholarship.org/content/qt8mh5p1mq/qt8mh5p1mq.pdf?t=qwtcig	English	null	Eclampsia	Journal of education & teaching in emergency medicine	2,021	cc-by	8,204	UC Irvine Journal of Education and Teaching in Emergency Medicine Title Eclampsia Permalink https://escholarship.org/uc/item/8mh5p1mq Journal Journal of Education and Teaching in Emergency Medicine, 6(3) Authors Yang, Thomas Sangal, Rohit Conlon, Lauren Publication Date 2021 DOI 10.5070/M56354256 Copyright Information Copyright 2021 by the author(s).This work is made available under the terms of a Creative Commons Attribution License, available at https://creativecommons.org/licenses/by/4.0/ Peer reviewed UC Irvine Journal of Education and Teaching in Emergency Medicine Title Eclampsia Permalink https://escholarship.org/uc/item/8mh5p1mq Journal Journal of Education and Teaching in Emergency Medicine, 6(3) Authors Yang, Thomas Sangal, Rohit Conlon, Lauren Publication Date 2021 DOI 10.5070/M56354256 Copyright Information Copyright 2021 by the author(s).This work is made available under the terms of a Creative Commons Attribution License, available at https://creativecommons.org/licenses/by/4.0/ Peer reviewed UC Irvine Journal of Education and Teaching in Emergency Medicine Title Eclampsia Permalink https://escholarship.org/uc/item/8mh5p1mq Journal Journal of Education and Teaching in Emergency Medicine, 6(3) Authors Yang, Thomas Sangal, Rohit Conlon, Lauren Publication Date 2021 DOI 10.5070/M56354256 Copyright Information Copyright 2021 by the author(s).This work is made available under the terms of a Creative Commons Attribution License, available at https://creativecommons.org/licenses/by/4.0/ Peer reviewed Copyright Information Copyright 2021 by the author(s).This work is made available under the terms of a Creative Commons Attribution License, available at https://creativecommons.org/licenses/by/4.0/ Peer reviewed SIMULATION Eclampsia Thomas J Yang, MD, Rohit B Sangal, MD, MBA and Lauren W Conlon, MD^ *Yale University, Department of Emergency Medicine, New Haven, CT ^University of Pennsylvania, Department of Emergency Medicine, Philadelphia, PA Correspondence should be addressed to Lauren W Conlon at lauren.conlon@uphs.upenn.edu Submitted: March 17, 2021; Accepted: June 17, 2021; Electronically Published: July 15, 2021; https://doi.org/10.21980/J8PS8R Copyright: © 2021 Kelley, et al. This is an open access article distributed in accordance with the terms of the Creative Commons Attribution (CC BY 4.0) Lic h // i /li /b /4 0/ ABSTRACT: Audience: Emergency medicine residents. Powered by the California Digital Library University of California eScholarship.org Audience: Emergency medicine residents. Introduction: Eclampsia is the development of a generalized seizure in pregnant patients with hypertension of pregnancy.1 Eclampsia exists on the spectrum of hypertension-related disorders in pregnancy, occurs in 1 out of 1,000-10,000 deliveries,1-3and is associated with significant maternal and fetal morbidity and mortality.4 Given the emergent nature of eclampsia and the benefit of rapid treatment, emergency medicine (EM) physicians need to quickly recognize and treat this rare pathology. Although residents have three to four years before becoming an attending, not all emergent pathologies may present clinically during their training. It is important to simulate rare, treatable conditions such as eclampsia to give learners exposure confidence in managing this disease. Educational Objectives: By the end of this simulation session, learners will be able to: 1. Demonstrate care of a gravid patient with altered mental status 2. Demonstrate care of a gravid patient with seizures 3. Recognize care involved in assessment of fetal status 4. Execute appropriate subspecialty consultation 5. Recognize the clinical signs and symptoms of eclampsia 6. Distinguish different treatment options for eclampsia 7. Identify magnesium toxicity and reversal agent 8. Differentiate the spectrum of preeclampsia Educational Objectives: By the end of this simulation session, learners will be able to: 1. Demonstrate care of a gravid patient with altered mental status 2. Demonstrate care of a gravid patient with seizures 3. Recognize care involved in assessment of fetal status 4. Execute appropriate subspecialty consultation 5. Recognize the clinical signs and symptoms of eclampsia 6. Distinguish different treatment options for eclampsia 7. Identify magnesium toxicity and reversal agent 8. Differentiate the spectrum of preeclampsia Educational Methods: As an educational strategy, simulation allows learners to partake in experiential learning. By creating a safe and supportive learning environment, simulation allows learners to facilitate deliberate practice and transfer learning in debriefing sessions. High-fidelity sessions involve software and technology to mimic realistic patient environments, which also activate learners’ affective states to aid in decision-making abilities in complex medical cases. This session was conducted using a high-fidelity mannequin, SimMom (Laerdal), and a controlling Laerdal 33 USER GUIDE (objectives 3 and 4). The consult will not be available immediately to allow the learner to continue working through the case. The learner needs to recognize the clinical signs and symptoms of eclampsia (objective 5). The learner should begin to treat the patient’s blood pressure (objective 6) and eventually assess the need for managing magnesium toxicity (objective 7). The simulation ends when the patient is admitted to the intensive care unit (ICU) or to the operating room (OR) for emergent delivery. The debriefing will review the objectives of the case, relevant clinical features of eclampsia, and a discussion of the spectrum of preeclampsia (Objectives 1-8). List of Resources: Abstract 33 User Guide 35 Instructor Materials 38 Operator Materials 48 Debriefing and Evaluation Pearls 52 Simulation Assessment 57 List of Resources: Abstract 33 User Guide 35 Instructor Materials 38 Operator Materials 48 Debriefing and Evaluation Pearls 52 Simulation Assessment 57 Linked objectives and methods: Simulation can be used for the purpose of practicing clinical care for rare or high-risk pathology, among a variety of other reasons.8 9 Simulation can help learners gain confidence and skills that help them perform in high acuity situations.9 This case is based on a pregnant patient who presents via ambulance with a clinical history and exam concerning for eclampsia. Learners need to begin management of a gravid patient with altered mental status and seizures (objective 1 and 2) They Learner Audience: Medical students, emergency medicine interns, junior residents, senior residents Time Required for Implementation: Instructor Preparation: 20-30 minutes Time for case: 15-20 minutes Time for debriefing: 10-30 minutes Recommended Number of Learners per Instructor: 3 Topics: Medical simulation, eclampsia, pregnancy, obstetrics, emergency medicine. Objectives: By the end of this simulation session, the learner will be able to: 1. Demonstrate care of a gravid patient with altered mental status 2. Demonstrate care of a gravid patient with seizures 3. Recognize care involved in assessment of fetal status 4. Execute appropriate subspecialty consultation 5. Recognize the clinical signs and symptoms of eclampsia 6. Distinguish different treatment options for eclampsia 7. Identify magnesium toxicity and reversal agent 8. Differentiate the spectrum of preeclampsia Recommended pre-reading for instructor: Recommended reading includes resources on eclampsia and emergency diagnosis and treatment.6 7 This can be supplemented with current guidelines from the American College of Obstetricians and Gynecologists, references within this case, and any hospital protocols related to eclampsia. Topics: Medical simulation, eclampsia, pregnancy, obstetrics, emergency medicine. Medical simulation, eclampsia, pregnancy, obstetrics, emergency medicine. Objectives: By the end of this simulation session, the learner will be able to: This simulation was taught to 18 EM residents in a four-year residency program. Prior to beginning the simulation, learners were oriented to the simulation center and high-fidelity mannequins. Learners were divided into groups by residency leadership to distribute skill levels across teams. Given limited decision-makers in this case, we recommend groups of three. Of note, we assigned groups of four or five given the presence of medical student rotators and limited time availability of simulation equipment. The operator of the mannequin can be anyone with a script to the case but should have basic knowledge of how to communicate through the mannequin and change digital vital signs. Finally, an EM faculty member observer was present in the room to evaluate the team as well as troubleshoot any unexpected issues. The faculty member had a list of predetermined critical actions on which to evaluate the team and a debriefing guide that outlined the objectives of the case. 6. Distinguish different treatment options for eclampsia 7. Identify magnesium toxicity and reversal agent 8. Differentiate the spectrum of preeclampsia SIMULATION LLEAP Software. Faculty-led debriefing followed the simulation case and included discussion regarding presentation, spectrum, and management of the obstetrical emergency.5 Research Methods: Resident participants completed an evaluation form consisting of questions on a 5-point Likert scale assessing the realism and usefulness of the simulation. Results: All 18 residents who participated in the simulation completed an evaluation form, and all agreed or strongly agreed the case was realistic and useful. Discussion: Incorporating high-stakes, low-frequency presentations through simulation can be readily applied in residency education and well-received by residents. Increasing the difficulty through adjusting the clinical history and exam may challenge learners further. Topics: Medical simulation, eclampsia, pregnancy, obstetrics, emergency medicine Topics: Medical simulation, eclampsia, pregnancy, obstetrics, emergency medicine. 34 USER GUIDE If available, such institutional protocols should be shown to learners for real-time reinforcement. Results and tips for successful implementation: This case was performed with a high fidelity simulator using SimMom (Laerdal). While this could be done with a standardized patient if a mannequin is unavailable, the prolonged seizure display may be challenging for a standardized patient. Standard emergency department supplies were available, including intravenous (IV) line supplies, IV fluids, code medications, defibrillator, and common ED medications. Imaging and laboratory data are available at learner request if appropriate for the case. Supply lists are provided within the case materials for set up. Linked objectives and methods: C lib i Si 10 Simulation can be used for the purpose of practicing clinical care for rare or high-risk pathology, among a variety of other reasons.8 9 Simulation can help learners gain confidence and skills that help them perform in high acuity situations.9 This case is based on a pregnant patient who presents via ambulance with a clinical history and exam concerning for eclampsia. Learners need to begin management of a gravid patient with altered mental status and seizures (objective 1 and 2). They should perform an initial diagnostic workup for the suspected pathology, fetal monitoring, and consult the appropriate service Yang T J, et al. Eclampsia. JETem 2021. 6(3):S33-61. https://doi.org/10.21980/J8PS8R 35 References/suggestions for further reading: 1. Fishel Bartal M, Sibai BM. Eclampsia in the 21st century. Am J Obstet Gynecol. 2020 Sep 24:S0002-9378(20)31128-5. Epub ahead of print. doi: 10.1016/j.ajog.2020.09.037. 1. Fishel Bartal M, Sibai BM. Eclampsia in the 21st century. Am J Obstet Gynecol. 2020 Sep 24:S0002-9378(20)31128-5. Epub ahead of print. doi: 10.1016/j.ajog.2020.09.037. 2. Ghulmiyyah L, Sibai B. Maternal mortality from preeclampsia/eclampsia. Semin Perinatol. 2012 Feb;36(1):56-9. doi: 10.1053/j.semperi.2011.09.011 3. Magpie Trial Follow-Up Study Collaborative G. The Magpie Trial: a randomised trial comparing magnesium sulphate with placebo for pre-eclampsia. Outcome for women at 2 years. BJOG 2007;114(3):300-09. Epub 2006 Dec 12. doi: 10.1111/j.1471-0528.2006.01166.x Positive: ● “Seems very bread and butter, known problem, known solution and a good review of the standard medications” (PGY 1) ● “I liked the monitoring for med toxicity/effects” (PGY 2) 4. Main EK, McCain CL, Morton CH, et al. Pregnancy-related mortality in California: causes, characteristics, and improvement opportunities. Obstetrics and gynecology 2015;125(4):938-47. doi: 10.1097/aog.0000000000000746 5. Rudolph JW, Simon R, Dufresne RL, Raemer DB. There's no such thing as "nonjudgmental" debriefing: a theory and method for debriefing with good judgment. Simulation in Healthcare : Journal of the Society for Simulation in Healthcare. 2006 ;1(1):49-55.doi 10.1097/01266021- 200600110-00006 Some suggested comments for improvement include “having a family to talk to,” “access to ultrasound images,” and “making IV access difficult.” Of note, the structure of the university ED has a separate OB triage managed by the OBGYN department; pregnancy-related complaints above 15 weeks are not often seen in the university ED. This likely influenced resident positive reception of the case and comments related to not seeing such pathology. If senior residents desire increased difficulty, this case could be modified using a postpartum patient or a past medical history of diabetes or epilepsy to present alternative treatment considerations for seizures. 6. Houry D, Salhi B. Acute Complications of Pregnancy. In: Marx J, Hockberger R, Walls R. Rosen’s Emergency Medicine. 8th ed. Philadelphia PA: Elsevier Saunders; 2014. 7. Young J. Maternal Emergencies After 20 Weeks of Pregnancy and in the Postpartum Period. In: Tintinalli J, Stapczynski J, Ma O, et al., eds. Tintinalli’s Emergency Medicine: A Comprehensive Study Guide. 8th ed. New York, NY: McGraw-Hill; 2016. 8. Abulebda K, Auerbach M, Limaiem F. Debriefing Techniques Utilized in Medical Simulation. [Updated 2020 Nov 21]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2021 Jan. Available from: https://www.ncbi.nlm.nih.gov/books/NBK546660/ USER GUIDE At the end of the debriefing, residents completed an evaluation form for the case. 18 out of 18 (100%) residents completed an evaluation. Six PGY-1, five PGY-2, two PGY-3, and five PGY-4s completed the surveys. Surveys were anonymous, and the University Institutional Research Board approved this study. All participants agreed (n =13) or strongly agreed (n=5) the simulation was realistic and useful. Selected comments are below: References/suggestions for further reading: Survey Instrument: 9. Al-Elq AH. Simulation-based medical teaching and learning. J Family Community Med. 2010 Jan;17(1):35-40. doi: 10.4103/1319-1683.68787 10. Ananth CV, Keyes KM, Wapner RJ. Pre-eclampsia rates in the United States, 1980-2010: age-period-cohort analysis. BMJ : British Medical Journal. 2013;347:f6564. doi: 10.1136/bmj.f6564 11. The Core Content Task Force II. 2016 Model of the Clinical Practice of Emergency Medicine. Available from: https://www.abem.org/public/docs/default- source/default-document-library/2016-em-model-website- document.pdf?sfvrsn=8b98c9f4_6 12. Marchi N, Tierney W, Alexopoulos AV, et al. The Etiological Role of Blood-Brain Barrier Dysfunction in Seizure Disorders. Cardiovascular Psychiatry and Neurology, 2011. Available at: https://doi.org/10.1155/2011/431470 3 Yang T J, et al. Eclampsia. JETem 2021. 6(3):S33-61. https://doi.org/10.21980/J8PS8R 36 U USER GUIDE 13. ACOG Practice Bulletin No. 202: Gestational Hypertension and Preeclampsia. Obstetrics and gynecology. 2019;133(1):1. doi: 10.1097/aog.0000000000003018 14. Sibai B. Preeclampsia and hypertensive disorders. In: Gabbe SB, Simpson JL, Landon MB, Galan HL, Jauniaux ER, Driscoll DA, eds. Obstetrics: Normal and Problem Pregnancies. 6th ed. Philadelphia: Elsevier Saunders; 2012:662-95. 15. Duley L, Gülmezoglu AM, Henderson-Smart DJ, et al. Magnesium sulphate and other anticonvulsants for women with pre-eclampsia. The Cochrane database of systematic reviews 2010;2010(11):Cd000025. doi: 10.1002/14651858.CD000025.pub2 16. Sibai BM. Magnesium sulfate prophylaxis in preeclampsia: Lessons learned from recent trials. American Journal of Obstetrics and Gynecology. 2004;190(6):1520-6. doi: 10.1016/j.ajog.2003.12.057 17. Kessler CS, Afshar Y, Sardar G, et al. A prospective, randomized, controlled study demonstrating a novel, effective model of transfer of care between physicians: the 5 Cs of consultation. Acad Emerg Med. 2012;19(8):968-74. doi: 10.1111/j.1553-2712.2012.01412.x Yang T J, et al. Eclampsia. JETem 2021. 6(3):S33-61. https://doi.org/10.21980/J8PS8R 37 INSTRUCTOR MATERIALS Case Title: Eclampsia Case Description & Diagnosis (short synopsis): A 24-year-old female with no known past medical history presents to the emergency department after having a witnessed seizure at home. Paramedics state the patient complained to her family about facial twitching, and then her whole body started shaking (but stops on arrival). Paramedics were not able to establish any IV lines or get the vital signs. She is now confused. The learner should first perform the primary survey to assess the stability of the patient. Then, the learner should obtain finger- stick blood glucose and perform a physical exam that reveals the patient is pregnant. The learner should recognize eclampsia and begin treatment with anti-hypertensive medications and magnesium sulfate. Simultaneously, learners should be able to consult the obstetrics and gynecology team for emergent evaluation and intervention of the patient, who will not be available temporarily. Monitoring and reversing magnesium toxicity are the final steps prior to disposition. Sim Exam - Plain clothed SimMom mannequin or other gravid simulator (presenting via EMS) Miscellaneous - Gloves - Alcohol/chloraprep - Plain clothed SimMom mannequin or other gravid simulator ( - Alcohol/chloraprep - Cardiac monitor Equipment or Props Needed: IV Supplies - Angiocatheters - IV tubing - Saline Airway Management - Non-rebreather mask - Nasal cannula - Laryngoscope - Bag-valve-mask - Endotracheal tube and stylet - Orogastric/nasogastric tube Medications - Etomidate - Succinylcholine - Epinephrine - Nicardipine - Magnesium sulfate - Hydralazine Yang T J, et al. Eclampsia. JETem 2021. 6(3):S33-61. https://doi.org/10.21980/J8PS8R 38 Confederates needed: The simulation operator can be a pharmacist or consultant available via phone. The faculty member in the room can be utilized as a family if desired. With multidisciplinary teams, nursing can help with IV access and patient monitoring. Otherwise, nursing tasks can be delegated to a member of the resident team. Stimulus Inventory: #1 Complete blood count (CBC) #2 Complete metabolic panel (CMP) #3 Finger-stick blood glucose #4 Venous blood gas #5 Urinalysis/Urine drug screen/Urine Pregnancy #6 Coagulation panel #7 Fetal heart tracing #8 Chest X-Ray #9 Magnesium #10 Brain CT #11 Uric Acid/LDH/Haptoglobin #12 Lactic Acid #13: Repeat Magnesium: Pending #14: Repeat Lactic Acid #15: Arrival EKG #16: Post-Magnesium EKG #17: Phosphorus Yang T J, et al. Eclampsia. JETem 2021. 6(3):S33-61. https://doi.org/10.21980/J8PS8R 39 INSTRUCTOR MATERIALS Background and brief information: EMS bring a 24-year-old female to a tertiary medical center confused after what appeared to be a seizure. Initial presentation: The patient arrives by EMS without family. She is an appropriately dressed female who has altered mental status. How the scene unfolds: On arrival, learners should assess the patient’s airway, breathing, and circulation while obtaining any additional information from EMS before they leave. They should attempt to perform a history and focused physical exam on the patient who will be unable to provide any additional information because of altered mental status. They should recognize the patient is gravid, and obtain IV access while performing a finger-stick blood glucose which will be normal. They should notify obstetrics and gynecologists who will be unavailable for a short period and recommend the learner provide treatment. Learners should perform fetal heart monitoring while giving anti-hypertensive medications and magnesium sulfate. As the patient is monitored in the ED, the patient exhibits symptoms of magnesium toxicity and will require reversal with calcium. The case ends with disposition to the OR with OB. General Appearance: Young, well-developed female, altered General Appearance: Young, well-developed female, altered Primary Survey: ● Airway: Patent ● Breathing: Clear to auscultation bilaterally ● Circulation: 2+ pulses throughout History: ● History of present illness: Critical actions: Critical actions: 1. Place IV, obtain vitals and a finger-stick glucose 2. Perform primary survey (airway, breathing, circulation) 3. Obtain a basic history of symptoms 4. Perform physical exam and recognize pregnancy 5. Verbalize “eclampsia” and call obstetrics consult early 6. Emergent blood pressure management (labetalol or hydralazine) 7. Treat seizure with magnesium 8. Recognize magnesium toxicity 9. Provide antidote for magnesium toxicity 10. Clearly communicate with consultants and nurses with the managem disposition 10. Clearly communicate with consultants and nurses with the management goals and disposition Yang T J, et al. Eclampsia. JETem 2021. 6(3):S33-61. https://doi.org/10.21980/J8PS8R 40 INSTRUCTOR MATERIALS Case Title: Eclampsia Chief Complaint: Altered Mental Status Vitals: Heart Rate (HR) 118 Blood Pressure (BP) 180/115 Respiratory Rate (RR) 24 Temperature (T) 99.0°F Oxygen Saturation (O2Sat) 95% on room air INSTRUCTOR MATERIALS Case Title: Eclampsia Vitals: Heart Rate (HR) 118 Blood Pressure (BP) 180/115 Respiratory Rate (RR) 24 Temperature (T) 99.0°F Oxygen Saturation (O2Sat) 95% on room air Vitals: Heart Rate (HR) 118 Blood Pressure (BP) 180/115 Respiratory Rate (RR) 24 Temperature (T) 99.0°F Oxygen Saturation (O2Sat) 95% on room air ● History of present illness: o Paramedic history: Fire rescue states they were called to the scene for a witnessed seizure at home. Per family, the patient had some facial twitching, and then the patient’s whole body was shaking, and she was unresponsive for about 2 minutes. She was incontinent of urine at the scene. She has no prior history of seizures or any other medical problems. No known trauma. o Patient history: Patient unable to provide history secondary to altered mental status initially only stating “my head hurts.” However, as the case progresses with correct actions the patient will become more awake and will complain of a mild headache, blurring of vision, right upper quadrant pain, and bilateral lower extremity swelling. o If asked: The patient is 31weeks pregnant by dates and has only had 1 prenatal appointment in the first trimester but nothing since then. medical history: No chronic medical problems. First pregnancy (G1P0) ● Past medical history: No chronic medical problems. First pregnancy (G1P0) ● Past surgical history: None ● Patient’s medications: Prenatal vitamins ● Allergies: None ●Social history: Drinks socially (last drink 6 months ago) no smoking or illicit drug use ● Social history: Drinks socially (last drink 6 months ago), no smoking or illicit drug use ● Family history: No history of seizures, intracranial aneurysms, or cancer ● Social history: Drinks socially (last drink 6 months ago), no smoking or illicit drug use ● Social history: Drinks socially (last drink 6 months ago), no smoking or illicit drug use ● Family history: No history of seizures, intracranial aneurysms, or cancer eturn: Calibri Size 10 Yang T J, et al. Eclampsia. JETem 2021. 6(3):S33-61. https://doi.org/10.21980/J8PS8R 41 Yang T J, et al. Eclampsia. JETem 2021. 6(3):S33-61. https://doi.org/10.21980/J8PS8R 41 Secondary Survey/Physical Examination: ● General appearance: Postictal appearing, confused, sleepy, smells of urine, moaning and groaning ● General appearance: Postictal appearing, confused, sleepy, smells of urine, moaning and groaning ●HEENT: ● HEENT: o Head: Within normal limits o Eyes: Pupils equally round and reactive 4mm ->2mm, no papilledema o Ears: No hemotympanum o Eyes: Pupils equally round and reactive 4mm ->2mm, no papillede o Ears: No hemotympanum o Nose: Within normal limits ● Neck: No stridor, trachea midline ● Heart: Tachycardic, regular rhythm, equal pulses, no murmurs ● Lungs: Clear to auscultation bilaterally, no respiratory distress, no wheezes/rhonchi/rales ● Abdominal/GI: Soft, mildly tender in the right upper quadrant, no distention, bowel sounds present, gravid uterus with fundus above umbilicus approximately 30 weeks, visible striae, no organomegaly ● Genitourinary: Within normal limits, no vaginal bleeding ● Rectal: Within normal limits ● Extremities: 1+ pitting edema in bilateral lower extremities, no muscle tenderness, full range of motion ● Back: Within normal limits ● Neuro: Glasgow Coma Scale (GCS)13, opens eyes to command, confused, follows commands). Oriented to self, pupils equally round and reactive, no facial droop, 3+ deep tendon reflexes, moving all extremities, able to squeeze hands equally, unable to further participate in the exam ● Skin: Normal color and turgor, no diaphoresis, no rash ● Lymph: Within normal limits ● Psych: Within normal limits Yang T J, et al. Eclampsia. JETem 2021. 6(3):S33-61. Secondary Survey/Physical Examination: https://doi.org/10.21980/J8PS8R 42 INSTRUCTOR MATERIALS Results: Complete blood count (CBC) White blood count (WBC) 12.9 x1000/mm3 Hemoglobin (Hgb) 9.7 g/dL Hematocrit (HCT) 24.6% Platelet (Plt) 95 x1000/mm3 Diff pending Complete metabolic panel (CMP) Sodium 136 mEq/L Chloride 108 mEq/L Potassium 3.8 mEq/L Bicarbonate (HCO3) 28 mEq/L Blood Urea Nitrogen (BUN) 24 mg/dL Creatine (Cr) 1.8 mg/dL Glucose 98 mg/dL Calcium 9.2 mg/dL Total bilirubin 1.3 mg/dL Direct bilirubin 0.8 mg/dL Indirect bilirubin 0.3 mg/dL Alkaline phosphatase 55 units/L Aspartate aminotransferase (AST) 95 units/L Alanine aminotransferase (ALT) 83 units/L Albumin 2.8 g/dL Finger-stick blood glucose 93 mg/dL Venous Blood Gas (VBG) pH 7.36 PaCO2 61 mmHg PaO2 45 mmHg Bicarbonate (HCO3) 22 mEq/L INSTRUCTOR MATERIALS Results: Complete blood count (CBC) White blood count (WBC) 12.9 x1000/mm3 Hemoglobin (Hgb) 9.7 g/dL Hematocrit (HCT) 24.6% Platelet (Plt) 95 x1000/mm3 Diff pending Complete metabolic panel (CMP) Sodium 136 mEq/L Chloride 108 mEq/L Potassium 3.8 mEq/L Bicarbonate (HCO3) 28 mEq/L Blood Urea Nitrogen (BUN) 24 mg/dL Creatine (Cr) 1.8 mg/dL Glucose 98 mg/dL Calcium 9.2 mg/dL Total bilirubin 1.3 mg/dL Direct bilirubin 0.8 mg/dL Indirect bilirubin 0.3 mg/dL Alkaline phosphatase 55 units/L Aspartate aminotransferase (AST) 95 units/L Alanine aminotransferase (ALT) 83 units/L Albumin 2.8 g/dL Finger-stick blood glucose 93 mg/dL Venous Blood Gas (VBG) pH 7.36 PaCO2 61 mmHg PaO2 45 mmHg Bicarbonate (HCO3) 22 mEq/L Yang T J, et al. Eclampsia. JETem 2021. 6(3):S33-61. https://doi.org/10.21980/J8PS8R 43 INSTRUCTOR MATERIALS Urinalysis (UA) Color yellow Appearance clear Specific gravity 1.0 pH 7.0 Glucose negative Bilirubin negative Ketones 0 Protein 2+ Leukocyte esterase negative Nitrites negative White blood cells (WBC) 0 WBCs/high powered field (HPF) Red blood cells (RBC) 0 RBCs/HPF Squamous epithelial cells 0-5 cells/HPF Urine drug screen Amphetamine negative Barbiturate negative Benzodiazepine negative Cocaine negative Marijuana negative Methadone negative Methamphetamine negative Opiate negative Phencyclidine negative Tricyclic antidepressants negative Urine Pregnancy positive Coagulation Panel Prothrombin time (PT) 12.3 seconds Partial thromboplastin time (PTT) 23.7 seconds INR 1.1 INSTRUCTOR MATERIALS Urinalysis (UA) Color yellow Appearance clear Specific gravity 1.0 pH 7.0 Glucose negative Bilirubin negative Ketones 0 Protein 2+ Leukocyte esterase negative Nitrites negative White blood cells (WBC) 0 WBCs/high powered field (HP Red blood cells (RBC) 0 RBCs/HPF Squamous epithelial cells 0-5 cells/HPF Urinalysis (UA) Color Appearance Specific gravity pH Glucose Bilirubin Ketones Protein Leukocyte esterase Nitrites White blood cells (WBC) Red blood cells (RBC) Squamous epithelial cells Yang T J, et al. Eclampsia. JETem 2021. 6(3):S33-61. https://doi.org/10.21980/J8PS8R 44 Fetal Heart Tracing Baseline fetal heart rate 130bpm with decrease in fetal heart rate from baseline lasting 30 seconds. Each decrease in fetal heart rate occurs after each contraction concerning for late decelerations Chest Radiograph (CXR) No acute disease in the chest Magnesium 1.8 mEq/L Brain Computed Tomography No acute intracranial abnormalities Uric Acid pending LDH pending Haptaglobin pending Lactic Acid 6.6 mmol/L Repeat Magnesium pending Repeat Lactic Acid 1.8 mmol/L Chest Radiograph (CXR) No acute disease in the chest Magnesium 1.8 mEq/L Brain Computed Tomography No acute intracranial abnormalities Uric Acid pending LDH pending Haptaglobin pending Lactic Acid 6.6 mmol/L Repeat Magnesium pending Repeat Lactic Acid 1.8 mmol/L Chest Radiograph (CXR) No acute disease in the chest Magnesium 1.8 mEq/L Brain Computed Tomography No acute intracranial abnormalities Uric Acid pending LDH pending Haptaglobin pending Lactic Acid 6.6 mmol/L Repeat Magnesium pending Repeat Lactic Acid 1.8 mmol/L Yang T J, et al. Eclampsia. JETem 2021. 6(3):S33-61. https://doi.org/10.21980/J8PS8R 45 INSTRUCTOR MATERIALS INSTRUCTOR MATERIALS Arrival Electrocardiogram (EKG) File NSR (CardioNetworks ECGpedia) is licensed under creative commo alike 3.0 imported (CC BY-SA 3.0) Arrival Electrocardiogram (EKG) File NSR (CardioNetworks ECGpedia) is licensed under creative commons attribution-share alike 3.0 imported (CC BY-SA 3.0) rrival Electrocardiogram (EKG) ile NSR (CardioNetworks ECGpedia) is licensed under creative commons attribution-share like 3.0 imported (CC BY-SA 3.0) Yang T J, et al. Eclampsia. JETem 2021. 6(3):S33-61. https://doi.org/10.21980/J8PS8R 46 INSTRUCTOR MATERIALS Post-Magnesium Electrocardiogram (EKG) Phosphorus 2.6 mEq/L INSTRUCTOR MATERIALS INSTRUCTOR MATERIALS Post-Magnesium Electrocardiogram (EKG) Phosphorus 2.6 mEq/L Post-Magnesium Electrocardiogram (EKG) 2.6 mEq/L Phosphorus Yang T J, et al. Eclampsia. JETem 2021. 6(3):S33-61. https://doi.org/10.21980/J8PS8R 47 eturn: Calibri Size 10 SIMULATION EVENTS TABLE: Minute (state) Participant action/ trigger Patient status (simulator response) & operator prompts Monitor display (vital signs) 0:00 (Baseline) Paramedic report. Place patient on the monitor Learner obtains focused physical exam (primary exam) Patient moaning, confused, states, “my head hurts,” but no other history (GCS 13). Primary Survey - Intact T 99.0°F (both temporal or rectal) HR 118 BP 180/115 RR 24 O2 95% RA 1:00-2:00 Give supplemental oxygen Obtain finger-stick blood glucose Peripheral IV, obtain basic history and undress patients/secondar y exam SPO2 will improve if given oxygen If glucose not obtained, nursing can prompt later in case when patient begins to seize Obtain labs (CMP, CBC, coags, HCG, LFTs) T 99.0°F (both temporal or rectal) HR 110 BP 186/112 RR 22 O2 98% on 2LNC 2:00-4:00 Recognizes patient is pregnant - request Fetal Heart Rate (FHR) monitor and OB consult Recognizes patient pregnant - request OB consult only Does not recognize patient pregnant Fetal heart tracing with HR in 130s, moderate variability, occasional late decelerations. OB resident in OR and is delayed. Patient able to provide limited history (if asked) OB resident in OR and is delayed. Patient still postictal Patient still postictal T 99.0°F HR 109 BP 205/112 RR 20 O2 94% on 2LNC (100% if started on NRB) OPERATOR MATERIALS eturn: Calibri Size 10 Yang T J et al Eclampsia JETem 2021 6(3):S33 61 https://doi org/10 21980/J8PS8R Minute (state) Participant action/ trigger Patient status (simulator response) & operator prompts Monitor display (vital signs) 4:00-6:00 Decide to treat HTN with labetalol (10-20mg IV) or hydralazine (5- 10mg IV, max bolus 20mg IV) Treat with nitroprusside Inappropriate dosing or forget to redose No treatment of BP BP improving initially Fetal cyanide poisoning, fetal distress with repetitive late decelerations, HR 90s on FHR monitor BP unresponsive BP slowly increases, patient with 30sec self-limited seizure HR 100 BP 190/98 Remaining vitals unchanged HR 100 BP 190/98 Remaining vitals unchanged Vitals unchanged T 99.0°F HR 115 BP 220/120 RR 20 O2 94% on 2LNC (100% if started on NRB) 6:00-8:00 Repeat dose of labetalol (20- 80mg IV to max of total 300mg IV) or hydralazine (5- 10mg IV to max dose 30mg or switch to new agent) Forget to redose BP continue to improve BP slowly increases T 99.0°F HR 90 BP 165/90 RR 20 O2 94% on 2LNC (100% if started on NRB) T 99.0°F HR 115 BP 220/120 RR 20 O2 94% on 2LNC (100% if started on NRB) Yang T J, et al. Eclampsia. JETem 2021. 6(3):S33-61. https://doi.org/10.21980/J8PS8R 48 Minute (state) 49 OPERATOR MATERIALS Minute (state) Participant action/ trigger Patient status (simulator response) & operator prompts Monitor display (vital signs) 8:00-9:00 Recognizes eclampsia and decides to treat with magnesium sulfate (4-6g over 20min, then 1- 2g/hr infusion) but does not do other anti-HTN treatments Treats BP but does not recognize eclampsia as cause of seizures Treats both eclampsia and hypertension Patient with some improvement in confusion and able to provide limited history and mild improvement in BP Seizures begin despite BP improvement and SPO2 drops requiring intubation No more seizure activity, BP as above and patient stabilizes T 99.0°F HR 115 BP 195/100 RR 20 O2 94% on 2LNC (100% if started on NRB) T 99.0°F HR 118 BP 165/90 RR 10 O2 84% T 99.0°F HR 102 BP 165/90 RR 18 O2 100% on NRB 9:00-10:00 Does not recognize patient is pregnant or no treatment of HTN/seizure and instead gets head CT If only benzodiazepines are given for seizures Patient has generalized tonic-clonic seizure in CT Seizures briefly improve but then continue. Respiratory rate drops with benzodiazepines, eventually hypoxia requiring intubation If FHR is on, HR to 130s with late decelerations with repeated benzodiazepines T 99.0°F HR 118 BP 165/90 RR 10 O2 84% on NRB T 99.0°F HR 108 BP 190/105 RR 10 O2 84% on NRB Participant action/ trigger OPERATOR MATERIALS Minute (state) Participant action/ trigger Patient status (simulator response) & operator prompts Monitor display (vital signs) 10:00-11:00 If appropriately managed thus far (magnesium and anti-HTN medications) Seizures stop and patient waking up but feels muscle aches, diffuse weakness, nausea and drowsiness. Patient able to provide full history. Loss of deep tendon reflexes T 99.0°F HR 105 BP 165/90 RR 18 O2 100% on NRB 11:00-13:00 Recognize magnesium toxicity and give calcium gluconate 1g IV Recognize magnesium toxicity and call renal for dialysis Does not recognize magnesium toxicity Patient feels better, symptoms resolve. No more muscle weakness, nausea or somnolence. Patient able to provide full history, interactive with staff. Deep tendon reflexes now present (if checked) Renal not in house and will see patient in the morning Patient starts to become bradycardic and hypotensive. Then, OBGYN team rushes in and take the patient to the OR and END the case. T 99.0°F HR 105 BP 165/90 RR 18 O2 100% on NRB T 99.0°F HR 40 BP 90/40 RR 18 O2 100% on NRB 13:00-15:00 (Case complete) If OB was never called Continues to have seizures refractory to treatment, intubation secondary to mental status and hypoxia. OB team then shows up and calls the OR to make arrangements for the patient T 99.0°F HR 125 BP 190/105 RR 18 O2 84% on NRB (Case Complete) Disposition to OR and ICU OB can arrive in ED and prompt learners. Also, family asks for the update and the plan for the patient care. This ends the scenario Minute (state) Yang T J, et al. Eclampsia. JETem 2021. 6(3):S33-61. https://doi.org/10.21980/J8PS8R 50 OPERATOR MATERIALS Eclampsia: Definition, Pathophysiology Eclampsia is one of the obstetrics emergency clinicians should be familiar with because it carries one of the highest mortality and morbidity rates for both the mother and the baby.10 It causes a mortality rate of 14% worldwide.4 According to the American Board of Emergency Medicine Model of Practice, pre-eclampsia and eclampsia are included as one of the critical pathologies emergency physicians should be able to manage.11 Thus, it is essential for Emergency Medicine residents to be familiar with the recognition and the management of eclampsia. Eclampsia is defined as “the occurrence of one or more generalized tonic-clonic convulsions unrelated to other medical conditions in women with hypertensive disorder of pregnancy.” 1 Although it is not precisely known, there are a few hypotheses why this disorder occurs. One of the theories involves the change in autoregulation in the central nervous system and the brain-blood barrier. Another theory involves the overregulation of the central nervous system and its result of vasoconstriction.12 Yang T J, et al. Eclampsia. JETem 2021. 6(3):S33-61. https://doi.org/10.21980/J8PS8R Yang T J, et al. Eclampsia. JETem 2021. 6(3):S33-61. https://doi.org/10.21980/J8PS8R 51 Eclampsia: Presentation, “It is a spectrum.” Emergency providers have difficulty recognizing this obstetrical emergency because of its atypical presentation and its wide spectrum of preeclampsia to eclampsia. Preeclampsia can also be divided further into a mild form and a severe form based on severe features and abnormal lab parameters. Chronic hypertension is the elevated blood pressure of at least 140/90 on 2 separate occasions at least 4 hours apart diagnosed before 20 weeks of gestation, while gestational hypertension is defined as high blood pressure after 20 weeks of gestations without proteinuria and without any features or preeclampsia. This is also important because about half of patients with gestational hypertension may develop preeclampsia or eclampsia.13 The mild form of preeclampsia is defined as gestational hypertension plus either of the following: The mild form of preeclampsia is defined as gestational hypertension plus either of the following: • Evidence of proteinuria defined as>300mg/24hour urine specimen (including 2 dipstick readings>2+ taken 6 hours apart), or protein to creatinine ratio >0.3.13 • Evidence of proteinuria defined as>300mg/24hour urine specimen (including 2 dipstick readings>2+ taken 6 hours apart), or protein to creatinine ratio >0.3.13 Yang T J, et al. Eclampsia. JETem 2021. 6(3):S33-61. https://doi.org/10.21980/J8PS8R 52 DEBRIEFING AND EVALUATION PEARLS The seizure prophylaxis is indicated in patients with preeclampsia with severe features. Since these patients’ likelihood of developing into eclampsia is high, the American College of Obstetrics and Gynecology recommends magnesium load.13 It is important to be familiar with the dosing of magnesium. Magnesium is loaded with 4-to-6 grams 10% magnesium sulfate in 100ml solution via IV over 15 minutes. Then, a continuous infusion of 1-2 grams/hour should follow. Studies have not demonstrated any mortality or morbidity benefit in patients with preeclampsia with mild features.1 Once the patient is actively seizing in the scenario, it is crucial to clear the secretions and prevent asphyxiation and aspiration. After that, seizure should be aborted as soon as possible. Learners should realize that the first line of seizure treatment in eclampsia is not anticonvulsants but rather magnesium. The dosing is the same as the seizure prevention. If seizures recur in real-life settings, physicians can give an additional dose of 2g bolus of magnesium sulfate.1 However, physicians should start to think of alternative diagnoses and administer anticonvulsants, lorazepam, or phenytoin, if seizures are refractory to eclampsia's corrective measures. The dosing of magnesium is significantly higher than what is typically used to treat other pathology. This may lead to some confusion for nurses where they may not feel comfortable administering the medication. Thus, it is essential to communicate the indication, the reason, and side effects to monitor for the medication. Symptoms of hypermagnesemia can be rapid and start to manifest around the level of 3.5. Symptoms observed include nausea and somnolence (level 3-4), the loss of deep tendon reflex and muscle weakness (level 4-8), respiratory failure (level 8-12), hypotension, bradycardia and cardiac collapse (level 12-15). EKG changes (prolonged PR, increased duration of QRS complex, prolonged QTc, increased amplitude of T waves and delayed conduction) vary and do not correlate with the level of magnesium. If patients have a normal patellar reflex, respiratory rate >12, and urine output >100cc, the magnesium maintenance therapy can be continued.15 If any side effects occur, physicians should stop the medication and give the antidote, calcium gluconate 10% 1.5-3g, via IV. DEBRIEFING AND EVALUATION PEARLS However, preeclampsia criteria is different in patients with chronic hypertension. About 20 to 50% of patients with chronic hypertension may develop preeclampsia or eclampsia. 13 It is defined as chronic hypertension, where the systolic blood pressure must have increased by 30mmHg or diastolic blood pressure increased by 15mmHg with mild proteinuria (as defined above).1 13 The severe form differs from the mild form with a higher blood pressure parameter and severe features. First, the blood pressure should be greater than or equal to the systolic blood pressure of 160 mmHg or the diastolic blood pressure of 110mmHg or higher on two separate occasions at least 4 hours apart on bed rest.13 Some severe feature findings include a) pulmonary edema or hypoxia, b) renal insufficiency, serum creatinine >1.1 mg/dL or doubling creatinine in the absence of kidney disease, c) new-onset headache, d) presence of visual disturbances, e) persistent epigastric or right upper quadrant pain, f) impaired liver function tests, g) generalized weakness, h) bleeding at intravenous insertions, i) thrombocytopenia <100,000 and j) altered mental state.14 Eclampsia differs from preeclampsia because it is the convulsive presentation of hypertensive obstetrical emergency.13 Seizures can manifest with tonic-clonic, focal or multifocal seizures without a history of seizures. Despite the differences in definition, preeclampsia and eclampsia can present any time between 20 weeks of gestation to 4 weeks postpartum. These hypertensive emergencies should be recognized and managed promptly since 1 in 400 women with preeclampsia with mild features and 1 in 50 women with preeclampsia with severe features develop eclampsia, which remains a significant cause of maternal death worldwide.13 A prompt recognition and management of this obstetrical emergency is paramount to decrease the morbidity and mortality associated with the condition. Pre-Eclampsia & Eclampsia: Management Part 1: Seizure Prophylaxis and Seizure treatment In an emergency, physicians should resuscitate with airway, breathing, and circulation before further management or investigation of the patient. The management of seizure can be divided into two-folds: prophylaxis and treatment. eturn: Calibri Size 10 Yang T J, et al. Eclampsia. JETem 2021. 6(3):S33-61. https://doi.org/10.21980/J8PS8R 53 The management of seizure can be divided into two-folds: prophylaxis and treatment. eturn: Calibri Size 10 Yang T J, et al. Eclampsia. JETem 2021. 6(3):S33-61. https://doi.org/10.21980/J8PS8R 53 Yang T J, et al. Eclampsia. JETem 2021. 6(3):S33-61. https://doi.org/10.21980/J8PS8R 53 DEBRIEFING AND EVALUATION PEARLS understand that the patient should be resuscitated and free of seizure before transport to the delivery room. Pre-Eclampsia & Eclampsia: Management Part 2: Calling Consultants Obstetricians should be called emergently with patients who present with both preeclampsia and eclampsia. This is to evaluate the fetus for fetal heart rate and monitoring and assess the need for an emergent delivery because it is the most definitive treatment of preeclampsia and eclampsia. Especially to patients who present with preeclampsia with severe features after 34 weeks of gestation, there are delivery criteria that obstetricians will look for: non-reassuring fetal heart activity, uncontrollable blood pressure, oliguria, markedly elevated creatinine, pulmonary edema, hypoxia, development of HELLP (hemolysis, elevated liver enzymes, low platelets), right upper quadrant tenderness, or any pregnancy complications such as ruptured membranes, oligohydramnios, intrauterine growth restriction. Most of the time, emergency providers will not have any supporting documents, such as lab values, because it takes time for returns. However, informing obstetricians of a potential obstetrics emergency based on other collaterals such as vital signs or symptoms can alert them to evaluate the patient and the fetus emergently.7 When calling consultants, learners need to be concise and direct. There is the “5C’s” model, which stands for: contact, communicate, core question, collaborate, and close the loop. This model was extensively studied in medical students but had the most extensive evidence of making a high-quality consult.17 Besides, learners should include pertinent information specific to each specialty. For example, in this simulated case, learners should include gravida/para status, gestational week, presenting issue, ultrasound/lab results (if present), and the last oral intake, since there might be a need for the patient to go to the operating room. Pre-Eclampsia & Eclampsia Management Part 3: Blood Pressure Management Since the definition of preeclampsia and eclampsia entails hypertension, learners should not only recognize but also correctly measure blood pressure. The blood pressure should be taken in either a sitting or semi-reclining position on the right arm with the right size cuff. Once learners realize that it is true hypertension, then there should be a few safe and effective anti- hypertensive agents that should come to mind to utilize in pregnant patients. Several medications can be used to lower the blood pressure effectively: hydralazine, labetalol, nifedipine, nicardipine, nitroglycerine, or nitroprusside. Although effective, providers should be cautious of the side effects medications can manifest. Labetalol is cautioned against patients with low heart rates because it blocks the atrioventricular node of the heart. DEBRIEFING AND EVALUATION PEARLS Also, since the medication is cleared via kidneys, the medication dose should be adjusted in renal failure patients—furthermore, some relative contraindications of the medication in myasthenia gravis, pulmonary edema, and renal edema.16 Besides the prophylaxis and chemical treatment, learners should also emergently consult the obstetrics team for definitive eclampsia management: delivery. However, learners need to Yang T J, et al. Eclampsia. JETem 2021. 6(3):S33-61. https://doi.org/10.21980/J8PS8R 54 DEBRIEFING AND EVALUATION PEARLS Hydralazine should be cautioned to use in renal patients because it is cleared more slowly in patients with acute kidney injury. Also, it is longer acting, so providers should be careful in redosing if blood pressure does not effectively reduce right away. Some agents like nitroprusside or nitroglycerine can be used; however, they can induce cyanide toxicity, so additional caution must be addressed. Since there is no consensus on the first-line agent, any available medications can be used.13 However, it is probably best to choose an agent that is familiar to the nursing team. Also, learners should communicate with nurses the goal and the titration of anti-hypertensives, especially if selecting a drip form. The goal of medication for severe preeclampsia is to lower the systolic blood pressure to less than 160 mmHg and diastolic to less than 110 mmHg to prevent progression to severe complications.13 Pre-Eclampsia & Eclampsia Management Part 3: Blood Pressure Management Yang T J, et al. Eclampsia. JETem 2021. 6(3):S33-61. https://doi.org/10.21980/J8PS8R 55 Assessment Timeline Assessment Timeline This timeline is to help observers assess their learners. It allows observer to make notes on when learners performed various tasks, which can help guide debriefing discussion. Other debriefing points: Besides the case’s flow and medical management, debriefing facilitators should also focus on teamwork and communication to cover attitudes. Communication • Was there a clear communication between the standardized patient and the participant? • Was there a clear consultation between the participant and the consultan Teamwork • Was there close-loop communications among team members? • Was there a shared mental model among team members? Wrap Up: Emergency medicine textbooks on preeclampsia as well as the American College of Obstetricians and Gynecologists can provide additional material for interested learners. Wrap Up: Emergency medicine textbooks on preeclampsia as well as the American College of Obstetricians and Gynecologists can provide additional material for interested learners. Yang T J, et al. Eclampsia. JETem 2021. 6(3):S33-61. https://doi.org/10.21980/J8PS8R 56 SIMULATION ASSESSMENT SIMULATION ASSESSMENT Eclampsia Learner: _ Critical Actions: Critical Actions: 1. Place IV, obtain vitals and a finger- stick glucose 2. Perform primary survey (airway, breathing, circulation) 3. Obtain a basic history of symptoms 4. Perform physical exam and recognize pregnancy 5. Verbalize “eclampsia” and call obstetrics consult early 6. Emergent blood pressure management (labetalol or hydralazine) 7. Treat seizure with magnesium 8. Recognize magnesium toxicity 9. Provide antidote for magnesium toxicity 10. Clearly communicate with consultants and nurses with the management goals and disposition 0 1. Place IV, obtain vitals and a finger- stick glucose 10. Clearly communicate with consultants and nurses with the management goals and disposition Yang T J, et al. Eclampsia. JETem 2021. 6(3):S33-61. https://doi.org/10.21980/J8PS8R 57 SIMULATION ASSESSMENT p i Learner: _ Critical Actions: Place IV, obtain vitals and a finger-stick glucose Perform primary survey (airway, breathing, circulation) Obtain a basic history of symptoms Perform physical exam and recognize pregnancy Verbalize “eclampsia” and call obstetrics consult early Emergent blood pressure management (labetalol or hydralazine) Treat seizure with magnesium Recognize magnesium toxicity Provide antidote for magnesium toxicity Clearly communicate with consultants and nurses with the management goals and disposition P O P V E T R P C Place IV, obtain vitals and a finger-stick glucose Perform primary survey (airway, breathing, circulation) Obtain a basic history of symptoms Perform physical exam and recognize pregnancy Verbalize “eclampsia” and call obstetrics consult early Emergent blood pressure management (labetalol or hydralazine) Treat seizure with magnesium Recognize magnesium toxicity Provide antidote for magnesium toxicity Clearly communicate with consultants and nurses with the management goals and disposition Standardized assessment form for simulation cases. JETem ã Developed by: Megan Osborn, MD, MHPE; Shannon Toohey, MD; Alisa Wray, MD Yang T J, et al. Eclampsia. JETem 2020. 6(3):S33-61. https://doi.org/10.21980/J8PS8R 5 Standardized assessment form for simulation cases. JETem ã Developed by: Megan Osborn, MD, MHPE; Shannon Toohey, MD; Alisa Wray, MD Yang T J, et al. Eclampsia. JETem 2020. 6(3):S33-61. https://doi.org/10.21980/J8PS8R Summative and formative comments: Standardized assessment form for simulation cases. JETem ã Developed by: Megan Osborn, MD, MHPE; Shannon Toohey, MD; Alisa Wray, MD Yang T J, et al. Eclampsia. JETem 2020. 6(3):S33-61. https://doi.org/10.21980/J8PS8R 58 58 SIMULATION ASSESSMENT Milestones assessment: Milestone Did not achieve level 1 Level 1 Level 2 Level 3 1 Emergency Stabilization (PC1) Did not achieve Level 1 Recognizes abnormal vital signs Recognizes an unstable patient, requiring intervention Performs primary assessment Discerns data to formulate a diagnostic impression/plan Manages and prioritizes critical actions in a critically ill patient Reassesses after implementing a stabilizing intervention 2 Performance of focused history and physical (PC2) Did not achieve Level 1 Performs a reliable, comprehensive history and physical exam Performs and communicates a focused history and physical exam based on chief complaint and urgent issues Prioritizes essential components of history and physical exam given dynamic circumstances 3 Diagnostic studies (PC3) Did not achieve Level 1 Determines the necessity of diagnostic studies Orders appropriate diagnostic studies. Performs appropriate bedside diagnostic studies/procedures Prioritizes essential testing Interprets results of diagnostic studies Reviews risks, benefits, contraindications, and alternatives to a diagnostic study or procedure 4 Diagnosis (PC4) Did not achieve Level 1 Considers a list of potential diagnoses Considers an appropriate list of potential diagnosis May or may not make correct diagnosis Makes the appropriate diagnosis Considers other potential diagnoses, avoiding premature closure Standardized assessment form for simulation cases. JETem ã Developed by: Megan Osborn, MD, MHPE; Shannon Toohey, MD; Alisa Wray, MD Yang T J, et al. Eclampsia. JETem 2020. 6(3):S33-61. https://doi.org/10.21980/J8PS8R 59 59 SIMULATION ASSESSMENT _________________________________________ Standardized assessment form for simulation cases. Standardized assessment form for simulation cases. JETem ã Developed by: Megan Osborn, MD, MHPE; Shannon Toohey, MD; Alisa Wray, MD Yang T J, et al. Eclampsia. JETem 2020. 6(3):S33-61. https://doi.org/10.21980/J8PS8R Summative and formative comments: JETem ã Developed by: Megan Osborn, MD, MHPE; Shannon Toohey MD; Alisa Wray MD Milestone Did not achieve level 1 Level 1 Level 2 Level 3 5 Pharmacotherapy (PC5) Did not achieve Level 1 Asks patient for drug allergies Selects an medication for therapeutic intervention, consider potential adverse effects Selects the most appropriate medication and understands mechanism of action, effect, and potential side effects Considers and recognizes drug-drug interactions 6 Observation and reassessment (PC6) Did not achieve Level 1 Reevaluates patient at least one time during case Reevaluates patient after most therapeutic interventions Consistently evaluates the effectiveness of therapies at appropriate intervals 7 Disposition (PC7) Did not achieve Level 1 Appropriately selects whether to admit or discharge the patient Appropriately selects whether to admit or discharge Involves the expertise of some of the appropriate specialists Educates the patient appropriately about their disposition Assigns patient to an appropriate level of care (ICU/Tele/Floor) Involves expertise of all appropriate specialists 9 General Approach to Procedures (PC9) Did not achieve Level 1 Identifies pertinent anatomy and physiology for a procedure Uses appropriate Universal Precautions Obtains informed consent Knows indications, contraindications, anatomic landmarks, equipment, anesthetic and procedural technique, and potential complications for common ED procedures Determines a back-up strategy if initial attempts are unsuccessful Correctly interprets results of diagnostic procedure Consistently evaluates the effectiveness of therapies at appropriate intervals 6 60 SIMULATION ASSESSMENT Milestone Did not achieve level 1 Level 1 Level 2 Level 3 20 Professional Values (PROF1) Did not achieve Level 1 Demonstrates caring, honest behavior Exhibits compassion, respect, sensitivity and responsiveness Develops alternative care plans when patients’ personal beliefs and decisions preclude standard care 22 Patient centered communication (ICS1) Did not achieve level 1 Establishes rapport and demonstrates empathy to patient (and family) Listens effectively Elicits patient’s reason for seeking health care Manages patient expectations in a manner that minimizes potential for stress, conflict, and misunderstanding. Effectively communicates with vulnerable populations, (at risk patients and families) 23 Team management (ICS2) Did not achieve level 1 Recognizes other members of the patient care team during case (nurse, techs) Communicates pertinent information to other healthcare colleagues Communicates a clear, succinct, and appropriate handoff with specialists and other colleagues Communicates effectively with ancillary staff Communicates pertinent information to other healthcare colleagues Standardized assessment form for simulation cases. Standardized assessment form for simulation cases. JETem ã Developed by: Megan Osborn, MD, MHPE; Shannon Toohey, MD; Alisa Wray, MD Yang T J, et al. Eclampsia. JETem 2020. 6(3):S33-61. https://doi.org/10.21980/J8PS8R 6 Summative and formative comments: JETem ã Developed by: Megan Osborn, MD, MHPE; Shannon Toohey, MD; Alisa Wray, MD Yang T J, et al. Eclampsia. JETem 2020. 6(3):S33-61. https://doi.org/10.21980/J8PS8R 61 61
https://openalex.org/W4391562883	https://thejas.com.pk/index.php/pjhs/article/download/1277/770	English	null	Exploring the Efficacy of Kinesio Taping as an Adjunct Treatment for Knee Osteoarthritis, Grade 1 & 2: A Quasi-Experimental Study	Pakistan journal of health sciences	2,024	cc-by	4,069	Corresponding Amna Khalid Faculty of Medical Sciences, Government College University Faisalabad, Faisalabad, Pakistan amnakhalid@gcuf.edu.pk th Received Date:9 January, 2024 th Acceptance Date: 29 January, 2024 st Published Date: 31 January, 2024 th Received Date:9 January, 2024 th Acceptance Date: 29 January, 2024 st Published Date: 31 January, 2024 th Received Date:9 January, 2024 th Acceptance Date: 29 January, 2024 st Published Date: 31 January, 2024 1 1 1 1 1 Danyal Ahmad , Hamza Zahid , Faiza Altaf , Ramish Sarfraz , Syeda Khadòa Kazmi and Amna ¹Faculty of Health Sciences, University of Management and Technology, Sialkot, Pakistan ¹Faculty of Health Sciences, University of Management and Technology, Sialkot, Pakistan ²Faculty of Medical Sciences, Government College University Faisalabad, Faisalabad, Pakistan ²Faculty of Medical Sciences, Government College University Faisalabad, Faisalabad, Pakistan Original Article Exploring the Efcacy of Kinesio Taping as an Adjunct Treatment for Knee Osteoarthritis, Grade 1 & 2: A Quasi-Experimental Study How to Cite: How to Cite: Ahmad, D., Zahid, H., Altaf, F., Sarfraz, R., Kazmi, S. K., & Khalid, A. (2024). Exploring the Eícacy of Kinesio Taping as an Adjunct Treatment for Knee Osteoarthritis, Grade 1 & 2: A Quasi-Experimental Study : Eícacy of Kinesio Taping for Knee Osteoarthritis . Pakistan Journal of Health Sciences, 5(01). https://doi.org/10.54393/pjhs.v5i01.1277 *Corresponding Author: Amna Khalid Faculty of Medical Sciences, Government College University Faisalabad, Faisalabad, Pakistan amnakhalid@gcuf.edu.pk Keywords: KinesioTapi Osteoarthritis (OA) is a prevalent chronic joint condition resulting in pain, stiffness, and reduced joint function. Kinesio taping (KT) has emerged as an adjunct treatment for OA. Objective: To examine the role of KT as an adjunctive intervention in the physiotherapy management of knee OA. Methods: A quasi-experimental investigation was conducted at Bethania Hospital, Pakistan, comparing the effectiveness of standard physiotherapy alone (Group 1) and standard physiotherapy plus KT (Group 2) in patients with knee OA grade I and II. By convenience sampling selected 50 participants aged above 40 with knee OA. Both groups received standard physiotherapy treatments, and KT was applied to Group 2 using a speciïc technique. Outcome measures included pain reduction Visual Analogue Scale (VAS), functional improvement using Western Ontario and McMaster Index (WOMAC), and presence of swelling and tenderness. Results: Demographic ïndings showed left-sided predominance of knee involvement, higher female prevalence, and a common age group of 40-45 years. Both groups demonstrated improvement in pain and functional outcomes post-intervention. The experimental group (Group 2) exhibited signiïcantly lower WOMAC scores (p <.001) and VAS pain scores (p=.011) compared to the control group indicating superior improvement. The incidence of swelling and tenderness around knee demonstrated no improvement in posttest analysis. Conclusions: Promising results are reported in KT group in management of knee OA in grade I and grade II as compared to only physiotherapy treatment. The study highlights the age speciïc considerations, tape application method and impact of kinesio tape intervention as an additional option in physiotherapy. I N T R O D U C T I O N One of the most common musculoskeletal ailment OA results in gradual decay of hyaline cartilage and symptoms of pain, stiffness and reduced range of motion are reported [1]. The weight-bearing joints especially knee are affected frequently and the sufferer are in millions globally especially in the elderly [2]. Once developed there is no absolute cure of the condition but various strategies are employed to reduce symptoms and reïne the standard of life [3]. KT as a treatment option in knee OA has gained popularity over the past few years [4]. This new technique was incepted by Dr. Kenzo, a Japanese chiropractor in 1970 that involved the use of elastic and adhesive tape in a speciïc manner to address the biomechanical dysfunctions in various soft tissues and joints. In 1970 [5]. Its clinical beneïts in terms of pain and dysfunctions have been studied with promising results by a number of clinicians [6, 7]. On the ðip side of this certain studies report it just as a subjective and temporary relief of pain perception with no deïnite superiority compared to placebo and with no longer beneïts [8, 9]. The non- invasive method of applying KT with no side effects has been investigated and this approach as an additional intervention supports the treatment outcome in addition to exercise, physical therapy and medication [10, 7]. The user friendly features in its application, its elasticity and porous texture with variable skin colors can make it a sound PJHS VOL. 5 Issue. 1 January 2024 90 DOI: https://doi.org/10.54393/pjhs.v5i01.1277 Eícacy of Kinesio Taping for Knee Osteoarthritis Ahmad D et al., Ahmad D et al., combination of Y-shaped and I-shaped strips was utilized in group 2. The initial Y-shaped band was positioned over the mid of rectus femoris with its end around patella towards tibial tuberosity. The subsequent Y-shaped band commenced just below the tibial tuberosity, with its tails encircling the patella, and the ends extending over the vastus medialis and vastus lateralis muscles with 0% tension at distal attachments and 10-15% in middle part. Two I-shaped bands were applied over the patella tendon transversely and the medial/lateral collateral ligaments transversely with partial superimposition of both with full tension [6]. The outcome measures were evaluated before and after the treatment to determine the effectiveness of this taping approach as an adjunctive therapy for knee osteoarthritis. M E T H O D S The research study was a quasi-experimental investigation that aimed to compare the effectiveness of two different treatment approaches in patients diagnosed with knee OA grade I and II according to the criteria set by the Kelgren Lawrence [12]. Both groups, group 1, non-taping & group 2, taping received same physiotherapy treatments whereas group 2 received an extra application of tape after each treatment session, concluding in 6 sessions on alternate days. The study was conducted at the physiotherapy OPD of Bethania Hospital Sialkot, a clinical aíliate of UMT Sialkot, Pakistan from May 2021 to May 2022. Before proceeding with the study, the ethical review committee of UMT Sialkot thoroughly examined the trial to ensure patient safety and approved its implementation on 23rd April 2021 vide letter with reference number “kuhs/pt/01-421”. Convenience sampling, a non-probability technique, was used to select the participants. The inclusion criteria for the study were individuals aged above 40 years, diagnosed with knee OA (grade I and II), and attending the hospital's physiotherapy OPD. Both genders were eligible for participation. Participants could have knee OA in one or both knees. The study excluded individuals with trauma, certain systemic conditions affecting joints and bones, having fragile skin, diagnosed with cancer, those who had received intra-articular injections within the last 3 months, individuals using analgesic medication, pregnant ladies, orthotics users, those with prior experience with the KT method, or individuals unwilling to participate were also excluded from the study. Sample size was calculated online, by reviewing previous literature and estimated inðow of cases in the hospital OPDs [13]. Fifty participants were selected and divided into two groups of 25 each. Both groups, received the same standard physiotherapy treatments, which included isometric knee exercises, interrupted therapeutic ultrasound therapy, transcutaneous electrical nerve stimulation (TENS), general range of motion exercises, and squatting [14-18]. A I N T R O D U C T I O N Data collection occurred both before and after the trial, and all outcome measures were recorded, tabulated, and compared using SPSS version 24.0 software to analyze the results. The primary outcome measure was the reduction in pain, which was assessed using the VAS [19]. Participants who scored 4 or 5 after the conclusion of the treatment sessions were categorized as “improvers”. Another outcome measure focused on the WOMAC index, indicating scores of disability from 0 to 96 [20]. The presence of swelling and tenderness, common symptoms in arthritis, were noted and analyzed before and after the trial to assess any changes associated with the treatment approaches. Decreased swelling and tenderness were evaluated through physical examination and by performing patellar ballottment test [21]. clinical tool in managing OA of knee [11]. The supportive role of KT in the management of knee OA is addressed in the study with its possible advantages, its biomechanical role and the evidence reinforcing its use in minimizing the sensations of pain, reducing disability and enhancing the quality of life. The tape application technique, its duration of use, possible side effects and its realistic implications can provide health care professionals an insight to address the treatment options to address this degenerative pathology. While KT is not a standalone solution for managing knee OA, its integration into a comprehensive treatment plan holds promise in optimizing patient outcomes. R E S U L T S Knee involvement was found to be more prominent on the left side. Interestingly, the study population exhibited a higher proportion of females compared to males, suggesting a potential gender-based difference in knee- related issues. The most prevalent age group affected was between 40 to 45 years, followed closely by the age groups of 50 to 55. The below data in Table 1 shows various important descriptive variables. Table 1: Demographic data. Non taping group (NTG), Taping group (TG) Variables Group 1 (NTG)(n= 25) Group 2 (TG)(n= 25) Sex(male/female) 9/16 12/14 Age(Mean ± SD) 44.90±7.26 45.40±7.46 BMI(Mean ± SD) 28.89±3.25 29.39±4.80 Affected Side(Right/Left) 9/14 7/16 Tenderness(positive/negative) 15/10 18/7 Swelling(positive/negative) 9/14 11/16 PJHS VOL. 5 Issue. 1 January 2024 Copyright © 2024. PJHS, Published by Crosslinks International Publishers 91 DOI: https://doi.org/10.54393/pjhs.v5i01.1277 Eícacy of Kinesio Taping for Knee Osteoarthritis Ahmad D et al., with mean of 35.24 and 34.88 and standard deviation of 4.83 and 3.19 respectively, measured at baseline. The posttest analysis shows decreased values in both groups but group 2 establishes more decrease in WOMAC score (M = 19.12.88, SD = 6.1) compared to the group 1 (M = 29.6, SD = 5.13) (Table 2). effectiveness of the intervention in reducing pain levels in group 2 as compared to group 1 (Table 5). Table 5: : t-test for Equality of Means of Pain (VAS) between groups Table 2: Group Statistics WOMAC Score Table 2: Group Statistics WOMAC Score Groups Pretest WOMAC Score(0-96) N Mean ± SD Group 1 (NTG) Group 2 (TG) 35.24 ± 4.83 34.88 ± 3.19 25 25 Posttest WOMAC Score(0-96) Group 1 (NTG) Group 2 (TG) 29.6 ± 6.01 19.12 ± 5.13 25 25 D I S C U S S I O N The present investigation highlighted the effectuality of kinesio taping technique in improving perception of pain and disability outcomes in individuals with grade 1 and grade 2 knee OA. Although both groups demonstrated signiïcant improvement in both primary treatment outcomes but tape use showed superiority over no tape along with standard physiotherapy treatments which both groups received. A systematic review demonstrated and acknowledged positive outcomes in terms of gain of muscular strength, psychological well-being and supportive effects on knee stability although these effects are not categorically reviewed upon in our study. Furthermore, a more cautious approach may be adopted before selecting such patients taking into account severity and duration of disease [7]. A randomized controlled trial established that the kinesio taping method resulted only in the subjective resolution of symptoms with no effects on overall mobility and function when compared to sham taping group. It is also crucial to note the large sample size and variable inclusion criteria in that study [8]. The ïndings from another inquiry demonstrated insigniïcant results in KT group and KT group with myofascial release to address joint range of motion in elder age group with knee OA. It is worth mentioning that we included participants across different age groups and with relatively mild disease of knee OA. The age related slowed healing processes may have been attributed to the study outcomes [22]. The demographic ïndings of the study indicate a higher The values of VAS score indicated the similar drop in its score in both groups post treatment but in group 2 these score decreased markedly. These ïndings further support our hypothesis of positive outcome in group 2 (M = 1.48, SD = 0.71) compared to the group 1 (M = 2.12, SD = 0.97) (Table 4). S o u r c e o f F u n d i n g All authors have read and agreed to the published version of the manuscript. All authors have read and agreed to the published version of the manuscript. R E F E R E N C E S Waldron T. Joint disease. Ortner's Identiïcation of Pathological Conditions in Human Skeletal Remains. 2019 Jan: 719-48. doi: 10.1016/B978-0-12-809738-0.0 0020-X. [1] Leifer VP, Katz JN, Losina E. The burden of OA-health services and economics. Osteoarthritis and Cartilage. 2022 Jan; 30(1): 10-6. doi: 10.1016/j.joca.20 21.05.007. [2] Leifer VP, Katz JN, Losina E. The burden of OA-health services and economics. Osteoarthritis and Cartilage. 2022 Jan; 30(1): 10-6. doi: 10.1016/j.joca.20 21.05.007. [2] Yang GY, Guo HL, Li T, Shang HB, Zhao YF, et al. The medial compartment and patellofemoral joint degenerate more severely in early stage knee osteoarthritis: a cross-sectional study. European Review for Medical & Pharmacological Sciences. 2020 Oct; 24(19): 9815-9823. doi: 10.26355/eurrev_ 202010_23191. [3] Mao HY, Hu MT, Yen YY, Lan SJ, Lee SD. Kinesio Taping Relieves Pain and Improves Isokinetic Not Isometric M u s c l e S t r e n g t h i n P a t i e n t s w i t h K n e e Osteoarthritis—A Systematic Review and Meta- Analysis. International Journal of Environmental Research and Public Health. 2021 Oct; 18(19): 10440. doi: 10.3390/òerph181910440. [4] The limitations of the study encompass a sample size that is relatively modest, patient reported pain and disability questionnaire and potential confounding variables such as prior level of activity and occupation that were not accounted for. Conducting research with a more extensive and varied sample, considering that well as the consideration of additional variables, could further strengthen the ïndings and provide a more comprehensive understanding of knee involvement and its management. [5] Noble MB, Noble SK, Shively SR, Jackson SB. Kinesio Taping for Bedside Pain Management. Bedside Pain Management Interventions. 2022 Nov: 159-167. doi: 10.1007/978-3-031-11188-4_17. [5] D I S C U S S I O N Table4::GroupStatisticsPain(VAS) Table 4: : Group Statistics Pain (VAS) Groups Parameters Pretest Visual Analogue Scale(0-10) N Mean ± SD Group 1 (NTG) Group 2 (TG) 3.84 ± 0.688 5.4 ± 0.5 25 25 Posttest Visual Analogue Scale(0-10) Group 1 (NTG) Group 2 (TG) 2.12 ± 0.971 1.48 ± 0.714 25 25 To check the signiïcant difference in pain scores, independent sample t-test was used after conïrming the assumption of homogeneity is met and variance in two samples is not signiïcantly different (p value>0.05) The t- test, with 95% CI and 48 df, supported the statistical signiïcance of this reduction (p = .011), emphasizing the To check the signiïcant difference in pain scores, independent sample t-test was used after conïrming the assumption of homogeneity is met and variance in two samples is not signiïcantly different (p value>0.05) The t- test, with 95% CI and 48 df, supported the statistical signiïcance of this reduction (p = .011), emphasizing the PJHS VOL. 5 Issue. 1 January 2024 Copyright © 2024. PJHS, Published by Crosslinks International Publishers 92 DOI: https://doi.org/10.54393/pjhs.v5i01.1277 Eícacy of Kinesio Taping for Knee Osteoarthritis Ahmad D et al., mild cases of knee OA. These ïndings hold substantial promise for managing knee-related degenerative issues, particularly in the age group of 40 to 65 years and grade I and grade II of Knee OA. Further research and exploration of gender-based and age differences and the inðuence of other factors on knee involvement will contribute to advancing knee health interventions and enhancing the quality of life. prevalence of knee involvement on the left side, a greater proportion of females affected, and the most common age group being between 40 to 45 years. This information is consistent with previous literature that suggests an increased susceptibility to knee issues in females and older individuals which might be attributed to hormonal and anatomical factors, warranting further investigation in future studies [23]. The post-intervention analysis demonstrated a statistically signiïcant decrease in pain and WOMAC scores in both the control and experimental groups. Notably, the experimental group exhibited a more signiïcant improvement, as evidenced by their lower WOMAC score and VAS pain scores compared to the control group. These ïndings align with previous research that highlights the potential beneïts of interventions in managing knee pain and function The treatment's success in improving functional outcomes can be attributed to its targeted approach, speciïcally tailored to address knee- related issues. D I S C U S S I O N Similar results have been reported in the study conducted by Anna but they used the sample of knee OA involving all severity levels whereas in our study we used grade I and II knee OA only [24]. The consistent presence of swelling in both groups throughout the trial indicates that the intervention did not signiïcantly inðuence the swelling aspect rather a study by Jarecki et al., has shown improvement in knee effusion post operatively in knee OA patients [25]. However, it is worth noting that this result might be inðuenced by other factors not directly targeted by the intervention. Future studies could explore additional strategies to address swelling in knee-related interventions. Regarding tenderness, the pretest values varied signiïcantly among the groups. Despite this initial difference, the posttest analysis revealed no signiïcant variation in tenderness presence between the control and experimental groups. This suggests that the intervention did not have a substantial impact on the tenderness aspect, which might be due to other underlying factors inðuencing tenderness levels in the participants, this ïnding has not been studied in detail in past. A u t h o r s C o n t r i b u t i o n Conceptualization: DA Methodology: FA Formal analysis: RS Writing-review and editing: DA, HZ, FA, SKK, AK All authors have read and agreed to the published version of the manuscript. A u t h o r s C o n t r i b u t i o n Conceptualization: DA Methodology: FA Formal analysis: RS Writing-review and editing: DA, HZ, FA, SKK, AK All authors have read and agreed to the published version of the manuscript. A u t h o r s C o n t r i b u t i o n Conceptualization: DA Methodology: FA Formal analysis: RS Writing-review and editing: DA, HZ, FA, SKK, AK All authors have read and agreed to the published version of the manuscript. C o n i c t s o f I n t e r e s t The authors declare no conðict of interest. S o u r c e o f F u n d i n g S o u r c e o f F u n d i n g C O N C L U S I O N S This study sheds light on the eícacy of the intervention in improving knee pain and functional outcomes in relatively PJHS VOL. 5 Issue. 1 January 2024 93 5 Issue. 1 January 2024 Copyright © 2024. PJHS, Published by Crosslinks International Publishers DOI: https://doi.org/10.54393/pjhs.v5i01.1277 Eícacy of Kinesio Taping for Knee Osteoarthritis DOI: https://doi.org/10.54393/pjhs.v5i01.1277 Eícacy of Kinesio Taping for Knee Osteoarthritis Ahmad D et al., Donec V and Kubilius R. The effectiveness of kinesio taping® for pain management in knee osteoarthritis: A randomized, double-blind, controlled clinical trial. Therapeutic Advances in Musculoskeletal Disease. 2019 Aug; 11: 1759720X19869135. doi: 10.1177/1759720 X19869135. [6] Nursing. 2021 Apr; 7: 2377960821993515. doi: 10.1177/ 2377960821993515. Wu Y, Zhu S, Lv Z, Kan S, Wu Q, Song W, et al. Effects of therapeutic ultrasound for knee osteoarthritis: a systematic review and meta-analysis. Clinical Rehabilitation. 2019 Dec; 33(12): 1863-75. doi: 10.1177/ 0269215519866494. [15] Melese H, Alamer A, Hailu Temesgen M, Nigussie F. Effectiveness of kinesio taping on the management of knee osteoarthritis: a systematic review of randomized controlled trials. Journal of Pain Research. 2020 May; 13: 1267-76. doi: 10.2147/JPR.S2 49567. [7] Wu Y, Zhu F, Chen W, Zhang M. Effects of transcutaneous electrical nerve stimulation (TENS) in people with knee osteoarthritis: a systematic review and meta-analysis. Clinical Rehabilitation. 2022 Apr; 36(4): 472-85. doi: 10.1177/02692155211065 636. [16] Donec V and Kubilius R. The effectiveness of Kinesio Taping® for mobility and functioning improvement in knee osteoarthritis: a randomized, double-blind, controlled trial. Clinical Rehabilitation. 2020 Jul; 34(7): 877-89. doi: 10.1177/0269215520916859. [8] Benner RW, Shelbourne KD, Bauman SN, Norris A, Gray T. Knee osteoarthritis: alternative range of motion treatment. Orthopedic Clinics. 2019 Oct; 50(4): 425-32. doi: 10.1016/j.ocl.2019.05.001. [17] Mohamed SH and Alatawi SF. Effectiveness of Kinesio taping and conventional physical therapy in the management of knee osteoarthritis: a randomized clinical trial. Irish Journal of Medical Science. 2023 Oct; 192(5): 2223-33. doi: 10.1007/s1184 5-022-03247-9. [9] Zhao Z, Wang R, Guo Y, Chen L, Wang K, Zhou H, et al. Static low-angle squatting reduces the intra- articular inðammatory cytokines and improves the performance of patients with knee osteoarthritis. BioMed Research International. 2019 Oct; 2019: 9617923. doi: 10.1155/2019/9617923. [18] Danazumi MS, Ibrahim SU, Yakasai AM, Dermody G, Bello B, Kaka B. A comparison between the effect of combined chain exercises plus kinesio taping with combined chain exercises alone in knee osteoarthritis: A randomized clinical trial. American Journal of Physical Medicine & Rehabilitation. 2021 Nov; 100(11): 1070-7. C O N C L U S I O N S doi: 10.1097/PHM.00000000000 01705. [10] Chiarotto A, Maxwell LJ, Ostelo RW, Boers M, Tugwell P, Terwee CB. Measurement properties of visual analogue scale, numeric rating scale, and pain severity subscale of the brief pain inventory in patients with low back pain: a systematic review. The Journal of Pain. 2019 Mar; 20(3): 245-63. doi: 10.1016/j.jpain.2018.07.009. [19] Jo H, Kim K, Im SC. Study of the Reliability and Validity of the WOMAC Index in Patients with Total Knee Replacement. Korean Society of Physical Medicine. 2023 May; 18(2): 93-101. doi: 10.13066/kspm.2023.18.2 .93. [20] de Brito Macedo L, Richards J, Borges DT, Melo SA, Brasileiro JS. Kinesio taping reduces pain and improves disability in low back pain patients: a randomised controlled trial. Physiotherapy. 2019 Mar; 105(1): 65-75. doi: 10.1016/j.physio.2018.07.005. [11] Rieck W and Loeffert J. Swelling from Down Under... The Patella. International Journal of Exercise Science: Conference Proceedings. 2021; 9(9): 4. [21] Olsson S, Akbarian E, Lind A, Razavian AS, Gordon M. Automating classiïcation of osteoarthritis according to Kellgren-Lawrence in the knee using deep learning in an unïltered adult population. BMC Musculoskeletal Disorders. 2021 Dec; 22(1): 1-8. doi: 10.1186/s12891-021-04722-7. [12] Waïq AZ and Yulianti A. Effectiveness of Combination Myofascial Release and Kinesio Taping Intervention on Range of Motion Improvement in Elderly with Knee Osteoarthritis. Physical Therapy Journal of Indonesia. 2020 May; 1(1): 13-6. doi: 10.51559/ptji.v1i1.4. [22] Mutlu EK, Mustafaoglu R, Birinci T, Ozdincler AR. Does Kinesio taping of the knee improve pain and functionality in patients with knee osteoarthritis?: a randomized controlled clinical trial. American Journal of Physical Medicine & Rehabilitation. 2017 Jan; 96(1): 25-33. doi: 10.1097/PHM.0000000000000 520. [13] Lu Y, Zheng ZL, Lv J, Hao RZ, Yang YP, Zhang YZ. Relationships between morphological changes of lower limbs and gender during medial compartment knee osteoarthritis. Orthopaedic Surgery. 2019 Oct; 11(5): 835-44. doi: 10.1111/os.12529. [23] Rahlf AL, Braumann KM, Zech A. Kinesio taping improves perceptions of pain and function of patients with knee osteoarthritis: a randomized, [24] Kangeswari P, Murali K, Arulappan J. Effectiveness of isometric exercise and counseling on level of pain among patients with knee osteoarthritis. SAGE Open [14] PJHS VOL. 5 Issue. 1 January 2024 Copyright © 2024. PJHS, Published by Crosslinks International Publishers 94 DOI: https://doi.org/10.54393/pjhs.v5i01.1277 Eícacy of Kinesio Taping for Knee Osteoarthritis DOI: https://doi.org/10.54393/pjhs.v5i01.1277 Eícacy of Kinesio Taping for Knee Osteoarthritis Ahmad D et al., controlled trial. Journal of Sport Rehabilitation. 2019 Jul; 28(5): 481-7. doi: 10.1123/jsr.2017-0306. Jarecki J, Sobiech M, Turżańska K, Tomczyk- Warunek A, Jabłoński M. A kinesio taping method applied in the treatment of postsurgical knee swelling after primary total knee arthroplasty. Journal of Clinical Medicine. 2021 Jul; 10(13): 2992. doi: 10.3390/jcm10132992. [25] controlled trial. Journal of Sport Rehabilitation. 2019 Jul; 28(5): 481-7. doi: 10.1123/jsr.2017-0306. C O N C L U S I O N S Jarecki J, Sobiech M, Turżańska K, Tomczyk- Warunek A, Jabłoński M. A kinesio taping method applied in the treatment of postsurgical knee swelling after primary total knee arthroplasty. Journal of Clinical Medicine. 2021 Jul; 10(13): 2992. doi: 10.3390/jcm10132992. [25] PJHS VOL. 5 Issue. 1 January 2024 5 Issue. 1 January 2024 Copyright © 2024. PJHS, Published by Crosslinks International Publishers 95
https://openalex.org/W1855564533	http://www.scielo.br/pdf/coluna/v9n2/a11v9n2.pdf	Portuguese	null	Prevalence and relationship of emotional and clinical factors in patients with degenerative disc disease	null	2,010	cc-by	5,586	RESUMEN ABSTRACT Objectives: to describe the psychosocial conditions, at the time of surgical indication, of patients with chronic low back pain caused by degenerative disc disease. Methods: in a series of 32 cases of degenerative disc disease elective surgery, a psychological evaluation was performed before the surgery. In this evaluation, socio-demographic data were collected: gender; age; present working status; occurrence of withdrawal due to disability; and clinical-psychological data: pain; physical restraint; use of psychiatric medication controlled; degree of anxiety; degree of depression; degree of hopelessness; expectations before the surgical goal. Results: the group was composed of 13 women and 14 men, mean age of 44.3 years old (23 to 65 years). The parameters of pain and physical restraint were compared to cases usually indicative of surgery, indicating no RESUMEN Objetivos: describir las condiciones psicosociales, en el momento de la in dicación quirúrgica, de pacientes con dolor lumbar crónico causado por dis copatia degenerativa del disco. Méto dos: en una serie de 32 casos de disco patia degenerativa del disco, se llevó a cabo una evaluación psicológica antes de la cirugía para la cirugía electiva. En esta evaluación, se recogieron datos socio-demográficos: de género; edad; situación laboral actual; la aparición de retiro debido a la discapacidad; y la clínica del dolor psicológico: la restric ción física; uso de medicación psiquiá trica controlada; el grado de ansiedad; el grado de depresión; el grado de la desesperanza; expectativas antes de la meta quirúrgica. Resultados: el grupo estaba compuesto por 13 mujeres y 14 hombres, edad media de 44,3 años (23 a 65 años). Los parámetros de dolor y de la restricción física están en la ac ción suele ser indicativa de la cirugía, lo que indica ninguna peculiaridad en SU O Objetivos: descrever as condições psicossociais, no momento da indica ção cirúrgica, dos pacientes com dor lombar crônica causada por discopatia degenerativa. Métodos: em uma série de 32 casos de discopatia degenerati va eletivos para cirurgia, foi realizada avaliação psicológica pré-operatória. Nesta avaliação, foram colhidos da dos demográficos e sociais: sexo, idade, situação ocupacional atual, ocorrência de afastamento por inva lidez; e dados clínico-psicológicos: dor, restrição física, utilização de me dicamento psiquiátrico controlado; grau de ansiedade; grau de depressão; grau de desesperança; expectativas frente ao objetivo cirúrgico. Resul tados: o grupo foi composto de 13 mulheres e 14 homens, com média de idade de 44,3 anos (23 a 65 anos). ARTIGO ORIGINAL / ORIGINAL ARTICLE ARTIGO ORIGINAL / ORIGINAL ARTICLE 1 Psicóloga do Instituto de Patologia da Coluna – IPC – São Paulo (SP), Brasil; Psicóloga da Irmandade da Santa Casa de Misericórdia de São Paulo – ISCMSP – São Paulo (SP), Brasil. 2 Mestre; Biomédico; Pesquisador do Instituto da Patologia de Coluna – IPC – São Paulo (SP), Brasil. 3 Biomédico; Pesquisador Coordenador do Instituto de Patologia da Coluna – IPC – São Paulo (SP), Brasil; Pós-graduando (Mestrado) do Departamento de Diag nóstico por Imagem da Universidade Federal de São Paulo – UNIFESP – São Paulo (SP), Brasil. 4 Doutor; Diretor Médico do Instituto de Patologia da Coluna – IPC – São Paulo (SP), Brasil; Professor da Universidade de San Diego – UCSD – San Diego (CA), Estados Unidos. Prevalência e relação de fatores emocionais e clínicos em pacientes com discopatia degenerativa Prevalence and relationship of emotional and clinical factors in patients with degenerative disc disease La prevalencia y la relación de los factores clínicos y los trastornos emocionales en pacientes con discopatia degenerativa del disco Prevalence and relationship of emotional and clinical factors in patients with degenerative disc disease Vivian Amaral1 Luis Marchi2 Leonardo Oliveira3 Luiz Pimenta4 Recebido: 20/03/2010 Instituto de Patologia da Coluna – IPC – São Paulo (SP), Brasil. Mestre; Biomédico; Pesquisador do Instituto da Patologia de Coluna – IPC – São Paulo (SP), Brasil. Aprovado: 25/06/2010 ; ; q g ( ), 3 Biomédico; Pesquisador Coordenador do Instituto de Patologia da Coluna – IPC – São Paulo (SP), Brasil; Pós-graduando (Mestrado nóstico por Imagem da Universidade Federal de São Paulo – UNIFESP – São Paulo (SP), Brasil. INTRODUÇÃO amigos. Esta situação conduz a uma espiral descendente física e emocional, que tem sido chamado de “descondi cionamento físico e mental”6. Já é bem conhecido, hoje em dia, que cerca de 80% da população mundial terá dor nas costas em algum momen to da vida1. Quase 99% destes pacientes conseguem re verter o quadro álgico com utilização de analgésicos ou tratamentos físicos conservadores2, por meio das diversas modalidades de correção postural ou de reforço da mus culatura paravertebral. Porém, o restante não se beneficia de práticas não-cirúrgicas, de modo que as dores sentidas perduram e se tornam problemas crônicos. Um fator fundamental para a reabilitação do paciente é a adesão ao tratamento pós-cirúrgico, como os cuida dos iniciais e os progressivos exercícios físicos. Caso o paciente esteja mentalmente ou socialmente desmotivado, ele provavelmente não se sentirá impelido a realizar o pro grama de reabilitação, comprometendo os resultados espe rados e realimentando o ciclo da doença. A depressão ocupa o 4º lugar na lista dos maiores pro blemas mundiais7 e, ainda, epidemiologistas alertam que até 2020 poderá ser a segunda causa de incapacitação para o trabalho8. Tendo em vista que pacientes com depressão apresentam quatro vezes mais dores restritivas do que pes soas sem alterações emocionais9, e que estas alterações se relacionam intimamente com os quadros álgicos, observa- se a necessidade de avaliação psicológica juntamente com a avaliação clínica. Nas síndromes de dor crônica, como dores de colu na e fribromialgia, existe alta prevalência de alterações emocionais importantes, como quadros de depressão e an siedade3,4. Pessoas que sofrem de dor crônica de coluna podem acabar desenvolvendo sintomas depressivos, uma vez que sentem dores constantes e incapacitantes que po dem, em alguns momentos, impedi-los de exercerem suas atividades profissionais, pessoais e sociais, e isso pode fa zer com que as limitações físicas acarretem em prejuízos emocionais. Tendo em vista esse preceito, a prevalência destes problemas emocionais foi comprovadamente maior em pessoas com diagnóstico de dor discogênica do que pessoas saudáveis5. Da mesma forma que os fatores emocionais, fatores sociais e financeiros influenciam na resolução de quadros de dor crônica, a compensação do trabalhador (em inglês, workers’ compensation) e afastamentos do trabalho por invalidez podem ser vistos pelos pacientes como recom pensas, e consequentemente o quadro crônico não é facil mente resolvido. Ainda, a sensibilidade à dor é aumentada em casos que envolvem incentivos fiscais e financeiros10. RESUMEN Estos parámetros indican la necesidad de una evaluación psicológica antes de la cirugía y el seguimiento, con miras a la rehabilitación del paciente. estudado. Foi evidenciada uma alta e correlacionada prevalência de al terações psicológicas nos pacientes: 50% em quadro depressivo, 54% em quadro de ansiedade e 22% em qua dro de desesperança. Além disso, foi possível evidenciar altos índices de afastamento do trabalho por invalidez física e correlação da presença de an siedade e/ou depressão com casos em que haviam expectativas deturpadas quanto ao resultado cirúrgico. Con clusões: dor lombar crônica é acom panhada de quadros de dor e de alte rações psicológicas, como ansiedade, depressão e desesperança. Esses parâ metros evidenciam a necessidade de avaliação psicológica pré-cirúrgica e acompanhamento posterior, visando à reabilitação do paciente. DESCRITORES: Psicología médica; Enfermedad crónica; Columna vertebral; Depresión; Ansiedad; Dolor de la región lumbar DESCRITORES: Psicologia médica; Doença crônica; Coluna vertebral; Depressão; Ansiedade; Dor lombar KEYWORDS: Psychology, medical; Chronic disease; Spine; Depression; Anxiety; Low back pain DESCRITORES: Psicologia médica; Doença crônica; Coluna vertebral; Depressão; Ansiedade; Dor lombar RESUMEN Os parâmetros de dor e restrição físi ca estão dentro de valores geralmente indicativos de cirurgia, não indican do nenhuma peculiaridade no grupo Recebido: 20/03/2010 Aprovado: 25/06/2010 Aprovado: 25/06/2010 Recebido: 20/03/2010 COLUNA/COLUMNA. 2010;9(2):150-156 Prevalência e relação de fatores emocionais e clínicos em pacientes com discopatia degenerativa 151 el grupo de estudio. Hemos demostra do una alta prevalencia y las correla ciones de cambios psicológicos en pa cientes: el 50% en la depresión, el 54% en el marco de la ansiedad y el 22% en el marco de la desesperanza. Además, era posible mostrar altas tasas de au sencia del trabajo debido a la discapa cidad y la correlación de la presencia física de la ansiedad y la depresión de los casos donde las expectativas eran engañosas en cuanto a los resultados. Conclusiones: el dolor lumbar crónico se acompaña de imágenes de dolor y los trastornos psicológicos tales como ansiedad, depresión y la desesperanza. Estos parámetros indican la necesidad de una evaluación psicológica antes de la cirugía y el seguimiento, con miras a la rehabilitación del paciente. peculiarity in the studied group. We demonstrated a high and correlated prevalence of psychological changes in patients: 50% of depression, 54% of anxiety and 22% of hopelessness. Furthermore, it was possible to show high rates of absence from work due to physical disability and relation to the presence of anxiety and/or depression with cases in which expectations were misleading according to the surgical objective. Conclusions: chronic low back pain is accompanied by clinical pictures of pain and psychological disorders such as anxiety, depression and hopelessness. These parameters indicate the need for pre-surgical psychological evaluation and posterior follow-up, aiming at the rehabilitation of the patient. el grupo de estudio. Hemos demostra do una alta prevalencia y las correla ciones de cambios psicológicos en pa cientes: el 50% en la depresión, el 54% en el marco de la ansiedad y el 22% en el marco de la desesperanza. Además, era posible mostrar altas tasas de au sencia del trabajo debido a la discapa cidad y la correlación de la presencia física de la ansiedad y la depresión de los casos donde las expectativas eran engañosas en cuanto a los resultados. Conclusiones: el dolor lumbar crónico se acompaña de imágenes de dolor y los trastornos psicológicos tales como ansiedad, depresión y la desesperanza. COLUNA/COLUMNA. 2010;9(2):150-156 MÉTODOS por meio da quantificação das condições clínicas, dor (ques tionário VAS) e limitação física (questionário ODI) (Tabela 2), foi possível observar que a média foi alta, característica de pa cientes com indicação a procedimentos cirúrgicos. Foi realizado um estudo descritivo prospectivo com 32 pa cientes, todos diagnosticados com dor crônica por conta de discopatia degenerativa do disco intervertebral (DDD). To dos os indivíduos aqui apresentados foram pacientes após análise médica no Instituto de Patologia da Coluna (IPC, São Paulo, Brasil) e, por não terem respondido a tratamentos conservadores, foram indicados a procedimentos cirúrgicos. A partir de questionários de avaliação psicológica, foi revelado um panorama que chamou a atenção dos pes quisadores: a maior parte dos pacientes se encontrava em Em visitas independentes e posteriores à indicação cirúr gica, um único psicólogo realizou durante entrevista a avalia ção de diversos fatores psicossociais destes pacientes e rea lizou aplicação de testes psicométricos direcionados. Dentre os itens avaliados, estão: situação ocupacional, ocorrência de tratamento com medicamento psiquiátrico controlado, ocorrência de acompanhamento psicológico, abrangência da dor sentida, expectativas sobre o resultado cirúrgico, grau de depressão (via questionário “Beck Depression Inventory” – BDI)11,12, grau de ansiedade (via questionário “Beck Anxiety Inventory” – BAI)13, grau de desesperança (via questionário “Beck Hopelessness Scale” – BHS)14, grau de dor (via ques tionário “Visual Analogue Scale” – VAS)15, e grau de restrição física (via questionário “Oswestry” – ODI)16. TABELA 1 – Dados demográficos da amostra TABELA 1 – Dados demográficos da amostra Variável Média±DP ou porcentagem Gê TABELA 1 – Dados demográficos da amostra Variável Média±DP ou porcentagem Gênero Masculino 53,1% Feminino 46,9% Idade 44,5±9,5 Níveis afetados por DDD L2L3 6,3% L3L4 12,5% L3L4/L4L5/L5S1 3,1% L4L5 34,4% L4L5/L5S1 28,1% L5S1 15,6% Situação ocupacional Afastado 43,8% Ativo 31,3% Do lar 9,4% Desempregado 6,3% Aposentado 9,4% DP: desvio padrão; DDD: discopatia degenerativa do disco intervertebral. Para categorizar os dados numéricos obtidos no ques tionário BDI, os intervalos de pontuações utilizados fo ram: resultados ≥0 e <11: ocorrência mínima; resultados ≥11 e <19: ocorrência leve; resultados ≥20 e <35: ocor rência moderada; resultados ≥35 e <63: ocorrência grave. Para categorizar os dados numéricos obtidos no ques tionário BAI, os intervalos de pontuações utilizados fo ram: resultados ≥0 e <10: ocorrência mínima; resultados ≥11 e <19: ocorrência leve; resultados ≥20 e <30: ocor rência moderada; resultados ≥31 e <63: ocorrência grave. INTRODUÇÃO A dor lombar crônica pode levar a uma diminuição da capacidade de participar de uma variedade de ativida des como trabalho, lazer e interação com os familiares e COLUNA/COLUMNA. 2010;9(2):150-156 Amaral V, Marchi L, Oliveira L, Pimenta L 152 Ainda na tabela 1, é possível observar um fenômeno alarmante: quase metade de todos pacientes do estudo es tava em situação de afastamento do trabalho por motivo de invalidez física (temporária ou permanente). Além disso, da parcela com situação ocupacional ativa, 25% já foram afastados alguma vez (dados não mostrados).i Sendo assim, o objetivo deste trabalho foi descrever e correlacionar a prevalência de fatores psicossociais em um grupo de pacientes encaminhados para cirurgia por diag nóstico de dor discogênica crônica. MÉTODOS desvio padrão; DDD: discopatia degenerativa do disco intervertebra Para categorizar os dados numéricos obtidos no ques tionário BHS, utilizaram-se os seguintes intervalos de pon tuações: resultados ≥0 e <4: ocorrência mínima; resultados ≥4 e <8: ocorrência leve; resultados ≥9 e <13: ocorrência moderada; resultados ≥14 e <20: ocorrência grave. TABELA 2 - Dados psicoclínicos TABELA 2 - Dados psicoclínicos Variável Média±DP ou porcentagem VAS 75,5±23,6 ODI 45,1±19,1 Depressão 52% Leve 24% Moderado 28% Ansiedade 56% Leve 28% Moderado 28% Desesperança 21,4% Leve 10,7% Moderado 10,7% Uso de medicação Psiquiátrica controlada 48,4% Expectativas incoerentes 38,7% DP: desvio padrão; VAS: Visual Analogue Scale; ODI: questionário Oswestry. TABELA 2 - Dados psicoclínicos Para análises estatísticas, foi utilizado o software Microsoft Office Excel®, usando como ferramentas o tes te t de Student, considerando resultados com valores de p≤0,05 estatisticamente significativos, e a regressão linear, considerando que valores de R2≥0,4 apontam que as variá veis estavam correlacionadas. Figura 4 Figura 4 Comparação do grau de dor com depressão ou ansiedade. As pontuações do questionário sobre dor (VAS – Visual Analogue Scale) de grupos de pacientes classificados como depressivos ou ansiosos (barras cheias) foram comparadas com as pontuações de grupos sem depressão ou ansiedade (barras vazias). Foram utilizados os questionários BDI (Beck Depression Inventory) e BAI (Beck Anxiety Inventory) para classificar os pacientes dentre os grupos de depressão e ansiedade, respectivamente. p<0,05. Figura 2 g Relação de níveis de depressão e desesperança. Cada ponto se refere às pontuações obtidas para cada sujeito pelos questionários BDI (Beck Depression Inventory) e BHS (Beck Hopelessnes Scale). Por meio da aplicação de regressão linear, foi obtido o valor do R2=0,5073. g Relação de níveis de depressão e desesperança. Cada ponto se refere às pontuações obtidas para cada sujeito pelos questionários BDI (Beck Depression Inventory) e BHS (Beck Hopelessnes Scale). Por meio da aplicação de regressão linear, foi obtido o valor do R2=0,5073. Fatores psicológicos em mulheres versus homens BDI / BAI / BHS 20,0 15,0 10,0 5,0 0,0 Depressão Ansiedade Desesperança Mulheres Homens Figura 5 Prevalência de depressão, ansiedade e desesperança. As pontuações do questionário sobre depressão (BDI – Beck Depression Inventory), ansiedade (BAI – Beck Anxiety Inventory) e desesperança (BHS – Beck Hopelessnes Scale) foram analisados separadamente entre grupos de mulheres (barras cheias) e de homens (barras vazias). p<0,003 e #p<0,02. Fatores psicológicos em mulheres versus homens BDI / BAI / BHS 20,0 15,0 10,0 5,0 0,0 Depressão Ansiedade Desesperança Mulheres Homens Figura 5 Figura 3 Relação de níveis de ansiedade e desesperança. Cada ponto se refere às pontuações obtidas para cada sujeito pelos questionários BAI (Beck Anxiety Inventory) e BHS (Beck Hopelessnes Scale). Por meio da aplicação de regressão linear, foi obtido o valor do R2=0,4698. Ansiedade versus Desesperança BHS BDI 14 12 10 8 6 4 2 0 R2 = 0,4698 0 5 10 15 20 25 30 35 F 3 Ansiedade versus Desesperança BHS BDI 14 12 10 8 6 4 2 0 R2 = 0,4698 0 5 10 15 20 25 30 35 g Prevalência de depressão, ansiedade e desesperança. As pontuações do questionário sobre depressão (BDI – Beck Depression Inventory), ansiedade (BAI – Beck Anxiety Inventory) e desesperança (BHS – Beck Hopelessnes Scale) foram analisados separadamente entre grupos de mulheres (barras cheias) e de homens (barras vazias). p<0,003 e #p<0,02. RESULTADOS Primeiramente, foi possível observar que a amostra de pa cientes utilizada foi homogeneamente distribuída entre os sexos (Tabela 1), o que evita qualquer interferência de gêne ro nos resultados globais aqui obtidos. A maior prevalência de DDD dentro do grupo está em L4L5, sendo ela unica mente neste nível ou se estendendo para L3L4 e/ou L5S1. COLUNA/COLUMNA. 2010;9(2):150-156 Prevalência e relação de fatores emocionais e clínicos em pacientes com discopatia degenerativa 153 quadros de ansiedade e/ou depressão. Constatou-se, ainda, que o quadro psicológico de desesperança estava presente em boa parcela do grupo estudado (Tabela 2). Por meio de análise cruzada entre as pontuações dos questionários de depressão, ansiedade e desesperança, foi possível mostrar que tais alterações emocionais estão correlacionadas nos pacientes (Figuras 1 a 3), ou seja, se o paciente tem um desses quadros psicológicos está predisposto a apresentar também os outros aqui estudados. Junto a esses dados de fatores psicológicos, verificou- se que a ocorrência atual ou anterior de tratamento com medicamentos psiquiátrico controlados era de quase meta de do grupo (Tabela 2). Outro dado relevante encontrado após a avaliação psi cológica foi que, apesar de ter passado em consultas clíni cas explicativas do tratamento cirúrgico que seria adotado e os respectivos riscos/benefícios, a impactante porcenta gem de 38,7% dos pacientes tinham expectativas deturpa das diante dos benefícios que eles poderiam obter (Tabela 2), sendo que quase a totalidade idealizava uma cura ex pansível e não limitada ao objetivo cirúrgico. i Figura 1 Relação de níveis de depressão e ansiedade. Cada ponto se refere às pontuações obtidas para cada sujeito pelos questionários BDI (Beck Depression Inventory) e BAI (Beck Anxiety Inventory). Por meio da aplicação de regressão linear, foi obtido o valor do R2=0,6558. Depressão versus Ansiedade BAI BDI 35 30 25 20 15 10 5 0 R2 = 0,6558 0 5 10 15 20 25 30 35 F 1 Depressão versus Ansiedade BAI BDI 35 30 25 20 15 10 5 0 R2 = 0,6558 0 5 10 15 20 25 30 35 Com os dados de fatores psicológicos estratificados em gêneros, pôde-se observar uma clara diferença en tre as prevalências em mulheres e homens, sendo que a Relação de dor com depressão/Ansiedade VAS 10 8 6 4 2 0 Depressão Ansiedade Presença de fator psicológico Ausência de fator psicológico Figura 4 Comparação do grau de dor com depressão ou ansiedade. RESULTADOS As pontuações do questionário sobre dor (VAS – Visual Analogue Scale) de grupos de pacientes classificados como depressivos ou ansiosos (barras cheias) foram comparadas com as pontuações de grupos sem depressão ou ansiedade (barras vazias). Foram utilizados os questionários BDI (Beck Depression Inventory) e BAI (Beck Anxiety Inventory) para classificar os pacientes dentre os grupos de depressão e ansiedade, respectivamente. p<0,05. Relação de dor com depressão/Ansiedade VAS 10 8 6 4 2 0 Depressão Ansiedade Presença de fator psicológico Ausência de fator psicológico Figura 4 Comparação do grau de dor com depressão ou ansiedade. As pontuações do questionário sobre dor (VAS – Visual Analogue Scale) de grupos de pacientes classificados como depressivos ou ansiosos (barras cheias) foram comparadas com as pontuações de grupos sem depressão ou ansiedade (barras vazias). Foram utilizados os questionários BDI (Beck Depression Inventory) e BAI (Beck Anxiety Inventory) para classificar os pacientes dentre os grupos de depressão e ansiedade, respectivamente. *p<0,05. Fatores psicológicos em mulheres versus homens BDI / BAI / BHS 20,0 15,0 10,0 5,0 Relação de dor com depressão/Ansiedade VAS 10 8 6 4 2 0 Depressão Ansiedade Presença de fator psicológico Ausência de fator psicológico Figura 4 Relação de dor com depressão/Ansiedade Figura 1 g Relação de níveis de depressão e ansiedade. Cada ponto se refere às pontuações obtidas para cada sujeito pelos questionários BDI (Beck Depression Inventory) e BAI (Beck Anxiety Inventory). Por meio da aplicação de regressão linear foi obtido o valor do R2=0,6558. g Relação de níveis de depressão e ansiedade. Cada ponto se refere às pontuações obtidas para cada sujeito pelos questionários BDI (Beck Depression Inventory) e BAI (Beck Anxiety Inventory). Por meio da aplicação de regressão linear, foi obtido o valor do R2=0,6558. Depressão versus Desesperança BHS BDI 14 12 10 8 6 4 2 0 R2 = 0,5073 0 5 10 15 20 25 30 35 Figura 2 Relação de níveis de depressão e desesperança. Cada ponto se refere às pontuações obtidas para cada sujeito pelos questionários BDI (Beck Depression Inventory) e BHS (Beck Hopelessnes Scale). Por meio da aplicação de regressão linear, foi obtido o valor do R2=0,5073. Depressão versus Desesperança BHS BDI 14 12 10 8 6 4 2 0 R2 = 0,5073 0 5 10 15 20 25 30 35 Fi 2 Depressão versus Desesperança Figura 4 DISCUSSÃO Segundo estudo na população em geral, a prevalência, du rante um ano, de distúrbios de ansiedade é de 10%17, dife rindo da prevalência de 56% que encontramos em pacientes com dor lombar crônica neste trabalho (Tabela 2). O mesmo acontece com os níveis de depressão, que estão em torno de 7%18,19 na população em geral, tendo sido revelada uma pre valência global de 52% neste estudo (Tabela 2). Esses dados confirmam que existe uma relação substancial de ansiedade e depressão com quadros de dor lombar crônica.i Além de fatores internos, algumas interferências exter nas também foram comprovadamente influenciadoras de resultados clínicos. Mostramos uma prevalência de 43,8% de afastamento do trabalho, corroborando dados que mos tram que a situação de afastamento e compensação do tra balhador é muito prevalente e de extrema importância em pacientes que sofrem de problemas na coluna35. É compro vado que estes pacientes tendem a apresentar piores prog nósticos36-38, fato que novamente evidencia a importância da identificação pré-operatória desses complicadores. Em outros trabalhos, são referidos ainda maiores graus de dor e piores resultados quando existe a presença de incentivos fiscais10, reforço por parte da família38,39, insatisfação ou processos para afastamento do trabalho40,41, solicitação de pensão42 e insatisfação quanto ao empregador43. Os quadros de depressão foram estratificados em pre valências de 3-5% para homens e 8-10% para mulheres na população em geral18,19 e, neste trabalho, observaram-se pre valências de 64% em mulheres e 28% em homens (dados não mostrados). É possível afirmar que a depressão aliada à dor crônica de coluna acomete um número maior de pessoas do sexo feminino, porém o aumento normalizado (mulher- mulher e homem-homem) em relação à depressão na popu lação em geral é o mesmo, ou seja, sete vezes maior. Várias pesquisas mostram que diversas alterações psicológicas pioram o quadro de dor e restrição física do paciente, mostrando que pacientes com depressão apre sentam predomínio de pensamentos negativos20, baixo limiar de dor21 e maiores restrições funcionais22. Além de modificar parâmetros somáticos, quadros emocionais nocivos dificultam ou mesmo impedem a reabilitação depois de diferentes tratamentos da coluna e podem ser preditivos de piores resultados de evolução clínica23-27. Estes e outros estudos comprovaram que, pela identifi cação pré-operatória de alguns aspectos psicológicos (como depressão, ansiedade, hostilidade e desesperança), os pacientes portadores desses fatores evoluem de forma Figura 4 Figura 3 Figura 3 Relação de níveis de ansiedade e desesperança. Cada ponto se refere às pontuações obtidas para cada sujeito pelos questionários BAI (Beck Anxiety Inventory) e BHS (Beck Hopelessnes Scale). Por meio da aplicação de regressão linear, foi obtido o valor do R2=0,4698. gu a 3 Relação de níveis de ansiedade e desesperança. Cada ponto se refere às pontuações obtidas para cada sujeito pelos questionários BAI (Beck Anxiety Inventory) e BHS (Beck Hopelessnes Scale). Por meio da aplicação de regressão linear, foi obtido o valor do R2=0,4698. COLUNA/COLUMNA. 2010;9(2):150-156 Amaral V, Marchi L, Oliveira L, Pimenta L 154 pior em relação aos outros pacientes, alertando quanto à necessidade de abordagens desta questão. depressão, a ansiedade e a desesperança apareceram com maior intensidade nas mulheres (Figura 4). No total, 69% de todos os casos de depressão e 57% de todos os casos de ansiedade eram mulheres (dados não mostrados). Outros parâmetros psicológicos, como a escala de psi castenia28 e as escalas de histeria e hipocondria29-31, tam bém comprovaram a influência negativa de fatores emo cionais em resultados após cirurgias de coluna. Posteriormente, fazendo uma correlação desses da dos clínicos com o estado psicológico dos pacientes, foi possível destacar a intensificação dos sintomas de dor e desabilidade física quando o paciente está ansioso em comparação a pacientes não-ansiosos (Figura 5). Existe a mesma tendência com pacientes em estado de depressão, apesar de não ter sido demonstrada estatisticamente devi do ao número relativamente reduzido de dados. Porém, existem processos emocionais que ajudam na recuperação, como a boa capacidade de enfrentamento, ou seja, pensamentos, sentimentos e comportamentos específicos do indivíduo para lidar com a dor de forma adaptativa32, mostrando resultados animadores para pa cientes com dores de coluna que utilizam essas ferramen tas aliadas à esperança e ao afastamento de pensamentos catastróficos33,34. Pelo que pudemos observar no presente trabalho, há uma alta prevalência de desesperança entre os pacientes com dor lombar crônica, fato que pode vir a atrapalhar os resultados clínicos no período pós-cirúrgico. 3. Ebmeier KP, Donaghey C, Steele JD. Recent developments and current controversies in depression. Lancet. 2006;367(9505):153-67. Comment in: Lancet. 2006;367(9518):1235. Lancet. 2006;367(9505):86. Lancet. 2006;367(9518):1235-6. Lancet. 2006;367(9518):1236. Lancet. 2006;367(9518):1236-7. 2. Scientific approach to the assessment and management of activity-related spinal disorders. A monograph for clinicians. Report of the Quebec Task Force on Spinal Disorders. Spine (Phila Pa 1976). 1987;12(7 Suppl):S1-59. CONCLUSÃO Como estabelecido anteriormente, a dor e seu alívio são parâmetros subjetivos, cada pessoa possui uma relação di ferente com eles, assim, as variáveis emocionais e sociais influeciam diretamente nessa dinâmica, que não é determi nada somente por terapias de intervenção somática. A grande maioria dos pacientes que sofrem de dor lom bar crônica apresenta problemas emocionais como depres são, ansiedade e desesperança, além de estar sujeita a fatores socioeconômicos determinantes. É necessária a identifica ção desses componentes antes de um procedimento terapêu tico, para que haja uma abordagem mais completa e eficaz. 3. Ebmeier KP, Donaghey C, Steele JD. 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https://openalex.org/W1972600158	https://www.scielo.br/j/eins/a/twdDFhF9R9YmSNW4Wss9HHr/?lang=en&format=pdf	English	null	Spectral characteristics of the hippocampal LFP during contextual fear conditioning	Einstein	2,012	cc-by	3,352	Study carried out at the Medical School, Universidade de São Paulo – USP, São Paulo (SP), Brazil. 1 Instituto do Cérebro – InCe, Hospital Israelita Albert Einstein – HIAE, São Paulo (SP), Brazil. 2 Faculdade de Medicina, Universidade de São Paulo – USP, São Paulo (SP), Brazil. 3 Associação Alberto Santos Dumont para Apoio à Pesquisa – São Paulo (SP), Brazil. Corresponding author: Birajara Soares Machado – Avenida Albert Einstein, 627/701 – Morumbi – Zip code: 05652-000 – São Paulo (SP), Brazil – Phone: (55 11) 2151-7354 – E-mail: birasm@gmail.com Received on: Sep 1, 2011 – Accepted on: Apr 24, 2012 Conflicts of interest: none. ORIGINAL ARTICLE ORIGINAL ARTICLE Spectral characteristics of the hippocampal LFP during contextual fear conditioning Características espectrais da atividade elétrica hipocampal durante o medo condicionado ao contexto Birajara Soares Machado1, Ana Carolina Bione Kunicki2, Edgard Morya3, Koichi Sameshima ABSTRACT delta-theta”, para caracterização dos diferentes comportamentos observados (vigília exploratória e congelamento) e tipos de memórias. Resultados: A razão delta-theta mostrou-se capaz de distinguir os grupos testados para as memórias recente e remota. Neste caso, observou-se, para o grupo testado 18 dias após, um valor menor do que para o grupo testado 1 dia após. Além disso, essa medida mostrou-se útil para distinguir os diferentes estados comportamentais. Conclusões: Os resultados sugerem que a dinâmica entre os ritmos delta e theta pode refletir o processamento da informação durante a evocação das memórias recente e remota. Objective: The hippocampus has an important role in the acquisition and recall of aversive memories. The objective of this study was to investigate the relationship among hippocampal rhythms. Methods: Microeletrodes arrays were implanted in the hippocampus of Wistar rats. The animals were trained and tested in a contextual fear conditioning task. The training consisted in applying shocks in the legs. The memory test was performed 1 day (recent memory) or 18 days (remote memory) after training. We proposed a measure based on the FFT power spectrum, denominated “delta-theta ratio”, to characterize the different behaviors (active exploration and freezing) and the memories types. Results: The delta-theta ratio was able to distinguish recent and remote memories. In this study, the ratio for the 18-day group was smaller than for the 1-day group. Moreover, this measure was useful to distinguish the different behavior states – active exploration and freezing. Conclusions: The results suggest delta-theta oscillations could reflect the demands on information processing during recent and remote memory recalls. Descritores: Hipocampo; Memória; Medo/fisiologia; Amnésia retrógrada INTRODUCTION The hippocampus has an important role in the acquisition of aversive memories. However, these memories are not stored permanently in the hippocampus. Hippocampal lesions, in humans and animals, produce serious deficits in recent memory, but not in remote memory(1-3). The current hypothesis suggests that the hippocampus is dynamically involved in a gradual process of reorganization and stabilization of memories in the neocortex(4-6). Keywords: Hippocampus; Memory; Fear/physiology; Amnesia retrograde METHODS We used 12 male Wistar rats from the School of Medicine, Universidade de São Paulo (USP), weighing approximately 300g at the beginning of the experiment. The animals were kept under controlled temperature (23±2°C) and 12 hour/12 hour light/dark cycle, in which the lights were turned on at 7am. Food and water were provided ad libitum. All procedures adopted were in accordance with the guidelines of the FMUSP Ethics Committee for Animal Research (CAPPesq 0948/09). The animals were habituated to the experimenter and to the environment, which was different from the conditioning place, with 5-minute sessions per day for 7 consecutive days. The training and test sessions were conducted during the light phase of the circadian cycle of the rat. In the first day (pre-exposure to the context), the animal was placed in the conditioning box for 5 minutes. In the second day (training), the animal was placed in the conditioning box and, after 2 minutes, received electric shocks in the legs. Each shock was of 0.7mA intensity and 2 seconds in duration. A total of 5 interleaved shocks were applied for 1 minute. The animals were reintroduced into the conditioning box for recall tests to asses recent (group G1) or remote (group G18) memories. The freezing time was recorded by the experimenter observing the animal’s behavior via the CCD video camera. ) The animals were weighed and anesthetized with ketamine hydrochloride (100mg/kg) and xylazine (4mg/kg) after previous induction with 5% halothane. The level of sedation was maintained with supplemental doses of 20% of the initial dose of ketamine per hour, or whenever the animal presented sensitivity to pain. After trichotomy and antisepsis of the surgical field, the rats were placed in a stereotaxic device (model 900, David Kopf Instruments, Tujunga, California) and a longitudinal skin incision in the midline of the skull was performed. A high speed dental drill was used to perform the craniotomy, opening three holes in which stainless steel microscrews were inserted. The microscrews fixed the implant in the skull with cyanoacrylate glue and served as contact points for the ground wire reference. The microelectrode arrays were implanted by stereotactic technique in the hippocampal CA1 region, with the center at the following anteroposterior (AP), mediolateral (ML) and dorsoventral (DV) coordinates from bregma: AP -3.60, ML 2.60, and DV 2.10mm. Behavior analysis was performed based on visual inspection of the video of the animals, frame by frame. RESUMO Objetivo: O hipocampo tem um importante papel na aquisição e evocação das memórias aversivas. Pretendeu-se, neste trabalho, investigar a dinâmica entre os ritmos hipocampais durante a evocação das memórias aversivas recente e remota. Métodos: Foram implantadas matrizes de microeletrodos em ratos Wistar. Os animais foram treinados e testados numa tarefa de condicionamento contextual de medo. O treino consistiu em aplicações de choques nas patas. O teste de memória foi realizado 1 dia (memória recente) ou 18 dias (memória remota) após o treino. Nesse trabalho, foi proposta uma medida baseada no espectro de potências, denominada “razão Evidence from experiments carried out by Bontempi et al.(7) and Frakland et al.(8) shows that several cortical regions such as the anterior cingulate cortex and pre and infralimbic cortices are involved in the processing of aversive memories, consistent with the proposal that remote memories are stored in distributed cortical networks. Numerous studies have investigated the einstein. 2012;10(2):140-4 Spectral characteristics of the hippocampal LFP 141 equipment (Multichannel Acquisition Processor, Plexon Inc., Dallas). The signals were pre-amplified (1000x), filtered through a 0.3Hz high-pass filter and a 400Hz low-pass filter, and digitized by means of a (National Instruments, Austin) 64-channel board at an acquisition frequency of 2kHz. cellular and molecular basis involved in the recall and storage of recent and remote memories(9-12). However, the electrophysiological mechanisms involved in the time course of the cortico-hippocampal circuit are not yet well known. y In rats, an excellent paradigm for the behavioral study of recent and remote memories is contextual fear conditioning(1,2). This paradigm involves placing the animal in a given context (pre-exposure) and applying electric shocks in the legs (training). The memory test is performed with two separate groups of rats trained with 5 shocks in the legs and tested 1 day (recent memory) or 18 days (remote memory) after training. Using this paradigm, we selected two behavioral states: active exploration, in pre-exposure, and freezing, a characteristic behavior observed in tests of memory. The rats were divided into two groups (G1 and G18, six rats per group). All animals underwent microelectrode implantation to record hippocampal electrical activity and were euthanized after the test – group G1 one day after training, and group G18 18 days after training. All animals were trained and tested in a contextual fear conditioning task. For this purpose, we used a conditioning box (26x21x27.5cm3), with black walls and transparent acrylic cover. RESUMO The floor consisted of a metal grid (with edges of 16.4mm in diameter and equally spaced every 1.2cm) connected to a discontinuous shock source. The conditioning box was placed in a Faraday cage (89.5x79x54cm3). On top of this cage we set up an infrared CCD video camera to record the animal’s behavior during the task. METHODS The animal’s behavioral state was classified as quiet awake, active exploration and freezing. The animal was considered in active exploration state when it presented intense exploratory locomotor activity, or in freezing state when it presented total absence of movements except those associated with respiratory movements. In this context, the quiet awake is behaviorally equivalent to freezing. Figure 1 shows the freezing rates in trained groups and control group (animals which did not receive shock in the legs). One week after the implant surgery, a recording was performed using a local field potential acquisition einstein. 2012;10(2):140-4 einstein. 2012;10(2):140-4 Machado BS, Kunicki AC, Morya E, Sameshima K 142 Figure 1. Freezing rates of trained and control animals. The trained groups are subdivided into those who underwent the test one day after training (recent memory) and 18 days after training (remote memory) Figure 2. Two-second segments of hippocampal electrical activity in behavioral states: (A) active exploration, (B) freezing 1 day after training and; (C) freezing 18 days after training. All signals range of ±0.5 mV A B C A Figure 2. Two-second segments of hippocampal electrical activity in behavioral states: (A) active exploration, (B) freezing 1 day after training and; (C) freezing 18 days after training. All signals range of ±0.5 mV Figure 1. Freezing rates of trained and control animals. The trained groups are subdivided into those who underwent the test one day after training (recent memory) and 18 days after training (remote memory) energies of delta (0.5 and 4Hz) and theta (4 and 10Hz) rhythms, as shown in figure 3. The estimator of the power spectrum used in this study was proposed by Welch(13). This procedure consists in dividing the time series data into overlapping segments. For each segment, the power spectrum is obtained, and the average of the estimates is calculated. Perfusion was performed through the left ventricle with paraformaldehyde in 0.1M buffer. Before opening the thoracic cavity, the animals were anesthetized with ketamine hydrochloride (350mg/kg) and xylazine (4mg/kg). After perfusion, the brain was removed and frozen at -80°C for subsequent tissue section and staining. Nissl staining using cresyl violet dye was conducted to identify the position of the electrodes. The nonparametric statistical tests for comparison of the delta-theta ratio were the Wilcoxon signed rank test (W-test), for paired comparisons, and the Mann- Whitney test (U-test), for comparison of independent samples. einstein. 2012;10(2):140-4 RESULTS RESULTS It is well established in the literature that, during active exploration, the predominant brain electrical activity in the hippocampus is the theta rhythm(15-17). Our results corroborate these findings and also show, during freezing reaction, a significant increase in delta rhythm in the typical brain activity when compared to the hippocampal theta rhythm. The delta-theta ratio allowed the characterization of recent and remote memories (p<0.005; U-test). In this case, the ratio for the tested animals after 18 days (3.231±0.970) was lower when compared with the tested group after 1 day (6.778±1.316), featuring a slower activity during recent memory recall. Moreover, this measure proved to be useful to distinguish different behavioral states – active exploration (0.314±0.099, G1 group; 0.370±0.208, G18 group) and freezing (p<0.05, for G1 e G18 groups; W-test). More specifically, the delta-theta ratio of animals in active exploration is lower when compared to animals in freezing. A significant difference was observed (p<0.005, U-test) in ρδθ-values test with the respective control groups (0.309±0.033, 1 day after; 0.341±0.050, 18 day after) from the quiet awake. Finally, an ANOVA test was applied in the ρδθ-values from the behavior states active exploration and quiet awake. No significant difference was not observed (p=0.241; F-test). Figure 4 summarizes the main results. A model to explain the oscillatory brain dynamics was proposed by Lakatos et al.(18). These researchers suggest that there is a hierarchical organization in brain electrical activity, in which the amplitude of each oscillatory frequency is modulated by the phase of a low frequency oscillation. Lakatos et al.(19) showed earlier that the phase of these oscillatory activities control the excitability of cortical neuronal populations and thus may influence sensory stimuli processing. Further studies on the dynamics of the interaction among brain rhythms are needed, since this may be an important mechanism to facilitate memory consolidation processes. In this study, we found that the dynamics between delta and theta waves changes depending on the type of memory recalled. Frankland et al.(8) reported that immediate early genes Fos and Zif-268 are strongly activated in the hippocampus after recent memory recall, but not after remote memory recall. The opposite was observed in the medial prefrontal cortex, which revealed a significant increase in the expression of these genes after remote memory recall, but not after recent memory recall. These results indicate that the recall of different types of memory is mediated by different mechanisms in different brain regions. RESULTS Figure 4. Box diagram of the delta-theta ratio during active exploration (VE) and 1 day freezing (C01D) and 18 days (C18D) after training The delta-theta ratio proved to be a useful measure in the characterization of behavioral states. Additionally, it differentiated groups of animals in freezing state during recent and remote memory recalls. Thus, the relationship between delta and theta rhythms may be an important parameter to be considered in the study of processes related to learning and memory. Figure 4. Box diagram of the delta-theta ratio during active exploration (VE) and 1 day freezing (C01D) and 18 days (C18D) after training Another relevant issue is the significant increase of the contribution of delta rhythm accompanied by a decrease in theta rhythm during recent memory recall. This may indicate a slowing of hippocampal activity. Knowing that the hippocampus has an important role in the acquisition of recent memory, but not of remote memory, it is suggested that the hippocampus coordinates the distribution of information to the neocortex over time. It is possible that the slowing of the hippocampus facilitates communication in the cortico-hippocampal circuitry, since the neocortex has a slower activity(20,21). It is proposed that the communication between the areas of the brain occurs by METHODS Due to sample size (six animals per group) it was decided not to conduct a normality test. The ρδθ values comparison for active exploration G1 and G18 and the two control groups was performed by one-way ANOVA (F-test). The level of significance for all statistical tests was 0.05. We selected segments (duration of 2 seconds) of the electrophysiological recordings in the three behaviors of interest: active exploration and freezing, besides the quiet awake. The first is related to the signals recorded before the animal was subjected to applications of shock in the legs. The freezing behavior was recorded in order to compare brain rhythms in the recall of recent and remote memories. Figure 2 illustrates examples of the three states of interest. Quiet awake records were obtained from the control group. Figure 3. Characteristic power spectra in behavioral states: (A) active exploration, and (B) freezing. Delta-theta ratios are 0.415 and 3.748, respectively A B To study the electrophysiological mechanisms involved in fear conditioning, we proposed a measure based on power spectrum ratios, considering the contributions of slow oscillations, in the other words ρδθ = Ēδ / Ēθ, where and ƒ(ν) is the Fourier transform of the signal ƒ(t). Therefore, Ēω can be interpreted as the normalized power spectrum of a specific electrophysiological signal ƒ(t) in a certain range of frequencies ω = [νs, νe]. The ρδθ measure is defined by the ratio between the average Figure 3. Characteristic power spectra in behavioral states: (A) active exploration, and (B) freezing. Delta-theta ratios are 0.415 and 3.748, respectively einstein. 2012;10(2):140-4 Spectral characteristics of the hippocampal LFP 143 CONCLUSION 11. Frankland PW, Bontempi B. The organization of recent and remote memories. Nat Rev Neurosci. 2005;6(2):119-30. The results suggest that the relationship between delta and theta rhythms may reflect the information processing during recent and remote memory recalls. The results suggest that the relationship between delta and theta rhythms may reflect the information processing during recent and remote memory recalls. 12. Takehara-Nishiuchi K, Nakao K, Kawahara S, Matsuki N, Kirino Y. Systems consolidation requires postlearning activation of NMDA receptors in the medial prefrontal cortex in trace eyeblink conditioning. J Neurosci. 2006; 26(19):5049-58. 13. Welch PD. The use of fast fourier transform for the estimation of power spectra: a method based on time averaging over short, modified periodograms. IEEE Trans Audio Electroacoust. 1967;15(2):70-3. DISCUSSION In the behavioral paradigm studied, the freezing level was used as an indication of the memory retention rate. There was a decrease in the freezing rate over time depending on the type of memory recalled. Similar results were described by Rudy and Wright-Hardesty(14). These authors showed that there is a direct relationship between the freezing level and the interval between training and testing of memory retention. Additionally, the delta-theta ratio also showed that the characteristic electrophysiological pattern of the hippocampus during freezing also decreased. In the behavioral paradigm studied, the freezing level was used as an indication of the memory retention rate. There was a decrease in the freezing rate over time depending on the type of memory recalled. Similar results were described by Rudy and Wright-Hardesty(14). These authors showed that there is a direct relationship between the freezing level and the interval between training and testing of memory retention. Additionally, the delta-theta ratio also showed that the characteristic electrophysiological pattern of the hippocampus during freezing also decreased. einstein. 2012;10(2):140-4 einstein. 2012;10(2):140-4 Machado BS, Kunicki AC, Morya E, Sameshima K 144 8. Frankland PW, Bontempi B, Talton LE, Kaczmarek L, Silva AJ. The involvement of the anterior cingulate cortex in remote contextual fear memory. Science. 2004;304(5672):881-3. means of synchronization events, and that these events would be facilitated by resonant activities. Finally, the subsequent restoration of hippocampal theta activity can be interpreted as a decrease of the hippocampal participation in the memory processes. 9. 9. Routtenberg A, Cantallops I, Zaffuto S, Serrano P, Namgung U. Enhanced learning after genetic overexpression of a brain growth protein. Proc Natl Acad Sci USA. 2000;97(13):7657-62. 10. Maviel T, Durkin TP, Menzaghi F, Bontempi B. Sites of neocortical reorganization critical for remote spatial memory. Science.2004;305(5680):96-9. ACKNOWLEDGEMENTS We thank the Permanent Forum of University-Industry Relations and the Foundation for Research Support of São Paulo (FAPESP). 14. Rudy JW, Wright-Hardesty K. The temporal dynamics of retention of a context memory: something is missing. Learn Mem. 2005;12(2):172-7. São Paulo (FAPESP). 15. Vanderwolf CH. Hippocampal electrical activity and voluntary movement in the rat. Electroencephalogr Clin Neurophysiol. 1969;26(4):407-18. 16. Gavrilov VV, Wiener SI, Berthoz A. Enhanced hippocampal theta EEG during whole body rotations in awake restrained rats. Neurosci Lett. 1995; 197(3):239-41. REFERENCES 1. Kim JJ, Fanselow MS. Modality-specific retrograde amnesia of fear. Science. 1992;256(5057):675-7. 17. Slawinska U, Kasicki S. The frequency of rat’s hippocampal theta rhythm is related to the speed of locomotion. Brain Res. 1998;796(1-2):327-31. 2. Anagnostaras SG, Maren S, Fanselow MS. Temporally graded retrograde amnesia of contextual fear after hippocampal damage in rats: within-subjects examination. J Neurosci. 1999;19(3):1106-14. 18. Lakatos P, Shah AS, Knuth KH, Ulbert I, Karmos G, Schroeder CE. An oscillatory hierarchy controlling neuronal excitability and stimulus processing in the auditory cortex. J Neurophysiol. 2005;94(3):1904-11. 3. Manns JR, Hopkins RO, Squire LR. Semantic memory and the human hippocampus. Neuron. 2003;38(1):127-33. 19. Lakatos P, Szilágyi N, Pincze Z, Rajkai C, Ulbert I, Karmos G. Attention and arousal related modulation of spontaneous gamma-activity in the auditory cortex of the cat. Brain Res Cogn Brain Res. 2004:19(1):1-9. 4. Zola-Morgan SM, Squire LR. The primate hippocampal formation: evidence for a time-limited role in memory storage. Science. 1990;250(4978):288-90. 5. Squire LR, Alvarez P. Retrograde amnesia and memory consolidation: a neurobiological perspective. Curr Opin Neurobiol. 1995;5(2):169-77. 20. Sirota A, Csicsvari J, Buhl D, Buzsáki G. Communication between neocortex and hippocampus during sleep in rodents. Proc Natl Acad Sci U S A. 2003; 100(4):2065-9. 6. Knowlton BJ, Fanselow MS. The hippocampus, consolidation and on-line memory. Curr Opin Neurobiol. 1998;8(2):293-6. 21. Mölle M, Yeshenko O, Marshall L, Sara SJ, Born J. Hippocampal sharp wave- ripples linked to slow oscillations in rat slow-wave sleep. J Neurophysiol. 2006;96(1):62-70. 7. Bontempi B, Laurent-Demir C, Destrade C, Jaffard R. Time-dependent reorganization of brain circuitry underlying long-term memory storage. Nature. 1999;400(6745):671-5. einstein. 2012;10(2):140-4
W2980467457.txt	https://pubs.rsc.org/en/content/articlepdf/2019/sc/c9sc03610e	en		A model for dinitrogen binding in the E<sub>4</sub>state of nitrogenase	Chemical science	2,019	cc-by	13,595	Chemical Science View Article Online Open Access Article. Published on 15 October 2019. Downloaded on 5/21/2024 11:05:43 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. EDGE ARTICLE Cite this: Chem. Sci., 2019, 10, 11110 All publication charges for this article have been paid for by the Royal Society of Chemistry View Journal \| View Issue A model for dinitrogen binding in the E4 state of nitrogenase† Albert Th. Thorhallsson,ab Bardi Benediktsson a and Ragnar Bjornsson ab Molybdenum nitrogenase is one of the most intriguing metalloenzymes in nature, featuring an exotic iron– molybdenum–sulfur cofactor, FeMoco, whose mode of action remains elusive. In particular, the molecular and electronic structure of the N2-binding E4 state is not known. In this study we present theoretical QM/ MM calculations of new structural models of the E4 state of molybdenum-dependent nitrogenase and compare to previously suggested models for this enigmatic redox state. We propose two models as possible candidates for the E4 state. Both models feature two hydrides on the FeMo cofactor, bridging atoms Fe2 and Fe6 with a terminal sulfhydryl group on either Fe2 or Fe6 (derived from the S2B bridge) and the change in coordination results in local lower-spin electronic structure at Fe2 and Fe6. These structures appear consistent with the bridging hydride proposal put forward from ENDOR studies and are calculated to be lower in energy than other proposed models for E4 at the TPSSh-QM/MM level of theory. We critically analyze the DFT method dependency in calculations of FeMoco that has resulted in strikingly diﬀerent proposals for this state. Importantly, dinitrogen binds exothermically to either Fe2 or Fe6 in our models, contrary to others, an eﬀect rationalized via the unique ligand ﬁeld (from the hydrides) at the Fe with an empty coordination site. A low-spin Fe site is proposed as being important to N2 binding. Furthermore, the geometries of these states suggest a feasible reductive elimination step that could follow, as experiments indicate. Via this step, two electrons are released, reducing the cofactor to yield a distorted 4-coordinate Fe2 or Fe6 that partially activates N2. We speculate that stabilization of an N2-bound Fe(I) at Fe6 (not found for Fe2 model) via reductive elimination is a crucial part of N2 activation in nitrogenases, possibly aided by the apical heterometal ion (Mo or V). By using protons from the Received 22nd July 2019 Accepted 14th October 2019 sulfhydryl group (to regenerate the sulﬁde bridge between Fe2 and Fe6) and the nearby homocitrate hydroxy group, we calculate a plausible route to yield a diazene intermediate. This is found to be more DOI: 10.1039/c9sc03610e favorable with the Fe6-bound model than the Fe2-bound model; however, this protonation is uphill in rsc.li/chemical-science energy, suggesting protonation of N2 might occur later in the catalytic cycle or via another mechanism. Introduction The mechanism of biological nitrogen reduction, catalyzed by the bacterial nitrogenase enzymes, has remained an unsolved problem in bioinorganic chemistry despite decades of research involving many experimental and theoretical research groups. The nitrogenases are metalloenzymes that catalyze the reduction of dinitrogen to two molecules of ammonia for each a Science Institute, University of Iceland, Dunhagi 3, 107 Reykjavik, Iceland b Department of Inorganic Spectroscopy, Max-Planck-Institut für Chemische Energiekonversion, Stistrasse 34-36, 45470 Mülheim an der Ruhr, Germany. E-mail: ragnar.bjornsson@cec.mpg.de † Electronic supplementary information (ESI) available: Further information on all broken-symmetry solutions calculated for all models. Details of the QM/MM model preparation. Spin populations of all models. Localized orbital analysis of selected models. Geometry analysis of E0 state calculated with diﬀerent functionals and electronic structure analysis. Cartesian coordinates for the QM regions of all optimized structures available as XYZ les. See DOI: 10.1039/c9sc03610e 11110 \| Chem. Sci., 2019, 10, 11110–11124 molecule of dinitrogen, impressively at ambient conditions.1,2 The molybdenum-dependent form is the most active and the best characterized, yet there is no consensus on the dinitrogen binding site on the cofactor nor is there a basic mechanism in place. The complete enzyme consists of a transient complex of the MoFe protein and the Fe protein. The function of the Fe protein is electron transfer to the MoFe protein via an [Fe4S4] cluster in an ATP-dependent process while dinitrogen reduction takes place in the MoFe protein. The active site of the MoFe protein, includes a complex [MoFe7S9C] cofactor (FeMoco, shown in Fig. 1) where dinitrogen binds. The kinetic studies by Lowe and Thorneley3 resulted in a model for dinitrogen catalysis linking diﬀerent redox states of the cofactor (denoted En, with n indicating the number of added electrons and protons) together. The obligatory formation of one molecule of H2 per molecule of N2 is an intriguing aspect of the model, suggesting the unusual stoichiometry and the apparent waste of 2 electrons and 4 molecules of MgATP to produce this extra molecule of H2.4 This journal is © The Royal Society of Chemistry 2019 View Article Online Open Access Article. Published on 15 October 2019. Downloaded on 5/21/2024 11:05:43 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Edge Article Top: Resting state structure of the iron–molybdenum cofactor (FeMoco) in the active site of MoFe protein of Mo-dependent nitrogenase. The cofactor is bound to the a-275Cys residue at Fe1 and the a442His residue at Mo. Bottom: A simpliﬁed Lowe–Thorneley scheme showing the early redox states of FeMoco up until dinitrogen binding. While H2 evolution can occur as side-reactions from the E2, E3 and E4 states, obligatory H2 evolution is proposed to occur concomitantly as N2 binds in the E4 state. Fig. 1 N2 + 8e + 8H+ + 16MgATP / 2NH3 + H2 + 16MgADP Curiously, in the Lowe–Thorneley model (a simplied scheme is shown in Fig. 1), dinitrogen does not bind to the iron–molybdenum cofactor until either the E3 or E4 state (aer 3 or 4 added electrons and protons to the resting state E0), concomitant with formation of one molecule of H2. This obligatory H2 evolution step (as N2 binds) is thus directly part of the mechanism (rather than being one of the known sidereactions).4 Most kinetic and spectroscopic studies have been directed towards the E4 state as it is EPR active, its population can be increased by specic mutations and it has been argued to be the primary state for N2 binding.3,4 The FeMoco cluster is a highly unusual metallocofactor with a molecular structure that took many years to characterize; it consists of 7 iron ions, 1 molybdenum ion, 9 suldes and an interstitial carbide5,6 (see Fig. 1). Its electronic structure is deeply complicated owing to the open-shell nature of the metal ions and their complex spin coupling in a highly covalent cluster (including an unusual carbide ligand).7 The iron oxidation states of the resting state FeMoco are formally Fe(II) and Fe(III) (typical in iron–sulfur clusters) while the Mo ion was determined via Mo K-edge and L-edge XAS spectroscopy to be in a Mo(III) oxidation state and theoretical calculations have shown it to be in an unusual spin-coupled low-spin state, likely due to metal–metal bonding interactions with the Fe ions.8,9 Recent Xray magnetic circular dichroism experiments demonstrated the This journal is © The Royal Society of Chemistry 2019 Chemical Science presence of the unusual spin-coupled state of Mo(III) in a related [MoFe3S4] model cubane.10 The charge of the cofactor in the resting state has been determined to be [MoFe7S9C]1 according to Mössbauer analysis, spatially resolved anomalous dispersion renement and comparison of calculated and crystallographic metal–metal distances.11–13 Recent studies of the electronic structure from broken-symmetry (BS) DFT studies reveals a complicated electronic structure featuring both antiferromagnetic coupling, mixed-valence delocalization and partial metal–metal bonding.8,11,13 Other redox states of FeMoco are less well-characterized but we note recent spectroscopic studies of the E1 state14,15 and the E2 state.16,17 The binding site of dinitrogen is far from obvious from inspection of the cluster in its resting state (E0) and since little is known about the structure of the E4 redox state (which has been argued to be the primary state for N2 binding during turnover3,4), this is one of the most pressing questions in nitrogenase research. Even less is known about the E3 state and its proposed N2 binding as the state is EPR silent. A correct structural model for E4 would arguably reveal (or at least strongly suggest) how dinitrogen can favourably bind to FeMoco and how this typically inert molecule is activated for protonation. An accumulated S ¼ 1/2 E4 state was originally probed by EPR spectroscopy in a mutant (a-70Val/Ile) of MoFe protein and ENDOR spectroscopy revealed that the structure contains 2 chemically near-equivalent bridging hydrides, likely storing the 4 reducing equivalents.18 Later, the EPR signal and ENDOR hyperne signals associated with this state were found in the wild-type protein as well.19 The reductive elimination of H2 from these two hydrides was furthermore proposed to explain the obligatory H2 evolution in the mechanism4,20 and how dinitrogen is activated for protonation and there is now ample experimental evidence for this proposal.19,21 There is, however, no consensus on the structure of the E4 state, neither the precise coordination of the hydrides, nor is the N2 binding site agreed upon. Mutation studies have previously implicated the Fe2–Fe3– Fe6–Fe7 face as a likely binding site for dinitrogen as no dinitrogen reduction is observed when the residue a-70Val is mutated into the bulkier a-70Ile.22 Recent joint experimental– computational studies have proposed E4 models featuring bridging hydrides23–25 (the favoured model features bridging hydrides between Fe3–Fe7 and Fe2–Fe6) while other computational studies have suggested mixed bridging/terminal hydride models26,27 or even, surprisingly, structures featuring a protonated carbide with/without bridging hydrides.28–31 Additionally, recent crystal structures of a CO-bound form32 of MoFe protein (CO being an inhibitor) and an XH-ligand-bound (X ¼ N or O) form33 of VFe protein have emerged, that show CO/XH replacing the Fe2–Fe6 bridging sulde in a bridging binding mode (we recently showed via QM/MM calculations that the XH ligand in the FeVco structure is more consistent with an OH).34 These crystal structures demonstrate the lability of the sulde bridges, which hints at a possible binding site of dinitrogen or perhaps a binding site of hydrides as discussed by Einsle et al.33,35 Overall, there is no consensus on the nature of the E4 state. The present study proposes new structural models for the E4 state of FeMoco as well as a model for dinitrogen binding based Chem. Sci., 2019, 10, 11110–11124 \| 11111 View Article Online Open Access Article. Published on 15 October 2019. Downloaded on 5/21/2024 11:05:43 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Chemical Science on theoretical calculations. A state-of-the-art QM/MM protocol using the broken-symmetry DFT approach is used that has been previously validated on the resting E0 state.13 We compare the new structural models to previous proposals by calculating all models at the same QM/MM level of theory. The DFT method dependence of the relative energies is discussed and is put into context with the ability of diﬀerent computational protocols to describe the electronic structure of the resting state correctly. Two energetically favourable models appear to be consistent with the bridging hydride proposal from experimental ENDOR studies and while favourable dinitrogen binding to both is possible, the E4 model featuring an open coordination site at Fe6 binds N2 slightly stronger. Concomitant H2 evolution with N2 binding via the reductive elimination proposal can be explained using our model due to close proximity of the hydrides (favoured over a hydride-proton reaction) and this leads to the stabilization of an N2-bound Fe(I) intermediate at Fe6 (or alternatively to an N2-bound Fe(II) intermediate at Fe2). A possible protonation step to yield a FeMoco-bound diazene intermediate is discussed. Computational details The QM/MM models for E4 were based on our model for the E0 resting state model that has been previously described.13 It is a spherical QM/MM model (42 Å radius and 37 000 atoms) centered on the carbide of FeMoco that includes roughly half of the tetrameric MoFe protein; see ESI† for details about the QM/ MM model preparation. In the QM/MM geometry optimizations of E4 models the active region consists of 1003 atoms and a QM region of 136 atoms while calculations of E0 used a smaller QM region of 54 atoms. All QM/MM calculations were performed in Chemshell36,37 using the built-in MM code DL_POLY38 with the CHARMM36 forceeld39 and ORCA version 4.0 (ref. 40) as QM code. The 136 atom QM region (see Fig. S4 in ESI†) contains the FeMoco cofactor, singly protonated homocitrate and the sidechains of residues a-70Val, a-96Arg, a-191Gln, a-195His, a-275Cys, a-278Ser, a-359Arg, a-380Glu, a-381Phe and a-442His. The QM region thus contains the main residues critical to describing the coordination, electrostatic environment, asymmetry, and hydrogen-bonding environment around FeMoco. This includes residues directly coordinating FeMoco (a-442His, a-275Cys), neighboring charged residues (a-96Arg, a-359Arg, a-380Glu), those capable of participating in hydrogen bonding (a-195His, a191Gln, a-278Ser), as well as spatially close residues (a-70Val, a381Phe). a-195His is calculated to be in the Nd protonation state, assuming the residue has donated a proton from N3 to the cofactor and has been reprotonated at Nd. All QM/MM calculations used electrostatic embedding, and hydrogen link atoms were used to terminate the QM–MM border together with the charge-shi procedure as implemented in Chemshell. The E4 QM calculations primarily used the TPSSh hybrid density functional,41,42 a ZORA scalar relativistic Hamiltonian,43,44 the relativistically recontracted def2-TZVP basis set45,46 on all metal, sulfur, carbide, homocitrate and hydride/SH/CH atoms (def2SVP on other atoms) and a D3BJ dispersion correction.47,48 The RIJCOSX approximation49,50 with a decontracted Coulomb 11112 \| Chem. Sci., 2019, 10, 11110–11124 Edge Article auxiliary basis set by Weigend51 was used. All DFT calculations used tight integration grids (Grid5 Finalgrid6 settings in ORCA). Single-point energy calculations of E4 models were performed at the TPSS,41 B3LYP52–55 and M06-2X56 levels using the same basis set. M06-2X calculations used a very large grid (Grid7 in ORCA) due to the grid-dependence associated with this functional.57 QM/MM geometry optimizations of the E0 state with other functionals always used the D3BJ dispersion correction except in the case of M06-2X where a D3(0) correction was used. Functionals used for E0 geometry optimizations were BP86,52,58 TPSS,41 B3LYP, PBE0,59,60 BHLYP,61 M06-2X and uB97MD3BJ.62,63 Broken-symmetry solutions of the E4 models were found by ipping spins on Fe atoms converging to the MS ¼ 1/2 solution. Four diﬀerent broken-symmetry solutions were explored (with Fe ions 235, 346, 247 or 147 spin down; atom numbering as in crystal structure) as discussed later. Vibrational frequencies of N2-bound models were calculated from a numerical QM/MM partial Hessian. While all QM/MM calculations minimized the QM/MM energy function, we primarily discuss the polarized QM energy rather than the total QM/MM energy as the former is less sensitive to the unrelated MM energy changes of the MM region. Results and discussion A. Computational modelling of the E4 state Theoretical modelling of the E4 state (here dened as the state aer addition of 4e and 4H+ to the resting state E0 of FeMoco) has been pursued recently by many groups and their models will be discussed and compared. It is helpful to rst discuss criteria that, in our view, a realistic model for the E4 state should ultimately full. These criteria are: (i) consistency with available spectroscopic data, (ii) the model being a thermodynamically viable state, (iii) demonstration of favorable N2 binding and consistency with isotope substitution experiments and, (iv) computational consistency. Regarding criterion i, regrettably, spectroscopic data for the E4 state are scarce and are primarily available in the form of EPR and ENDOR data, and primarily on a mutant form of the protein (which appears though to have the same spectroscopic signature as the wild type19). The EPR data indicates a spin state of S ¼ 1/2 and analysis of the 1H hyperne tensors from 1H ENDOR spectroscopy led to the proposal that the state contains two near-identical metal-bound hydrides, that are bridging rather than terminal and bound to Fe ions rather than the Mo ion according to 95Mo ENDOR.64 Furthermore, 57Fe ENDOR indicates that the overall iron redox level of E4 is the same as in the E0 state,65 which suggests that the two hydrides are acting as carriers of the four added electrons. A recent high-resolution ENDOR study of the same state has additionally revealed signals from two other hydrogens, likely present as protons bound to suldes.25 As quantum chemical calculations of FeMoco are currently mostly limited to broken-symmetry DFT approaches (where pure spin states are not calculated) and spin projection schemes are problematic for non-Heisenberg systems like FeMoco, this unfortunately makes a direct comparison of the calculations to magnetic spectroscopy data This journal is © The Royal Society of Chemistry 2019 View Article Online Open Access Article. Published on 15 October 2019. Downloaded on 5/21/2024 11:05:43 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Edge Article from EPR and ENDOR diﬃcult. However, an indirect comparison is still possible, i.e. a comparison of computed structures to the bridging hydride structural motif suggested by the ENDOR analysis. Regarding thermodynamic stability i.e. criterion ii, kinetic studies of MoFe protein under turnover, indicates the E4 state is only eetingly stable. The state can be freeze-trapped in a turnover sample with a small population but will evolve H2 with fast rates to fall back to an E2 state (that in turn further evolves H2 and falls back to E0).66 The eeting nature of this state and the fact that the mechanisms of reduction and protonation are not entirely clear, indicates that thermodynamics alone cannot be the only guiding principle for diﬀerentiating between models for E4; however, thermodynamic stability must still be relevant to its formation. Criterion iii implies that the model should ideally demonstrate favourable N2 binding. Experimental studies indicate N2 binding to be unfavourable in the early redox states, suggesting that the unknown E4 state features a structural component that makes N2 binding favourable (e.g. an empty coordination site), unlike redox states E0–E2 (E3 has also been proposed to bind N2). Furthermore, a model for E4 should oﬀer a plausible explanation or mechanism for the obligatory H2 evolution resulting from the reductive elimination of H2 from two hydrides (as shown by isotope substitution studies) as N2 binds to FeMoco. Finally, we propose computational consistency as criterion iv, that the computational E4 model should satisfy. By this, we mean that the computational protocol used to propose the model should also be shown to be consistent with other important experimental data of the system such as data for the other redox states. The 1.0 Å crystal structure of the E0 state6 is in our view the most accessible experimental data for gauging the quality of the computational protocols. The quality of the crystal structure of MoFe protein has improved steadily in recent years and has a bond length uncertainty of 0.02 Å. We consider a satisfactory agreement of the computed resting state structure to the crystal structure to be vital to any computational protocol that is used to suggest new redox state models of FeMoco. All models that we consider in this study were structurally optimized at the same QM/MM level of theory that we have previously used to describe the resting state E0 and we considered multiple broken-symmetry states for each model. As recently discussed by Raugei et al.,24 the protein environment and the quality of the computational model can make a surprisingly large diﬀerence regarding the relative stability of an E4 isomer. Raugei et al. demonstrated e.g. that a protonatedcarbide model for E4 was only stable for small cluster models of the active site but became unstable when larger cluster models were considered. As our QM/MM model accounts for the explicit protein environment from the beginning (and avoids potentially articial constraints on residues) and furthermore utilizes a large QM-region (including important charged and hydrogenbonding residues near FeMoco), our calculations should suﬀer much less from such artifacts. Our QM/MM methodology has previously been validated by detailed comparison of the calculated molecular structure of the cofactor in the resting state to This journal is © The Royal Society of Chemistry 2019 Chemical Science the available high-resolution crystal structure, giving better agreement for the basic metal framework than cluster models.13 As before, we primarily utilize the TPSSh level of theory (a 10% Hartree–Fock (HF) exchange TPSS hybrid), a ZORA scalar relativistic Hamiltonian, dispersion correction and a exible polarized triple-zeta basis set (on the cofactor). The functional dependency of calculations is discussed in chapter C. Due to the complex open-shell nature of the [MoFe7S9C] cluster, the spin coupling problem of FeMoco is far from trivial. The broken-symmetry DFT methodology used here, ips individual spins on atoms and converges to broken-symmetry SCF solutions with localized alpha and beta spins on diﬀerent atoms. These BS states likely correspond well to some of the low energy electronic states of FeMoco, but the methodology cannot capture the full multiplet spectrum of such a complex spincoupled metal-sulfur cluster. Additionally, as discussed in recent multicongurational wavefunction theory studies, simplied model Hamiltonians (Heisenberg and Double Exchange Hamiltonians) are likely too simple to give a realistic description for spin-coupled iron–sulfur systems,67 preventing the use of spin-projection schemes. For the resting state, the BS7 class of solutions (that appears to maximize antiferromagnetic interactions) has been consistently found to be most favourable in multiple studies5,8,68–71. The three BS7 solutions, here labelled according to which Fe ions are spin-down (crystal structure numbering): BS-235, BS-346 and BS-247 have been found to be within 1 kcal mol1 of each other (QM/MM-TPSSh level of theory) and in previous work13 we found that the BS-235 solution was in better agreement with the crystal structure than the other spin isomers. As E4 is a diﬀerent redox state, however, the spin coupling may well have changed completely due to binding of hydrides and in fact the experimental spin state changes from S ¼ 3/2 (E0) to S ¼ 1/2 (E4). Thus a similar BS state to the one in E0 cannot simply be assumed to apply to E4. Cao et al.27 have shown, however, in their work where many diﬀerent E1–E4 models were considered (featuring many of the models studied herein) that the BS solutions that are consistently lowest in energy are the BS7 class of solutions, thus supporting the hypothesis that maximizing antiferromagnetic coupling remains important, even when hydrides are bound. In this work we have primarily considered the BS7 solutions (BS-235, BS-346 and BS-247) as the most relevant broken-symmetry solutions as well as the BS-147 solution. The BS7 solutions are similar in energy and feature a very similar electronic structure in the resting state E0 (as discussed in ref. 11 and 13); the diﬀering positions of spin-down Fe atoms though has the eﬀect of changing the iron oxidation state at each site, as the mixedvalence pairs or localized ferric/ferrous irons will then involve diﬀerent atoms in the structure. This local oxidation state interpretation of the diﬀerent broken-symmetry states has been previously discussed.11,13,15 A BS-147 solution features ferromagnetic alignment of atoms Fe2 and Fe6. As some of the models in this study feature structural changes at these Fe ions, it seems possible that such a solution could become more favourable than other BS solutions. To aid in the computational problem involving many models and many BS states at an expensive level of theory, we have restricted ourselves to Chem. Sci., 2019, 10, 11110–11124 \| 11113 View Article Online Chemical Science performing geometry optimizations with the BS-235, BS-247, BS-346, BS-147 solutions to explore the energetics of all models in Fig. 2. We additionally explored all 35 BS solutions of two models (l and o) at the single-point level (see ESI, Fig. S5 and S6† respectively). All models assumed a nal MS ¼ 1/2 spin state, consistent with the experimental S ¼ 1/2 spin state of E4. Open Access Article. Published on 15 October 2019. Downloaded on 5/21/2024 11:05:43 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. B. QM/MM calculations of models of the E4 state Fig. 2 shows the molecular structures of various proposed E4 models from previous work and new ones, featuring a variety of hydride binding modes and/or sulde/carbide protonation scenarios. We are aided here by the work of Cao et al.,27 who performed a systematic study of a large number of protonation positions in the E0–E4 redox states. Thus, we have included the most stable E4 models found in their study (the study lacks, however, open sulde-bridge models) according to the levels of theory employed in their study (TPSS and B3LYP), as well as models from other groups. All models, shown in Fig. 2, can be grouped according to whether they contain a protonated carbide (model a, c and d),27,29,30 whether they feature terminal/ bridging hydrides with an intact sulde bridge (models e, f, g and h)24,26,27 or whether they feature hydrides with an open sulde bridge in the form of a terminal sulydryl group (models b, i, j, k, l, m, n, o). Edge Article Models n, m and o were previously discussed as models close in energy to the favoured model e in work by Raugei et al. In this study we introduce models l and model o (though a model like o has been discussed by Raugei et al.24) as plausible candidates for E4 as well as models j and k, being similar to models m and n, and model b featuring two terminal sulydryl groups and two bridging hydrides. Model i also has a terminal sulydryl group like l but has a diﬀerent sulde protonation state.72 The models with a terminal sulydryl group at Fe2/Fe6 (rather than at Fe4/Fe5 or Fe3/Fe7) seem consistent with crystal structures of ligand-bound states32,33 lacking S2B (that bridges Fe2 and Fe6), suggesting some lability of this particular sulde bridge. Fig. 2 and Table 1 contains data for the lowest energy BS solution but data for all broken-symmetry solutions can be found in Tables S1–S5† along with Mulliken spin populations for each state. As shown in Fig. 2 and Table 1, the relative energies (polarized QM energies at QM/MM geometries) at the TPSSh level of theory indicate models featuring protonated carbides (a, c, d) are strongly disfavoured, appearing much too high in energy (17–32 kcal mol1 higher in energy than the lowest energy model n) to be likely candidates for the E4 state. Additionally, models e, f, g, h, featuring an intact sulde bridge also appear rather high in energy ranging from 13–22 kcal mol1. Only open sulde bridge models, j–o, featuring a terminal sulydryl group Fig. 2 Structures and relative polarized electronic energies (EQM in kcal mol1) of all FeMoco models for the E4 state considered in this study. All models were minimized using the same QM/MM level of theory using the TPSSh-D3 functional, ZORA–def2-TZVP basis set and a ZORA scalar relativistic Hamiltonian. See Tables 1 and S1 and S2† for information on all BS solutions tested. A large QM region of 136 atoms was used in the calculations but only the cofactor geometry is shown here. Hydrides are colored in light blue and carbide/sulﬁde-bound protons in magenta. 11114 \| Chem. Sci., 2019, 10, 11110–11124 This journal is © The Royal Society of Chemistry 2019 View Article Online Edge Article Chemical Science Calculated relative energies (kcal mol1) of the E4 models investigated (shown in Fig. 2) with diﬀerent functionals, with the lowest energy broken symmetry solution indicated for each model. For TPSSh, both the polarized QM energies (MM point charges included in the QM calculation) and total QM/MM energies are given. Single-point (polarized) QM calculations with TPSS, B3LYP and M06-2X used the TPSSh geometries. See Tables S2–S5 in the ESI for data on all BS solutions calculated Open Access Article. Published on 15 October 2019. Downloaded on 5/21/2024 11:05:43 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Table 1 TPSSh Model (BS-state) a (147) b (247) c (247) d (147) e (147) f (346) g (346) h (247) i (247) j (147) k (147) l (147) m (346) n (346) o (147) EQM 31.46 20.40 17.14 16.96 16.88 21.80 17.60 12.56 12.55 4.99 3.29 3.75 2.47 0.00 2.48 EQM/MM 23.67 17.66 14.73 17.58 11.23 22.55 13.02 11.37 10.91 3.35 1.95 2.44 2.04 0.00 2.25 Single-point QM energies (EQM) Model TPSS Model a (235) 37.77 a (147) b (247) 15.77 b (147) c (247) 36.81 c (147) d (147) 26.12 d (346) e (147) 12.81 e (235) f (346) 3.43 f (235) g (346) 4.07 g (235) h (346) 2.87 h (247) i (247) 12.08 i (247) j (235) 3.50 j (147) k (235) 0.37 k (147) l (235) 2.28 l (147) m (235) 0.21 m (346) n (346) 6.73 n (346) o (147) 0.00 o (346) at Fe2 or Fe6 are found to be similarly low in energy, suggesting that it is generally thermodynamically favourable to alter the coordination of a protonated sulde bridge to aid stabilizing hydrides (bridging, terminal or dihydrogen-like). Model b with two open sulde bridges is an exception to this trend. This energetic analysis suggests that models j–o are the most viable models for the E4 state (at the TPSSh-QM/MM level). Open sulde-bridge models like j–o were not considered in the study by Cao et al.27 but some open-sulde bridge models were discussed by Raugei et al.24 and were found to be similar in energy as model e, which, however, is not in agreement with our results. We attribute this disagreement to diﬀerent modelling aspects, i.e. cluster modelling vs. QM/MM modelling, as well as BS-solution dependence and functional dependence, which will be discussed in the next section. C. Computational protocol dependence As has recently been discussed in the literature, by Raugei et al.24 and especially by Cao et al.27,73 there is a considerable functional dependency present in calculations of FeMoco, aﬀecting e.g. whether carbide protonation is favoured or not, or the structure of FeMoco. Thus, when hybrid density functionals with $20% HF exchange such as B3LYP (20% HF exchange) and M06-2X (54% HF exchange) have been used in the computational protocol, carbide protonation models have been found to be favoured over models with only hydrides. This is at odds with our relative energy comparison at the TPSSh level (10% HF exchange), shown in Fig. 2. We have been able to conrm some of this behaviour by performing single-point energy calculations using these functionals with the TPSSh-optimized geometries. As shown in Table 1, the energy comparison of models is dramatically altered when these hybrid functionals are considered. Meanwhile, results from the non-hybrid functional TPSS are more similar to the TPSSh results. As an example of this strong functional dependency, model c (a protonated carbide model found in the study by Cao et al.), is predicted to be only This journal is © The Royal Society of Chemistry 2019 B3LYP 36.83 26.84 2.46 15.70 34.70 43.19 38.21 33.04 19.60 12.34 13.18 14.52 0.00 1.40 12.96 Model a (147) b (346) c (147) d (147) e (235) f (235) g (147) h (247) i (247) j (247) k (147) l (147) m (346) n (235) o (346) M06-2X 59.26 47.33 0.00 41.68 61.35 114.39 104.64 100.05 58.24 31.41 57.42 55.05 10.29 26.54 44.50 2.5 kcal mol1 higher in energy than the lowest energy model (m) at the B3LYP level of theory but 17 kcal mol1 higher in energy than the lowest model (n) at the TPSSh level of theory. Structural optimization at the same level of theory would likely further stabilize model c. This eﬀect of using a higher HF exchange hybrid is further magnied when using the M06-2X functional (having 54% HF exchange); this even leads to model c becoming the most stable model as seen in Table 1 (and the energy gap between models becoming 114 kcal mol1). The M06-2X functional has been used to suggest carbide protonation as an important aspect of the mechanism.29 The large energy changes seen for diﬀerent functionals echo the results of Cao et al.73 We also note the pronounced sensitivity w.r.t. which BS-solution is calculated (see Tables S1–S2†), underlining the importance of studying multiple BS states in calculations of FeMoco. Such a large functional dependency of the relative energies (even when the same TPSSh geometries are used) naturally calls into question the reliability of the DFT calculations to distinguish energetically between E4 models of these complex systems. In fact, these results strongly suggest that the diﬀerent functionals are describing the electronic structure of the FeMoco E4 state very diﬀerently. Such a diﬀerent description of electronic structure with diﬀerent functionals for FeMoco in the E4 state should be apparent in the E0 state as well (despite an absence of hydrides and protonated suldes/carbide). In fact, when diﬀerent functionals are used to describe the electronic structure of FeMoco in the E0 state, there are notable diﬀerences in the spin populations and isosurface plots of the spin density (see ESI, Fig. S13 and S14, Tables S9 and S11†). These diﬀerences can partly be attributed to the diﬀering delocalization of electrons in FeMoco as well as the strength of Mo–Fe interactions (as discussed in previous studies the Mo electrons are partially delocalized towards the Fe ions, suggesting some Mo–Fe bonding7,8,11,13). A functional such as B3LYP for example shows a slightly more localized description of unpaired electrons in FeMoco and this eﬀect is magnied Chem. Sci., 2019, 10, 11110–11124 \| 11115 View Article Online Open Access Article. Published on 15 October 2019. Downloaded on 5/21/2024 11:05:43 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Chemical Science when going to functionals with more HF exchange like BHLYP or M06-2X. In fact, when QM/MM geometry optimizations of the E0 state are performed with hybrid functionals with HF exchange $20% (such as B3LYP, BHLYP and M06-2X), large structural changes occur. This can be seen for Mo–Fe, Fe–Fe and Fe–C distances in Fig. 3 which shows the mean deviation of specic atom–atom distances w.r.t. crystal structure (more data available in the ESI, Fig. S10–S12†). Especially noteworthy is the up to 0.8 Å deviation seen for the Mo–Fe1 distance (see ESI, Fig. S10†) that suggests that the basic structure of the cofactor is very badly described at some of these same hybrid levels. As discussed further in the ESI,† the largest structural deviations involve Mo–Fe distances, and for BHLYP and M06-2X, this can be attributed to a completely diﬀerent electronic structure on the molybdenum, giving rise to a high-spin Mo(IV) instead of the non-Hund Mo(III) conguration. The oxidation state of Mo as Mo(III) in FeMoco is now rmly established from Mo K-edge and L-edge XAS spectroscopy and theoretical calculations.8,9 Additionally, the unusual spin-coupled non-Hund Mo(III) conguration, rst proposed by theoretical calculations8 has now experimental support via recent Mo L-edge and XMCD spectra.10 Thus, computational protocols giving rise to long Mo–Fe5/Fe6/ Fe7 distances (>3 Å) by stabilizing a high-spin Mo(IV) ion are not only incompatible with the experimental molecular structure data (high resolution crystallography) but experimental electronic structure data (XAS and XMCD) as well. The QM/MM B3LYP results in Fig. 3 and the ESI† show considerable deviations with respect to the crystal structure (though not as large as the BHLYP and M06-2X results); however, this eﬀect is further magnied if the QM/MM model is replaced by a simpler cluster model instead. The B3LYP cluster model data shown in the gures are from Siegbahn74 and give deviations as bad as the Edge Article BHLYP and M06-2X QM/MM data. As shown in the ESI† there are also dramatic changes in the electronic structure of the B3LYP cluster model, especially regarding the oxidation state of Mo, again indicating a Mo(IV) oxidation state. It is also worth noting that overestimated Fe–C bond lengths (suggesting destabilized Fe–C chemical bonds) are seen from functionals/ protocols that show more favourable carbide protonation according to Table 1 and these functionals/protocols have been used in studies that suggest carbide protonation occurring in reduced states of FeMoco. The non-hybrid functionals, BP86 and TPSS, give structures in better agreement with the experimental crystal structure (though showing underestimation of distances instead of overestimation). TPSS shows relative energies of E4 models more similar to TPSSh than the hybrid functionals, at least regarding the stability of carbideprotonation models. FeMoco is thus an interesting example of where there is quite a strong relationship between electronic structure (and hence reaction energies) and molecular structure. The good agreement seen between our TPSSh-QM/MM E0 structure and the high resolution crystal structure of E0 strongly suggests that we are describing the electronic structure of the system more correctly with TPSSh, clearly much better than the higher exchange hybrids and also better than non-hybrid functionals such as BP86 and TPSS. We note that our calculations include dispersion corrections, a large polarized triplezeta basis set (on the cofactor) that should be close to the basis set limit, as well as scalar relativistic eﬀects (via ZORA) and we account for the explicit protein environment from the beginning; the good agreement seen with our TPSSh calculations should thus primarily be due to a more correct description of the electronic structure rather than being due to accidental Mean deviations (Å) of Fe–Fe, Mo–Fe, Fe–C, Fe–S and Mo–S distances of resting state FeMoco (relative to crystal structure), calculated with various functionals using the same QM/MM protocol. Also shown is a B3LYP cluster model from Siegbahn.74 Fig. 3 11116 \| Chem. Sci., 2019, 10, 11110–11124 This journal is © The Royal Society of Chemistry 2019 View Article Online Open Access Article. Published on 15 October 2019. Downloaded on 5/21/2024 11:05:43 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Edge Article error cancellations of both model and method errors. Whether this ability to describe the resting state FeMoco well, carries over to describing the relative stability of hydrides and protonation of a metal-carbide is not as clear. We note though that the TPSSh functional has been shown to be a reliable functional for many problems in inorganic and bioinorganic chemistry giving both good structures75 and reaction energies.76 It appears most likely to us that the higher HF exchange in hybrids such as B3LYP, BHLYP and M06-2X is responsible for introducing severe artifacts in the basic electronic structure of FeMoco, that would carry over to the description of all redox states. Results from these functionals, such as a tendency to favour protonated carbides (not found with functionals that describe the E0 structure well), should therefore be regarded with high suspicion, in our view, as 20% or higher HF exchange appears to destabilize metal–ligand and metal–metal bonding in FeMoco (resulting even in a wrong Mo oxidation state) that is arguably crucial to its reactivity. In our view, the relative energy comparison at the TPSSh level is at this point the most reliable as only this level of theory describes the electronic structure of the cofactor accurately in the resting state. This comparison indicates E4 models j–o as the most energetically favourable E4 models. We note that the stability of such open sulde-bridge models is for the most part also found at the non-hybrid level of theory (TPSS) while the higher HF exchange hybrids predict a very diﬀerent energy landscape. Geometry optimizations with these hybrid functionals (B3LYP, M06-2X) would no doubt further change the energy landscape of E4 models (likely further stabilizing protonated carbide models), however, this would not lead to any useful comparison, in view of the aforementioned electronic structure artifacts. Thus, models j–o appear to be viable choices for the E4 state of FeMoco. We hesitate to distinguish further between these models based on their energetics. There is ample uncertainty regarding the reliability of relative energies with our DFT level of theory but it is also worth noting that the E4 state is thermodynamically not stable with respect to H2 formation (that leads back to the E2 state) according to experiments. The E4 state must then be a state that is kinetically trapped behind a barrier, and not necessarily at the lowest energy conguration (prior to H2 dissociation). The E4-n model is the lowest in energy according to our energetic comparison but this is a state where H2 has already formed, bound to Fe2 with a Kubas-type metaldihydrogen geometry. Our calculations indicate an almost barrierless H2 dissociation from this state (a path leading to the E2 redox level) and this H2 formation (involving hydrides) would thus occur without N2 involvement which is inconsistent with isotope-substitution experiments. Those experiments showed that N2 is necessary for H2 formation via the hydrides (criterion iii from Section A.). Additionally, the E4-n model does not feature bridging hydrides, at odds with the structural interpretation of the ENDOR studies (criterion i from Section A.). The E4-n state, while energetically favourable, may thus be a state never formed under experimental conditions, it most likely represents an alternative path towards E4/E2 relaxation, but likely one that would not be seen experimentally. This journal is © The Royal Society of Chemistry 2019 Chemical Science If we focus on models that seem broadly consistent with the ENDOR proposal of two almost chemically equivalent bridging hydrides then models E4-l and E4-o (see Fig. 4) appear to be the most appealing models as these models feature two almost equivalent hydrides with considerable bridging character. These two models are also among the most thermodynamically stable models according to our TPSSh protocol and there is very likely a kinetic barrier towards H2 formation involving these hydrides that could be aﬀected by N2 binding, making a reductive elimination step possible. On the other hand, model E4-e has been previously suggested by Hoﬀman and coworkers to be a structure that is consistent with the ENDOR data. In a very recent study,25 a case was made for this model being in the best agreement with even higher resolution ENDOR data while a structure similar to model E4-o was considered less likely. This comparison was based on calculated hyperne tensor orientations with a simplied point-dipole approximation where the metal ions are assumed to all be high-spin and to behave as localized “spherical balls of spin”. The latter seems questionable, in our view, considering the strong delocalization of electrons seen in calculations of FeMoco and we also note that calculated spin populations of almost all E4 models (including E4-l, E4-o and E4e, see Tables S2–S5†) show reduced spin populations on some Fe ions, calling into question the high-spin nature of all Fe ions in the E4 models. A direct calculation of the 1H hyperne tensors of the hydrides of these models via multireference wavefunction theory may in fact be required to condently tell apart models based on the hyperne tensors. Such calculations are unfortunately still out of reach but may become possible in the near future. Our QM/MM calculations of E4-e do not suggest the state as a thermodynamically favourable model for the E4 state compared to open-sulde bridge models (with no functional tested). Further studies will be required to determine whether such a model could be favourable under some conditions or even whether such a model represents a kinetically trapped state. The energetically favourable E4-l and E4-o models (at TPSSh level of theory), featuring bridging hydrides (in agreement with ENDOR analysis), will be studied further in the next section. The energy surface and barriers that connects all of the states j–o will be reported on later; at present it is not clear whether all of these states are accessible to each other and this requires careful mapping of the minimum energy paths between them. D. Molecular and electronic structure of E4-l and E4-o The two models E4-l and E4-o both feature the same bridging hydride structure with the hydrides between Fe2 and Fe6 but the models diﬀer in the position of the terminal sulydryl group (Fe2 vs. Fe6) derived from sulde S2B (Fig. 4). Sulde S5A is also protonated in these models but remains bridging. The two hydrides in these models are mostly but not completely bridging between Fe2 and Fe6. For model E4-l, both hydrides are slightly more associated with Fe6 (Fe6–H distances of 1.61 and 1.63 Å) than Fe2 (Fe2–H distances of 1.73 and 1.67 Å) and for model E4-o, we see similarly stronger binding to Fe6 as well, Chem. Sci., 2019, 10, 11110–11124 \| 11117 View Article Online Chemical Science Edge Article Open Access Article. Published on 15 October 2019. Downloaded on 5/21/2024 11:05:43 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 4 Close-up view of the hydride structures in E4-l (left) and E4-o (right) with Fe–H bond lengths (Å) indicated. with Fe6–H distances of 1.61 and 1.59 Å and Fe2–H distances of 1.71 and 1.74 Å. The coordination of these two new ligands to Fe2 and Fe6 change the geometry of these Fe ions from approximate tetrahedral to ve- and six-coordinate geometries (distorted octahedral geometries), which naturally has a pronounced eﬀect on the electronic structure of both Fe ions. In fact, the electronic structure of the two models reveal a change in the local spin state of both Fe ions in the lowest energy solution found for both, BS-147. With hydrides known to act as strong-eld ligands and the change in four-coordinate local tetrahedral geometry to ve or six-coordinate geometry this is perhaps not surprising. The change in local spin state is revealed in the change of spin population at Fe2 and Fe6 (from >3 to 2.4–2.5, see Table S6† for spin populations) and the localized orbitals reveal more clearly spin-pairing occurring at Fe2 and Fe6. The electronic structure appears consistent with intermediate spin S ¼ 3/2 Fe(III) ions but this will require further study. In this context it is worth noting that such a doublehydride bridging geometry between two Fe ions has precedent in the form of synthetic dimeric Fe compounds from the groups of Peters77 and Holland78 though these synthetic compounds have a planar Fe–H–Fe–H geometry. A bridging hydride structure, with two hydrides between Fe2 and Fe6, has also been discussed by Einsle and coworkers as a possible E4 structure.33,79 However, that structure assumed an absent sulde, in line with the hypothesis that the sulde S2B leaves to open up a binding site, as crystal structures have shown that the sulde can be displaced by CO or NH/OH.12,33 Importantly, in our E4 models, the S2B sulde is protonated and remains present as a terminal sulydryl group on either Fe2 or Fe6. Dance has recently explored the thermodynamic feasibility of completely dissociating S2B in the form of SH or H2S and found it not to be favourable80 and our own preliminary results suggest this as well. It is presently not clear under what conditions (or when in the cycle) complete sulde removal from the cofactor (as shown by the crystal structures) occurs. Based on the similarity of models E4-l and E4-o in terms of structure and energy, we cannot easily distinguish between them. Both appear reasonable candidates for the E4 state; only one of them may be formed under turnover conditions, however, and this may depend on the precise way that protons and electrons get introduced into the active site. Unfortunately, these mechanisms are not well established. E. Dinitrogen binding to E4-l and E4-o With the change in coordination by introduction of bridging hydrides, one of the Fe ions in each state is coordinatively unsaturated with respect to a six-coordinate octahedral 11118 \| Chem. Sci., 2019, 10, 11110–11124 geometry. This makes N2 binding to Fe6 (in E4-l) or Fe2 (in E4-o) a feasible scenario. In fact the synthetic Fe(II)–(mH)2–Fe(II) dimer by Peters and coworkers featured a bound N2 ligand on one of the Fe ions and 1-electron reduction to give a Fe(II)–Fe(I) dimer resulted in a large increase in the binding aﬃnity of a second N2.77 There is thus precedent for a similar bridging-hydride dimer structure to be capable of favorable N2 binding. We explored N2 binding to the empty coordination site at Fe6/Fe2 for both E4-l and E4-o models to give N2-bound models E4-l-N2 and E4-o-N2 (shown in Fig. 5). N2 binding is found to be thermodynamically favourable (w.r.t. free N2) for both E4-l (bound to Fe6) and E4-o (bound to Fe2) states, with an electronic binding energy of DE ¼ 13.5 kcal mol1 for the E4-l-N2 state and a slightly weaker binding of DE ¼ 10.2 kcal mol1 is found for the E4-o-N2 state. We note that accounting for translational entropy (10.7 kcal mol1 based on gas phase statistical mechanics) would reduce these electronic binding energies by that amount, leading to slightly endothermic 0.5 kcal mol1 binding for E4-o and exothermic 2.8 kcal mol1 for E4-l. Diﬀerent broken-symmetry solutions (BS-235, BS-346, BS-247 and BS-147) were tested for the N2-bound states E4-l-N2 and E4-o-N2 that turned out to be crucial, as the energies of diﬀerent states diﬀered by up to 14 kcal mol1 (see ESI, Table S6†), with some BS states not showing favorable N2 binding. For E4-l-N2 and E4-o-N2 models, the BS-235 and BS-346 solutions were lowest in energy, respectively. We note that these results are in sharp contrast with a recent study by Raugei et al.24 that found N2 binding (in a bridging geometry between Fe2 and Fe6) to be endothermic by 5 kcal mol1 (DE). N2 binds end-on to Fe6/Fe2 in these E4-l-N2 and E4-o-N2 structures resulting in an unusual distorted octahedral geometry at the participating Fe. This favorable binding of N2 to a FeMoco redox state is notable; N2 will unsurprisingly not bind to FeMoco at the E0 redox level in our calculations (no minimum found) but the same applies to a 1-electron reduced state. Our model for the E1 redox state (one of two favoured models from a recent joint EXAFS-QM/MM study15) involves an added electron to the [MoFe3S3C] sub-cubane and a protonated S2B sulde bridge (multiple BS states with MS ¼ 2 were calculated). Even when the E1-N2 optimization is started from a structure with a terminal sulydryl group bound to Fe2 and N2 in close proximity to Fe6, then N2 spontaneously dissociates. Thus neither 1-electron reduction of the cofactor or protonation of the sulde bridge alone is suﬃcient for favorable N2 binding to occur at the cofactor. N2 will also not bind favorably to other Fe ions in the E4-l model; while a stable minimum is found when N2 is placed at Fe7, the binding is endothermic (5.5 kcal mol1). This journal is © The Royal Society of Chemistry 2019 View Article Online Open Access Article. Published on 15 October 2019. Downloaded on 5/21/2024 11:05:43 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Edge Article Chemical Science Fig. 5 N2 binding of E4-l and E4-o models compared to models of earlier redox states according to QM/MM calculations. The E1 model features a 1-electron reduced FeMoco (MS ¼ 2) with S2B protonated while the E2 model (BS-235, MS ¼ 3/2) is analogous to the E4-l model with a bridging hydride between Fe2 and Fe6 and a terminal sulfhydryl group on Fe2. N2 binding energies (kcal mol1) are relative to free N2 and are electronic energies. Accounting for translational entropy, (10.7 kcal mol1, based on gas phase statistical mechanics), would decrease the binding energies by that amount. These results suggest that the hydride ligands at Fe2 and Fe6 in E4-l and E4-o are responsible for the favorable formation of the N2-binding states, E4-l-N2 and E4-o-N2, likely due to the unique ligand-eld now found at these Fe ions. In support of this, we have also calculated a model for the E2 state of FeMoco, analogous to E4-l (with a sulydryl group at Fe2) but with only one bridging hydride between Fe2 and Fe6. This state (BS-235 and MS ¼ 3/2) interestingly features favorable N2 binding (6.9 kcal mol1), about half of the binding energy to the E4-l state. The calculated N2 binding of this E2 model may, however, not be enough to overcome entropy and implies that the presence of two hydrides at the same Fe may be required for favorable dinitrogen binding. Electronic structure aspects of dinitrogen binding of FeMoco will be explored in more detail in a future study. Our current interpretation of the electronic structure behind the favorable Fe6–N2 binding in the E4-l/E4-l-N2 step is that the low-spin conguration found at Fe6 in E4-l-N2 (spin population of 0.46) is a vital aspect of the N2 binding. The localized orbital analysis of Fe6 in E4-l-N2 reveals that this iron can be interpreted as a low-spin octahedral S ¼ 1/2 Fe(III) ion with doubly occupied dxz and dyz orbitals. This low-spin conguration must arise via This journal is © The Royal Society of Chemistry 2019 the strong-eld hydride ligands as well as due to the N2 ligand. The double occupation of the dxz and dyz orbitals should allow increased backbonding to the N2 p orbitals and the localized orbitals reveal some N2 character in these orbitals (see Fig. S8†). Taken together, the results in this section imply that a change from a high-spin to a low-spin Fe conguration may be vital to N2 being able to bind to FeMoco in the rst place. The same argument holds for the E4-o-N2 model. While the N2 ligand binds more strongly to Fe6 in our E4-l-N2 structure than to Fe2 in the E4-o-N2 structure, the diﬀerence is not large enough to condently tell the two scenarios apart. The N2 ligand has a slightly elongated N–N bond length of 1.109 and 1.112 Å (for E4-l-N2 and E4-o-N2 respectively), a shi of 0.014/ 0.017 Å compared to free N2 at the same level of theory. The shi in N–N vibrational frequency (compared to free N2 at our level of theory) of 172 cm1 (E4-l-N2) and 202 cm1 (E4-o-N2) also indicates weak N2 activation for both states. F. Reductive elimination As is now well established from experiments, H2 is eliminated from the E4 state as N2 binds, via a reductive elimination mechanism involving the hydrides.4,19–21 Due to the proximity of Chem. Sci., 2019, 10, 11110–11124 \| 11119 View Article Online Open Access Article. Published on 15 October 2019. Downloaded on 5/21/2024 11:05:43 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Chemical Science the two hydrides in the E4 and E4-N2 models discussed in the previous chapters, one can imagine how such a reductive elimination step could proceed from our models. Isotope substitution experiments indicate that H2 evolution via reductive elimination can only proceed in the presence of N2. Experiments performed in the absence of N2, indicate that a regular hydride-proton reaction is responsible for the H2 formed from the E4 state (leading to the unproductive E4/E2 side-reaction). This implies that as N2 binds to the E4 state, either the H2 formation via reductive elimination is favoured thermodynamically over a hydride-proton reaction or it is kinetically favoured by lowering of the barrier for reductive elimination (relative to a hydride-proton reaction). A future study will explore in detail the reaction barriers for H2 formation via both reductive elimination and hydrideproton mechanisms from our E4 models. However, there is a simple argument in favour of reductive elimination over a more normal hydride-proton reaction that applies to both E4-lN2 and E4-o-N2 models. The simplest hydride-proton reaction would involve combining one of the bridging hydrides with the proton from a sulydryl group (the closest accessible proton). The required deprotonation of the terminal sulydryl group (to act as proton donor) would be highly unfavoured as the terminal deprotonated sulde is prevented from recombining with Fe6/Fe2 to reform the sulde-bridge due to the presence of the other hydride in this position (in fact, calculations indicate such a step to be uphill by 15.1 kcal mol1). The dinitrogen ligand would furthermore block a hydride-proton pathway involving the hydroxy proton from the Mo-bound homocitrate and possibly other proton donors. The molecular structures of the E4-l-N2 and E4-o-N2 models thus appear to oﬀer an intuitive explanation for why the reductive elimination step can only occur in the presence of N2. Without the N2 ligand (i.e. in the absence of N2 gas), a hydride-protonation reaction (i.e. a E4/E2 step) should be more likely to occur, probably due to a lower barrier of a hydride-proton reaction compared to a hydride– hydride reaction. This could occur e.g. by hydride protonation via the hydroxy group on Mo-bound homocitrate or via the sulydryl group (and a possible transformation of the remaining bridging hydride to a terminal hydride and reformation of the sulde bridge). With the N2 ligand present, however, reductive elimination could be the only favoured H2 evolution pathway. This reductive elimination step involving two hydrides (formally storing four electrons) releases two electrons in the form of H2 and makes two electrons now available to the 0 0 cofactor in states we call E4 -l-N2 and E4 -o-N2 (see Fig. 6). Our calculations predict an electronic energy change (DE) of +3 kcal mol1 uphill. While this step is predicted to be mildly endothermic according our calculations, this result is not in strong disagreement with experiment. In fact, kinetic studies show this step to be reversible,81 meaning the step is close to thermoneutral. Additionally, the translational entropy contribution to the free energy would favour the elimination of H2. We have opted for not adding gas phase translational entropy corrections (8.4 kcal mol1 for H2 elimination) to our energies as the entropic contribution could be more complicated for 11120 \| Chem. Sci., 2019, 10, 11110–11124 Edge Article a complex condensed phase macromolecular system such as nitrogenase (as has been discussed by Reiher and coworkers82), however, the entropy contribution would likely be the same magnitude as the gas phase value. The fact that the 0 E4 -N2 /E4 -N2 step is predicted to be close to thermoneutral (DE) according to our calculations is due to the nature of the reductive elimination. A regular H2 formation step should be quite exothermic (our calculations for hydride-proton reactions for an E4 / E2 predict exothermicity of 20 kcal mol1). The 0 lack of strong exothermicity for E4 -N2 /E4 -N2 step stems from the fact that the metal cofactor is reduced (by 2 electrons) during the reaction, as previously the four electrons were stored in the form of the hydrides and two leave in the form of H2. This rather unfavorable two-electron reduction of the metal cofactor may thus be oﬀset via the exothermicity of the H2 formation. 0 The molecular and electronic structure of the E4 -l-N2 and the 0 E4 -o-N2 states is thus of interest as the metal ions of the cofactor are now more reduced than before and the structures lack the hydrides that previously helped bind N2. The local Fe geome0 0 tries of the Fe2/Fe6 ions in E4 -l-N2 and E4 -o-N2 are unusual. A terminal sulydryl group is still present on Fe2 or Fe6. Attempts to reform the sulde bridge (with sulde still protonated) were not successful as the system returns to a geometry with the terminal sulydryl group. Clearly, the distorted geometries at Fe2/Fe6 are stable, though this is presently not well understood. Using the s4 and s04 structural metrics83,84 for 4-coordinate 0 compounds, we nd that the Fe2 and Fe6 in both E4 -l-N2 and 0 E4 -o-N2 models are about halfway between a tetrahedral geometry and a seesaw geometry (see ESI, Tables S7 and S8,† for calculated s4 and s04 parameters for all Fe ions in multiple E4 models). We note that such geometries have previously been found in DFT calculations of FeMoco.85,86 Analysis of the electronic structure suggests that the dis0 torted N2-bound Fe2 ion in E4 -o-N2 can be described as an S ¼ 1 0 Fe(II) ion. However, remarkably, the distorted Fe6 ion in E4 -l-N2 best ts the description of an S ¼ 3/2 Fe(I) ion according to the localized orbitals (see ESI, Fig. S9†), revealing a stark diﬀerence between the two states and the nature of the diﬀerent binding 0 0 site. Both E4 -l-N2 and E4 -o-N2 states feature doubly occupied dxz and dyz orbitals that likely account for the favorable dinitrogen binding via metal backbonding (similarly to the E4-l-N2 and E4o-N2 models despite the absence of hydrides). While distinguishing between the E4-l and E4-o pathways is not quite clear based on the calculated energies, it is tempting 0 to attribute the presence of Fe(I) in E4 -l-N2 as a potentially important aspect of the mechanism when going forward. The interpretation of the reductive elimination is then that the electrons in the reductive elimination step are used to reduce the N2-binding Fe6 ion to an Fe(I) ion. Compared to the resting state E0, the mixed-valent delocalized Fe6(2.5)–Fe7(2.5) pair has then been reduced to a pair of localized Fe6(I) and Fe7(II) ions. Thus, the reductive elimination step could be imagined as a specic mechanism towards stabilizing an Fe(I)–N2 species without going to the strongly negative potentials such as those required for mononuclear complexes. Indeed, low-valent mononuclear iron complexes from Jonas Peters and coworkers have been found to act as catalysts for N2 reduction; This journal is © The Royal Society of Chemistry 2019 View Article Online Open Access Article. Published on 15 October 2019. Downloaded on 5/21/2024 11:05:43 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Edge Article Chemical Science Fig. 6 N2 binding, reductive elimination and N2 protonation reactions for the E4-l and E4-o structures. State energies are relative to the E4-o 0 0 model. A reductive elimination of H2 via the bridging hydrides releases 2 electrons to give a doubly-reduced cofactor in states E4 -l-N2 and E4 -o-N2 . A possible subsequent protonation step of N2 via the sulfhydryl group and hydroxy group of homocitrate, reforms the sulﬁde bridge between Fe2 and Fe6 to form diazene-bound intermediates E4-l-N2H2 or E4-o-N2H2 at either Fe2 or Fe6. this is accomplished via the use of strong reductants (KC8) to access low (Fe(I), Fe(0) and Fe(I)) oxidation states that bind and activate N2.87–89 Reductive elimination could thus enable reduction of an Fe ion to Fe(I) while using the same low potential of the Fe protein. 0 0 The N2 ligand in E4 -l-N2 and E4 -o-N2 is only weakly activated as seen in a small increase in N–N bond length of 0.021 and 0 0 0.023 Å for E4 -l-N2 and E4 -o-N2 respectively, when compared to free N2 and by the decrease in N–N vibrational frequency of 242 0 0 and 255 cm1 for E4 -l-N2 and E4 -o-N2 respectively, when compared to free N2. The N2 activation is greater though than in the E4-l-N2 and E4-o-N2 models (N–N bond length shis of 0.014 and 0.017 Å and frequency shis of 172 cm1 and 202 cm1). A high-spin Fe(I)–N2 complex was rst synthesized by Harman 0 and coworkers.90 Unlike the E4 -N2 states proposed here, the synthetic Fe(I)–N2 complex has C3v symmetry and the dinitrogen activation is stronger with a N–N stretching frequency shi of 372 cm1 compared to free N2. 0 While activation of the N2 ligand in the in E4 -l-N2 and 0 E4 -o-N2 states is thus still considered weak, and protonation might be considered unlikely, we tested simple protonation reactions by transferring nearby protons to the nitrogen atoms of the ligand. Two possible protonation agents seem likely, one being the terminal sulydryl group on Fe2/Fe6 and the hydroxy group of homocitrate another. The hydroxy proton on homocitrate was found to be present in the resting state FeMoco according to a previous QM/MM study by us13 and by Cao et al.91 In this context it is of note that homocitrate is experimentally known to be important for dinitrogen reduction.92 Transferring a proton from the hydroxy group to either the distal or proximal 0 0 nitrogen of either E4 -l-N2 or E4 -o-N2 resulted in high energy intermediates of 34.3–36.3 kcal mol1. If a double protontransfer step is calculated, however, using both the sulydryl group and the hydroxy group, we get lower-energy diazene intermediates E4-l-N2H2 and E4-o-N2H2 instead. While the E4-lN2H2 diazene intermediate is predicted to be higher in energy 0 than the E4 -l-N2 state by 5.6 kcal mol1, it is interestingly much more favorable than the E4-o-N2H2 intermediate (uphill by This journal is © The Royal Society of Chemistry 2019 20.1 kcal mol1), demonstrating very diﬀerent reactivity of the 0 Fe sites in the two models. Furthermore, the E4 -l-N2 /E4 -l-N2 H2 pathway, being in close proximity to the hydroxy group appears more plausible for direct proton transfer. These results indicate that protonation of the N2 ligand may not occur until later in the cycle (e.g. in the E5 redox state) or possible via another protonation mechanism not considered here. Experimentally, via a quench-cryoannealing relaxation protocol and varying H2/N2 concentrations, a reaction intermediate was shown to accumulate following reductive elimination, at the same redox level as E4.81 While this intermediate is denoted as E4(2N2H) by Hoﬀman and coworkers and discussed as “a state in which FeMo-co binds the components of diazene, which may be present as N2 and two [e/H+] or as diazene itself”81 EPR and ENDOR studies of this same intermediate93,94 showed 15N hyperne coupling (using 15N2) but no hyperne coupling from hydrogens was seen. A single 15N hyperne signal was found, suggesting end-on binding which ts well 0 with our E4 -N2 models. A recent EPR/ENDOR study of synthetic mononuclear Fe–N2 compounds by Peters and coworkers of states featuring either a single and double protonated N2 ligand: Fe–N2H and Fe–N2H2 (distal protonation) revealed a clear hyperne signal from the hydrogens.95 These experiments may thus be an indication that the experimental dinitrogen-bound intermediate, “E4(2N2H)” is still unprotonated. Based on our computational modelling, it is possible that the experimental intermediate “E4(2N2H)” could corre0 0 spond to the E4 -l-N2 model (or possibly E4 -o-N2 ) rather than a diazene-bound intermediate like E4-l-N2H2/E4-o-N2H2. An alternative explanation is that the hyperne couplings from the hydrogens in a diazene intermediate may be too weak to be measured. Additional spectroscopy is required to clarify the nature of this intermediate. Conclusions We have presented QM/MM calculations of possible models for the E4 state of nitrogenase and how dinitrogen can bind to some Chem. Sci., 2019, 10, 11110–11124 \| 11121 View Article Online Open Access Article. Published on 15 October 2019. Downloaded on 5/21/2024 11:05:43 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Chemical Science of these models. In view of the thermodynamic stability of our model for the E4 state and the favourable dinitrogen binding we have presented, our work looks promising as a step towards a mechanistic understanding of biological dinitrogen reduction. Questions remain about the reliability of calculated reaction energies for this complicated cofactor and a stronger connection to experiment would be much desired, requiring more spectroscopy on nitrogenase intermediates. In this context, we should note that a recent ENDOR study25 combined with calculations has presented evidence in favour of another model (model e in article) as the structure of the E4 state. Our QM/MM calculations of that model suggest it to be higher in energy than all open-sulde bridge models (with all calculated functionals) and this model has not been found to bind dinitrogen favorably.24 Additional spectroscopic data on the E4 state to sort out this disagreement would therefore be desirable. Our proposed mechanism for binding of dinitrogen and reductive elimination suggests a role for many of the components of the complicated cofactor of nitrogenase. The size of the cofactor and the nature of the fused iron–sulfur double-cubane may play a primary role of favourably accepting electrons and storing them as hydrides at the same potential as provided by the Fe protein; the stability of the hydride geometries being facilitated by a labile sulde bridge between cubanes. Furthermore, our calculations suggest the strong-eld nature of the hydrides to aid the binding of dinitrogen by a local high-spin/ low-spin electronic structure change at Fe6 or Fe2. The reductive elimination step then maintains the low-spin structure while doubly reducing the cofactor and partially activating the N2 ligand. The carbide may play a role in tuning the redox potential for the reduction steps but it may also play a role in either binding or activation of N2. The carbide being approximately 0 trans to the N2 ligand in our E4 -N2 =E4 -N2 structures, suggests a link to the model chemistry of Peters and coworkers87,88 where carbide and boride-containing mononuclear trigonal bipyramidal Fe complexes were found to be active catalysts for dinitrogen reduction. Similar to the model compounds, the carbide in FeMoco may aid in pushing electron density into the paccepting orbitals of the N2 ligand. The molybdenum ion likely also has an eﬀect on the redox potential of the cofactor (the redox potential can in fact be tuned by heterometal substitution as known by synthetic [XFe3S4] chemistry where X ¼ Mo, V, W96,97). We speculate that the role of the molybdenum may also 0 be vital in stabilizing the N2-bound Fe(I) ion in E4 -l-N2 , formed aer the reductive elimination step, making the electrons available for partial activation of the N2 ligand. The homocitrate ligand likely plays a role in protonation steps and the proton on the Mo-bound alcohol group of homocitrate is in an ideal position to protonate the N2 ligand when bound to Fe6 in the 0 E4 -l-N2 state; it may also help deliver protons in the early redox states (becoming protonated suldes or hydrides). Finally, we emphasize that this work presents only a preliminary mechanism for dinitrogen binding to FeMoco that relies primarily on calculated energies with density functional theory approximations (where a large functional dependency is seen) and where only a small part of chemical space has been 11122 \| Chem. Sci., 2019, 10, 11110–11124 Edge Article explored. More experimental data on the E4 and E4-N2 states are urgently needed to further constrain the mechanistic possibilities of FeMoco as well as to help benchmark the theoretical methodology employed. The precise way in which the N2 ligand is activated for protonation is also not clear from our results. At the very least this computational study has presented falsiable ideas about the mechanism of biological nitrogen reduction that can be conrmed or ruled out by suitable experiments. Conﬂicts of interest There are no conicts to declare. Acknowledgements RB acknowledges support from the Icelandic Research Fund, Grants No. 141218051 and 162880051 and the University of Iceland Research Fund. The Max Planck society is acknowledged for funding. Serena DeBeer is thanked for support. Some of the computations were performed on resources provided by the Icelandic High Performance Computing Centre at the University of Iceland. References 1 B. K. Burgess, Chem. Rev., 1990, 90, 1377–1406. 2 L. C. Seefeldt, B. M. Hoﬀman and D. R. Dean, Annu. Rev. Biochem., 2009, 78, 701–722. 3 R. N. F. Thorneley and D. J. Lowe, in Molybdenum Enzymes, ed. T.G. Spiro, Wiley, New York, 1985, pp. 221–284. 4 B. M. Hoﬀman, D. Lukoyanov, Z. Yang, D. R. Dean and L. C. Seefeldt, Chem. Rev., 2014, 114(8), 4041–4062. 5 K. M. Lancaster, M. Roemelt, P. Ettenhuber, Y. Hu, M. W. Ribbe, F. Neese, U. Bergmann and S. DeBeer, Science, 2011, 334, 974–977. 6 T. Spatzal, M. Aksoyoglu, L. Zhang, S. L. A. Andrade, E. Schleicher, S. Weber, D. C. Rees and O. 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https://openalex.org/W3028558183	https://www.nature.com/articles/s41598-020-65507-x.pdf	English	null	Author Correction: WDR1 is a novel EYA3 substrate and its dephosphorylation induces modifications of the cellular actin cytoskeleton	Scientific reports	2,020	cc-by	593	www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Author Correction: WDR1 is a novel EYA3 substrate and its dephosphorylation induces modifications of the cellular actin cytoskeleton x OPEN Published: xx xx xxxx Mihaela Mentel, Aura E. Ionescu, Ioana Puscalau-Girtu, Martin S. Helm, Rodica A. Badea, Silvio O. Rizzoli & Stefan E. Szedlacsek Correction to: Scientific Reports https://doi.org/10.1038/s41598-018-21155-w, published online 13 February 2018 The Article contains errors. The Article contains errors. The Article contains errors. The image for Lysates/WDR1 in Figure 6B is a duplication of another image from Figure 3A. Additionally, images for Lysates/Src and Lysates/Actin in Figure 6B show incorrect sections of the membrane. The correct Figure 6B appears below as Figure 1. The original gels for Lysates samples appear below as Figure 2. Figure 1. The original gels for Lysates samples appear below as Figure 2. Scientific Reports \| (2020) 10:8638 \| https://doi.org/10.1038/s41598-020-65507-x www.nature.com/scientificreports/ The correction does not affect the conclusions of the Article. 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) 2020 Figure 2. The correction does not affect the conclusions of the Article. 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) 2020 Figure 2. Fi 2 Figure 2. Figure 2. The correction does not affect the conclusions of the Article. 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) 2020 Scientific Reports \| (2020) 10:8638 \| https://doi.org/10.1038/s41598-020-65507-x
https://openalex.org/W1994867863	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0070552&type=printable	English	null	Species-Specific and Cross-Reactive IgG1 Antibody Binding to Viral Capsid Protein 1 (VP1) Antigens of Human Rhinovirus Species A, B and C	PloS one	2,013	cc-by	9,481	Abstract Competing Interests: The authors have declared that no competing interest exist. * E-mail: belinda@ichr.uwa.edu.au Species-Specific and Cross-Reactive IgG1 Antibody Binding to Viral Capsid Protein 1 (VP1) Antigens of Human Rhinovirus Species A, B and C Jua Iwasaki1,2, Wendy-Anne Smith1, Shane R. Stone1, Wayne R. Thomas1, Belinda J. Hales1,2* 1 Telethon Institute for Child Health Research, Centre for Child Health Research, University of Western Australia, Perth, Australia, 2 School of Paediatrics and Child Health, University of Western Australia, Perth, Australia Abstract Background: Human rhinoviruses (HRV) are associated with upper and lower respiratory illnesses, including severe infections causing hospitalization in both children and adults. Although the clinical significance of HRV infections is now well established, no detailed investigation of the immune response against HRV has been performed. The purpose of this study was to assess the IgG1 antibody response to the three known HRV species, HRV-A, -B and -C in healthy subjects. Methods: Recombinant polypeptides of viral capsid protein 1 (VP1) from two genotypes of HRV-A, -B and -C were expressed as glutathione S-transferase (GST) fusion proteins and purified by affinity and then size exclusion chromatography. The presence of secondary structures similar to the natural antigens was verified by circular dichroism analysis. Total and species-specific IgG1 measurements were quantitated by immunoassays and immunoabsorption using sera from 63 healthy adults. Results: Most adult sera reacted with the HRV VP1 antigens, at high titres. As expected, strong cross-reactivity between HRV genotypes of the same species was found. A high degree of cross-reactivity between different HRV species was also evident, particularly between HRV-A and HRV-C. Immunoabsorption studies revealed HRV-C specific titres were markedly and significantly lower than the HRV-A and HRV-B specific titres (P,0.0001). A truncated construct of HRV-C VP1 showed greater specificity in detecting anti-HRV-C antibodies. Conclusions: High titres of IgG1 antibody were bound by the VP1 capsid proteins of HRV-A, -B and -C, but for the majority of people, a large proportion of the antibody to HRV-C was cross-reactive, especially to HRV-A. The improved specificity found for the truncated HRV-C VP1 indicates species-specific and cross-reactive regions could be defined. Editor: Ellen R. Goldman, Naval Research Laboratory, United States of America Received April 23, 2013; Accepted June 23, 2013; Published August 7, 2013 Copyright: 2013 Iwasaki et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by a National Health and Medical Research Council of Australia grant (458513). J. Iwasaki was supported by an Australian Postgraduate Award, a University of Western Australia Scholarship, a Stan and Jean Perron Scholarship and an Asthma Foundation of Western Australia Scholarship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Introduction Neutralising antibodies provided the key observations that first distinguished HRV-A from HRV-B but, because they are directed to a limited number of capsid structures that frequently mutate [8], they have limited application for sero-epidemiology and profiling the history of immune responses to repeated infections. The neutralising antibodies induced by experimental HRV inoculation appear 2–3 weeks after infection, well after the clearance of virus and resolution of the symptoms [9,10], and at least for experimental infection can no longer be detected after 9 months [11]. As found for other respiratory virus infections, it is likely that innate immunity and T-cell responses are the most important responses for the resolution of infection, while neutralising antibodies protect against short-term reinfection [12]. Antibodies have been shown to interact with T-cells in adaptive immune responses to clear viruses, but interestingly these were non- neutralising antibodies [13,14] that are also likely to be more abundant. Such interaction between non-neutralising antibodies Human rhinovirus (HRV) infection is not only the main cause of the common cold [1] but is strongly associated with exacerbations of asthma, acute bronchitis, chronic obstructive pulmonary disease in adults, as well as pneumonia in immunocompromised patients [2–4]. The biologically and molecularly distinct HRV-C species, which has been found in hospitalised patients, has attracted attention for a role in asthma exacerbation of children [5] and for serious disease in adults and the elderly, including transplant patients [4]. HRV-C has been difficult to grow in vitro [6], so its detection has relied on the sequencing of viral RNA in tissue samples, the method now most commonly used to detect HRV-A and HRV-B. The prevalence of RNA detection for all species has been higher in hospitalized patients [3] but has also been high in healthy subjects often in similar abundance [7]. August 2013 \| Volume 8 \| Issue 8 \| e70552 1 PLOS ONE \| www.plosone.org August 2013 \| Volume 8 \| Issue 8 \| e70552 Antibody Binding to Human Rhinovirus Antigens Table 1. Amino acid sequence identity for six HRV VP1 and HPV Sabin 1 VP1. HRV-A HRV-B HRV-C HPV 34 1B 14 69 3 5 Sabin HRV-A 34 77.7 35.2 35.5 41.4 44.6 35.2 1B 36.9 37.2 39.4 43.6 33.9 HRV-B 14 71.1 32.9 33.1 40.5 69 33.8 36.2 42.2 HRV-C 3 61.9 28.3 5 32.0 HPV Sabin doi:10.1371/journal.pone.0070552.t001 doi:10.1371/journal.pone.0070552.t001 and HRV-C3 (EF186077 [20]) and HRV-C5 (EF582386 [2]) of HRV-C species (Table S1). Ethics Statement Written informed consent was obtained from all participants. Ethics approval was provided by The Princess Margaret Hospital Human Ethics Committee. Introduction The VP1 of another enterovirus, human poliovirus (HPV) Sabin VP1 (AY184219.1) was produced as a control to determine specificity in antibody binding to HRV. The amino acid sequence identities of the VP1 proteins are shown in Table 1. and T-cells might also be expected to affect immunopathology, for example in aiding the capture of virus by monocytes and thereby enhancing inflammatory cytokine production [15–17]. The objectives of this study were to use antibody-binding to defined HRV antigens to determine whether immune responses to the different HRV species could be distinguished, and further, if the antibody responses to the different species had different characteristics. The antigen examined was the VP1 capsid protein that exhibits the highest surface exposure of the four HRV capsid proteins and forms part of neutralising antigenic determinants [18]. It, along with VP2 and VP3, exhibits the amino acid sequence variation found amongst genotypes of each species. Comparing the VP1 of HRV-A and HRV-B, most isolates form a cluster with 58–99% amino acid sequence identity within each species, contrasting with the 35–44% identity found between HRV-A and HRV-B [19]. Similarly, the VP1 of HRV-C genotypes has 59–90% amino acid sequence identity within HRV-C, and about 45% and 36% identity between HRV-C and HRV-A and HRV-B respectively [2]. Our studies here show high IgG1 antibody binding to recombinant full length VP1 antigen constructs for all species, with high titres of species-specific antibody for HRV-A and HRV-B, in contrast to a low prevalence and titre for HRV-C. A construct of HRV-C made without the N- terminal alpha-helical domain showed a lower degree of antibody binding but, significantly, was restricted to sera from subjects with species-specific antibodies. Study Population For expression of VP1, an overnight culture diluted 1:20 was grown to OD600 nm 0.6 and induced with 0.1 mM IPTG at 30uC for 2 hours. The E.coli pellets were resuspended in 5 ml/g Buffer A (150 mM NaCl, 50 mM NaH2PO4, 1% Tween-20, 1 mM PMSF, pH 8) with the addition of lysozyme (1 mg/ml, Sigma-Aldrich, St Louis, MO), sonicated and clarified at 18,000 rpm for 60 min. The soluble supernatant was then purified in accordance with the manufacturer’s protocols (Sigma, USA) with modifications. Briefly, glutathione agarose was pre-equilibrated with Buffer B (150 mM NaCl, 50 mM NaH2PO4, 0.1% Tween-20, pH 8). The clarified lysate was bound to the matrix and the column was washed with 106column volume with Buffer B. Bound protein was eluted with Buffer C (Buffer B +10 mM reduced glutathione). Fractions collected from the column containing recombinant protein were pooled, concentrated and passed over a high resolution S300 26/ 60 column (GE Healthcare, Uppsala, Sweden). Sera from 63 adults with no evidence of current respiratory illness from the general population in Perth, Western Australia were examined (40 women and 23 men; age ranged from 20–53; median age 31). Expression and Purification of Recombinant HRV VP1 The nucleotide sequences encoding VP1 cDNAs were synthe- sized with codon optimization for expression in E.coli by GenScript (Piscataway, NJ). They were subsequently engineered for expres- sion as fusion proteins with glutathione S-transferase (GST) at the N-terminus and a hexa-histidine tag on the C-terminus. The genes were amplified by PCR from cDNA in pUC57 as a template. Specific PCR primers were designed to amplify the VP1 coding sequence and the addition of six histidine residues. PCR was performed using high-fidelity Pfu DNA polymerase (Promega, Madison, WI) using the following conditions: 1 cycle at 95uC for 5 min; 35 cycles at 95uC for 1 min, 55uC for 30 s, and 74uC for 3 min; and finally 74uC for 7 min. The PCR products were extracted from a 1% agarose gel using the Gel Purification Kit (Qiagen, Hilden, Germany). The amplified DNA fragment was digested with BamHI and EcoRI, and ligated into a BamHI/EcoRI digested pGEX-2T expression vector (GE Healthcare). After transformation into chemically competent TOP 10 E.coli, positive clones were verified by DNA sequencing (AGRF, Australia). The recombinant plasmid was subsequently transformed into the E.coli expression strain BL21. A GST control was produced directly from pGEX-2T. HRV Antigens VP1 antigens from two genotypes of HRV-A, -B and -C were produced in Escherichia coli (E.coli) as they represent genetically disparate variants within each species. The following HRV VP1 proteins were produced: HRV-A34 (GenBank accession number FJ445189.1) and HRV-A1B (D00239.1) of HRV-A species; HRV- B14 (NC001490) and HRV-B69 (FJ445151) of HRV-B species; The purity of the recombinant proteins were analysed by size exclusion chromatography and SDS-PAGE analysis using a 12.5% August 2013 \| Volume 8 \| Issue 8 \| e70552 August 2013 \| Volume 8 \| Issue 8 \| e70552 PLOS ONE \| www.plosone.org 2 Antibody Binding to Human Rhinovirus Antigens Figure 1. Purification and SDS-PAGE analysis of recombinant HRV-C3 VP1 pGEX-2T. (A) Chromatograph of HRS300 size exclusion chromatography immediately following affinity chromatography (Run 1). The void volume and the peak at 660 kDa are indicated above each of the main peaks. Each fraction has a 5 ml elution volume. Fractions containing the 660 kDa peak from Run 1 were pooled and concentrated for a second run and third run on the HRS300. The bar indicates fractions used for subsequent runs and for binding analysis. (B) A 10 mL aliquot of the pooled fractions containing the 660 kDa peak collected from the first run of gel filtration (as indicated by ) was analysed by SDS-PAGE. The MW of the protein standard is indicated. doi:10.1371/journal.pone.0070552.g001 Figure 1. Purification and SDS-PAGE analysis of recombinant HRV-C3 VP1 pGEX-2T. (A) Chromatograph of HRS300 size exclusion chromatography immediately following affinity chromatography (Run 1). The void volume and the peak at 660 kDa are indicated above each of the main peaks. Each fraction has a 5 ml elution volume. Fractions containing the 660 kDa peak from Run 1 were pooled and concentrated for a second run and third run on the HRS300. The bar indicates fractions used for subsequent runs and for binding analysis. (B) A 10 mL aliquot of the pooled fractions containing the 660 kDa peak collected from the first run of gel filtration (as indicated by ) was analysed by SDS-PAGE. The MW of the protein standard is indicated. doi:10 1371/journal pone 0070552 g001 (NH4)2SO4 buffer (pH 8) and circular dichroism (CD) spectrosco- py performed as outlined in Hales et al. [21]. Briefly, CD spectral measurements were performed on an OLIS DSM-1000 spectro- photometer over the wavelength range 260–190 nm operating at 20uC in a 1-mm path length cell (Starna). Circular Dichroism Analysis Purified protein preparations were diluted to a final concentra- tion of 3 mM in 10 mM potassium phosphate, 100 mM Table 2. Percentages of the secondary structure of the circular dichroism (CD) spectra of HRV VP1 antigens following subtraction of GST. a-helix b-sheet b-turns Disordered HRV-A34 VP1 10 30 24 36 HRV-B14 VP1 10 36 25 27 HRV-C3VP1 13 33 25 29 Truncated HRV-C3 VP1 12 33 26 30 Analysed using DiChroWeb Server: CDSSTR algorithm, reference set 4. doi:10.1371/journal.pone.0070552.t002 HRV Antigens The analysis of protein secondary structure from CD spectra was performed using the DiChroWeb Server [22] and the CDSSTR algorithm and reference sets 4, 7 and SP175 (short) [23]. electrophoretic gel and GelCode Blue Safe Protein Stain (Thermo Scientific). Protein concentrations were calculated using OD280 nm and extinction coefficients calculated for each fusion protein. August 2013 \| Volume 8 \| Issue 8 \| e70552 Results Expression of Recombinant GST-fusion HRV VP1 Antigens Recombinant GST-fusion HRV and HPV Sabin VP1 with a C- terminal histidine tag was produced in E.coli as a soluble protein. Following glutathione-agarose affinity chromatography and se- quential gel exclusion chromatography, the final gel filtration isolated a multimeric protein estimated to be 660 kDa (Figure 1A) distinct from the aggregated protein that was excluded from the gel. Under denaturing conditions, the SDS-PAGE revealed a single band of 59 kDa (Figure 1B). IgG1 Competitive Inhibition Studies The plates were read using a Wallac Victor 3 plate reader (Wallac, Oy). blocking buffer (0.5% bovine serum albumin in 50 mM Tris-HCl, 0.9% NaCl, 0.05% sodium azide (pH 7.4) buffer with 0.01% Tween-20) and incubated overnight at 4uC with shaking. The coated plates were washed 5 times with 50 mM Tris-HCl, 0.9% NaCl (pH 7) buffer with 0.01% Tween-20. This washing step was conducted after each incubation step. The plates were blocked with 200 mL of blocking buffer for 2 hours at room temperature with shaking. 100 mL of sera was then added to the antigen-coated wells and incubated for 2 hours at room temperature with shaking. The wells were then incubated with 100 mL of biotinylated anti- human IgG1 (BD Pharmingen, USA) diluted 1:2000 in assay buffer (Wallac, Oy) for 2 hours at room temperature with shaking. This was then followed by incubation for 30 minutes with 100 mL Europium-labelled streptavidin diluted 1:1000 in assay buffer, with shaking. The final washing step involved washing the plates 8 times before the addition of 100 mL enhancement solution. The plates were read using a Wallac Victor 3 plate reader (Wallac, Oy). Antibody Binding to Human Rhinovirus Antigens Antibody Binding to Human Rhinovirus Antigens Figure 2. Total IgG1 antibody binding (ng/ml) to HRV and HPV Sabin VP1 antigens in 63 healthy adults. The geometric mean and 95% confidence interval are indicated. Human myeloma IgG1 was used as a negative control to determine non-specific binding and identification of negative control sera. Three negative control sera for each antigen and a titration of reference sera were included on every plate for the quantitation of IgG1 binding and to assess reproducibility of the assay. doi:10.1371/journal.pone.0070552.g002 determine non-specific binding and the identification of negative control sera (Figure S1). Three negative control sera for each antigen were included on every plate. The absolute quantity of antigen-specific IgG1 (ng/ml) in the reference sera was interpo- lated from a titration curve constructed with equivalent concen- trations of recombinant GST-fusion Der p 2 and a standardised humanised anti-Der p 2 chimeric IgG1 (Indoor Biotechnologies, Charlottesville, USA) [25,26]. Wells coated with the same quantity of antigen were used, as determined by the binding of anti- glutathione-S-transferase antibody. The lower limit of detection for IgG1 binding was 500 ng/ml and thus negative values were assigned 50% of the lower limit of detection (250 ng/ml). Structural Characterization of HRV VP1 Antigens Structural Characterization of HRV VP1 Antigens Purified GST-fusion proteins were subjected to subtractive CD analysis in which the signal from the GST control was subtracted from the fusion proteins. The CD spectrum of the GST dimer showed predominantly a-helical structure analogous to published data [28]. The CD spectra of HRV-A34, HRV-B14 and HRV-C3 VP1 demonstrated the presence of well-folded secondary struc- ture, with broad minima between 208–220 nm indicative of mixed b-sheet and a-helical structure. Minima at 194 nm, close to the expected 198 nm minimum, also suggested random coil confor- mation (Figure S2). Secondary structure calculations of HRV-A34 Statistical Analysis The absolute antibody binding to each VP1 antigen was log- transformed to approximate a normal distribution. Differences in antibody binding to different antigens were compared by the paired t-test. A P value ,0.05 was considered significant. The geometric mean and 95% confidence intervals were calculated. Correlations between antibody responses were analysed by the Spearman correlation. All analyses were performed using GraphPad Prism Software (La Jolla, USA). IgG1 Competitive Inhibition Studies Sera from individuals with high IgG1 binding to two HRV species were diluted 1:50 with blocking buffer and then mixed 1:2 with different dilutions of purified VP1 antigens to give final concentrations ranging from 0.004 to 8 mg/ml of the inhibiting protein. Following overnight incubation at 4uC, the mixtures were centrifuged at 14,000 g for 10 min and 100 mL aliquots of the supernatant were added to the antigen-coated plates for the DELFIAH assays. The assay was then developed as per the IgG1 immunoassay. An irrelevant recombinant antigen, either Fel d 3 or the bacterial protein pneumococcal surface protein (Psp)-C, was used as a negative control to illustrate that any cross-reactivity that existed was specific to HRV species and, where present to HPV Sabin. Recombinant Fel d 3 and Psp-C were both expressed in an E.coli expression system and purified by multiple steps of purification. Details of the antigen preparations have been described previously [24,27]. Figure 2. Total IgG1 antibody binding (ng/ml) to HRV and HPV Sabin VP1 antigens in 63 healthy adults. The geometric mean and 95% confidence interval are indicated. Human myeloma IgG1 was used as a negative control to determine non-specific binding and identification of negative control sera. Three negative control sera for each antigen and a titration of reference sera were included on every plate for the quantitation of IgG1 binding and to assess reproducibility of the assay. doi:10.1371/journal.pone.0070552.g002 doi:10.1371/journal.pone.0070552.g002 blocking buffer (0.5% bovine serum albumin in 50 mM Tris-HCl, 0.9% NaCl, 0.05% sodium azide (pH 7.4) buffer with 0.01% Tween-20) and incubated overnight at 4uC with shaking. The coated plates were washed 5 times with 50 mM Tris-HCl, 0.9% NaCl (pH 7) buffer with 0.01% Tween-20. This washing step was conducted after each incubation step. The plates were blocked with 200 mL of blocking buffer for 2 hours at room temperature with shaking. 100 mL of sera was then added to the antigen-coated wells and incubated for 2 hours at room temperature with shaking. The wells were then incubated with 100 mL of biotinylated anti- human IgG1 (BD Pharmingen, USA) diluted 1:2000 in assay buffer (Wallac, Oy) for 2 hours at room temperature with shaking. This was then followed by incubation for 30 minutes with 100 mL Europium-labelled streptavidin diluted 1:1000 in assay buffer, with shaking. The final washing step involved washing the plates 8 times before the addition of 100 mL enhancement solution. Immunoassays to Determine Total IgG1 Antibody Binding Table 2. Percentages of the secondary structure of the circular dichroism (CD) spectra of HRV VP1 antigens following subtraction of GST. g The DELFIAH assay is described in detail elsewhere [24] and was performed with modifications. In brief, microtitre plates (Nunc, Roskilde, Denmark) were coated with 100 mL of purified VP1 antigens diluted to 0.125–4 mg/ml in 50 mM Na2CO3/ NaHCO3 (pH 9.6) at 4uC overnight. The concentration of antigen used for coating was standardised by titrating the coating concentrations using monoclonal anti-glutathione-S-transferase antibody (Sigma-Aldrich, St Louis, MO) to measure the amount adhering to the well. Optimal concentrations were determined by referencing to a recombinant GST-fusion Der p 2 standard that was later used for quantitation of antibody binding (see below). Each test sera was diluted to a final concentration of 1:100 in Analysed using DiChroWeb Server: CDSSTR algorithm, reference set 4. doi:10.1371/journal.pone.0070552.t002 August 2013 \| Volume 8 \| Issue 8 \| e70552 PLOS ONE \| www.plosone.org 3 Immunoabsorption Assays to Determine Species-Specific IgG1 Antibody Binding Immunoabsorption assays were conducted to determine the species-specific IgG1 antibody binding and were performed as per the immunoassay above, except that each test sera was pre- incubated in a lysate mixture of E.coli producing the other two HRV species and HPV Sabin VP1 to absorb out cross-reactive binding. The lysates (produced using soluble supernatants following sonication as described above) were used at a final concentration of 1:250 shown by pilot experiments to be an excess amount to ensure complete inhibition where present. Quantitation of IgG1 Antibody Binding The percentages of secondary structure of recombinant HRV-B14 VP1 was also similar to published data on native HRV-B14 VP1 ([30]; pdb: 1NCQ). Quantitation of IgG1 Antibody Binding A titration of reference sera was included on every plate to construct a standard curve and act as a positive control to assess reproducibility. The correlation of variation was less than 5% between plates. The standard curve was then used to quantitate the IgG1 binding to the VP1 antigens. Human myeloma IgG1 (Sigma-Aldrich, St Louis, MO) was used as a negative control to August 2013 \| Volume 8 \| Issue 8 \| e70552 PLOS ONE \| www.plosone.org 4 Antibody Binding to Human Rhinovirus Antigens Figure 3. Strong IgG1 cross-reactivity between two HRV genotypes of the same species. Each graph represents percentage of inhibition in individual sera. Subjects that had high total IgG1 titres to testing antigens were selected for competitive inhibition assays and example inhibition assays are shown. (A) Level of inhibition between HRV-A34 and HRV-A1B (HRV-A species). (B) Level of inhibition between HRV-B14 and HRV-B69 (HRV- B species). (C) Level of inhibition between HRV-C3 and HRV-C5 (HRV-C species). The inhibition of binding by an irrelevant recombinant antigen (Fel d 3), used as a negative control, are also indicated. doi:10.1371/journal.pone.0070552.g003 Figure 3. Strong IgG1 cross-reactivity between two HRV genotypes of the same species. Each graph represents percentage of inhibition in individual sera. Subjects that had high total IgG1 titres to testing antigens were selected for competitive inhibition assays and example inhibition assays are shown. (A) Level of inhibition between HRV-A34 and HRV-A1B (HRV-A species). (B) Level of inhibition between HRV-B14 and HRV-B69 (HRV- B species). (C) Level of inhibition between HRV-C3 and HRV-C5 (HRV-C species). The inhibition of binding by an irrelevant recombinant antigen (Fel d 3), used as a negative control, are also indicated. doi:10 1371/journal pone 0070552 g003 than HRV-B (83% for HRV-B14 and 89% for HRV-B69) and HRV-C (70% for HRV-C3 and 79% for HRV-C5). VP1 using the CDSSTR algorithm and reference set 4 indicated the presence of 10% a-helix and 30% b-sheet content (Table 2), comparable to the published secondary structure of HRV-A1A VP1 ([29]; pdb: 1R1A). The percentages of secondary structure of recombinant HRV-B14 VP1 was also similar to published data on native HRV-B14 VP1 ([30]; pdb: 1NCQ). VP1 using the CDSSTR algorithm and reference set 4 indicated the presence of 10% a-helix and 30% b-sheet content (Table 2), comparable to the published secondary structure of HRV-A1A VP1 ([29]; pdb: 1R1A). Competitive Inhibition Studies To examine cross-reactivity between the genotypes, the cross inhibition of the two VP1 proteins representing each species was examined. Sera from two high responder subjects were selected for each species and reciprocal competitive inhibition assays conduct- ed with antigen from the two genotypes for each species. The genotype VP1 for the HRV-A strongly cross inhibited each other (Figure 3A) as did the genotypes for HRV-B (Figure 3B). The inhibition for HRV-C was similar, although the inhibition of binding to HRV-C5 by HRV-C3 plateaued at 80% (Figure 3C). The inhibitions all reached a maximum with antigen concentra- tion of 4 mg/ml or less. Total IgG1 Antibody Binding to HRV VP1 Antigens Total IgG1 Antibody Binding to HRV VP1 Antigens Initially, total IgG1 antibody binding to each of the HRV VP1 antigens was measured. This provided a measure of IgG1 binding to each of the species, including any reactivity that may be directed to cross-reacting epitopes shared with other HRV species, or with other enteroviruses. The VP1 antigens bound IgG1 antibodies from the 63 adult sera at high titres (Figure 2). There was no significant difference between HRV-A, -B, -C or HPV Sabin VP1 titres. However, the prevalence of total IgG1 reactivity was higher for HRV-A variants (97% for HRV-A34 and 94% for HRV-A1B) Cross inhibition measured with VP1 between the species had a high degree of variation with different sera. The reciprocal August 2013 \| Volume 8 \| Issue 8 \| e70552 August 2013 \| Volume 8 \| Issue 8 \| e70552 PLOS ONE \| www.plosone.org 5 Antibody Binding to Human Rhinovirus Antigens Figure 4. Cross-reactivity between HRV-A and HRV-C. (A) An example of strong cross-inhibition in HRVA34 and HRV-C3 double-positive sera. (B) An example of poor cross-inhibition between HRV-A34 and HRV-C3 observed in a minority of subjects who had high IgG1 titres to both HRV-A and HRV-C. The inhibition of binding to HRV-A34 and HRV-C3 by an irrelevant recombinant antigen (bacterial protein, Psp-C), used as a negative control, are also indicated. doi:10.1371/journal.pone.0070552.g004 Figure 4. Cross-reactivity between HRV-A and HRV-C. (A) An example of strong cross-inhibition in HRVA34 and HRV-C3 double-positive sera. (B) An example of poor cross-inhibition between HRV-A34 and HRV-C3 observed in a minority of subjects who had high IgG1 titres to both HRV-A and HRV-C. The inhibition of binding to HRV-A34 and HRV-C3 by an irrelevant recombinant antigen (bacterial protein, Psp-C), used as a negative control, are also indicated. doi:10.1371/journal.pone.0070552.g004 The same procedure was used for the antigens from the other species. Each assay included three negative control sera, as determined by human myeloma IgG1, and reference sera. The species-specific titres determined, for each of the two genotypes of HRV-A, HRV-B and HRV-C, show the very high titres of antibody to the HRV-A species compared to usually low titres for HRV-C, and much higher prevalence of the species-specific binding antibodies to HRV-A than HRV-C (Figure 7; P,0.0001). Only 19/63 subjects had antibodies specifically attributable to HRV-C (HRV-C3) and the titres of the responders did not reach the titres found for HRV-A. Antibody Responses to Truncated HRV-C VP1 Antibody Responses to Truncated HRV-C VP1 To measure IgG1 binding to truncated HRV-C VP1, HRV-C3 VP1 was cloned and expressed from residue 14 to 275 in the pGEX-2T expression system. The purification protocol was the same as for full-length HRV-C3 VP1 and showed the same characteristics. CD analysis revealed similar structures to full- length HRV-C3 VP1, with the truncated HRV-C3 VP1 being composed of 33% b-strand and 12% a-helix (Table 2). IgG1 binding to truncated HRV-C3 VP1 was measured in 30 subjects who had high IgG1 titres to full-length HRV-C3 VP1. The IgG1 titres to truncated HRV-C3 VP1 were significantly lower than total IgG1 to full length VP1 (Figure 9A, P,0.0001) with 9/30 subjects having no reactivity to the truncated construct of HRV-C3 VP1. The sera with detectable antibody to truncated HRV-C3 VP1 were however, interestingly, the same as those with an antibody response to full-length HRV-C3 VP1 remaining after Total IgG1 Antibody Binding to HRV VP1 Antigens The HRV-B titres were similar in size and prevalence to the HRV-A. The correlation of the antibody binding between the VP1 antigens of each genotype within each species was very high (Figure 8). inhibition with the serum shown in Figure 4A shows almost 100% reciprocal inhibition between HRV-A34 genotype antigen of HRV-A and the HRV-C3 genotype of HRV-C, while the inhibition between HRV-A and HRV-C shown in Figure 4B in another subject, was low. Inhibition of IgG1 binding to HRV VP1 was negligible using the bacterial protein, Psp-C as a control (Figure 4), and HRV VP1 was similarly unable to inhibit IgG1 binding to Psp-C. Further competitive inhibitions as exemplified for the individual in Figure 5 showed there were also lesser inhibitions between all the VP1 including with HRV-B and HPV Sabin. The binding to HPV Sabin was strongly cross inhibited by the HRV-B14 genotype of HRV-B, while inhibition by HRV-A and HRV-C was minimal (Figure 5D). The pattern, however, was variable when other subjects were examined. Despite the variation, absorption assays using sera of 30 subjects who had high IgG1 titres to HRV-C (HRV-C3) revealed a much greater inhibition of antibody binding to HRV-C with HRV-A, than with HRV-B or HPV Sabin. Here, absorption with HRV-A lysate resulted in complete inhibition of binding to HRV-C3 in the majority of subjects, showing frequent high cross-reactivity (Figure 6). In comparison, absorption with a mixture of HRV-B and HPV Sabin lysates produced lower and less frequent inhibition. August 2013 \| Volume 8 \| Issue 8 \| e70552 Species-specific IgG1 Antibody Binding to HRV VP1 Antigens In the light of the competitive inhibition studies, the species- specific titres were determined following absorption of each sera with lysates containing the VP1 of the other species. Thus antibodies to the HRV-C genotypes were measured after absorption with a mixture of HRV-A, HRV-B and HPV Sabin. August 2013 \| Volume 8 \| Issue 8 \| e70552 PLOS ONE \| www.plosone.org 6 Antibody Binding to Human Rhinovirus Antigens Figure 5. An example of the level of cross-reactivity found between HRV species and HPV Sabin VP1 in an individual’s serum. (A) HRV-A VP1 antigen inhibited by HRV-B, HRV-C and HPV Sabin antigens. (B) HRV-B VP1 antigen inhibited by HRV-A, HRV-C and HPV Sabin antigens. (C) HRV-C VP1 antigen inhibited by HRV-A, HRV-B and HPV Sabin antigens. (D) HPV Sabin antigen inhibited by antigens representing the three HRV species. An irrelevant recombinant antigen (Fel d 3) was used as a negative control for each competitive inhibition assay (data not shown). doi:10.1371/journal.pone.0070552.g005 Figure 5. An example of the level of cross-reactivity found between HRV species and HPV Sabin VP1 in an individual’s serum. (A) HRV-A VP1 antigen inhibited by HRV-B, HRV-C and HPV Sabin antigens. (B) HRV-B VP1 antigen inhibited by HRV-A, HRV-C and HPV Sabin antigens. (C) HRV-C VP1 antigen inhibited by HRV-A, HRV-B and HPV Sabin antigens. (D) HPV Sabin antigen inhibited by antigens representing the three HRV species. An irrelevant recombinant antigen (Fel d 3) was used as a negative control for each competitive inhibition assay (data not shown). doi:10.1371/journal.pone.0070552.g005 HRV-A absorption. There was a strong correlation between the titres of full-length HRV-C3 VP1 which had cross-reactivity with HRV-A species removed and the truncated construct (Figure 9B). although as expected from the sequences, the competition was highest between HRV-A and -C. Given the variability between subjects observed in the pilot competitions, the assays were further developed by absorbing sera with an excess of the VP1 antigens from the heterologous HRV species and HPV Sabin and then conducting the titrations against the target VP1. The results showed a high prevalence of species-specific antibodies to HRV-A, HRV-B and HPV Sabin. It should be noted that the Sabin VP1 amino acid sequence (35% and 25% identical to HRV-A and C, respectively) can have nearly 60% identity to some Echoviruses, so this is an important consideration for future studies of HPV Sabin. Species-specific IgG1 Antibody Binding to HRV VP1 Antigens The standout result here was the low prevalence of HRV-C specific titres even though most subjects had high total IgG1 antibody binding. The two genotypes examined for each HRV species gave the same results with high correlations between the species-specific titres that concurred with the competitive inhibi- tion studies showing complete cross-competition with low doses of antigen. The pairs of genotypes that were selected to represent disparate sequences within each species (22%, 29% and 38% amino acid disparity within the HRV-A, -B and -C species, respectively) had the same species-specific binding especially for HRV-A, and where present, also to HRV-C. The response specific to an infecting genotype has not been measured in this study. Future studies could accomplish this by producing an antigen with the sequence of the known HRV genotype infecting the patient, and matching it to the patient anti-serum response. However, given the large shared IgG1 binding and the limited amino acid PLOS ONE \| www.plosone.org Discussion IgG1 binding to HRV-C (HRV-C3 and HRV-C5) antigens were significantly lower than HRV-A, HRV-B and HPV Sabin titres (P,0.0001) as indicated by . Human myeloma IgG1 was used as a negative control to determine non-specific binding and identification of negative control sera. Three negative control sera for each antigen and a titration of reference sera were included on every plate for the quantitation of IgG1 binding and to assess reproducibility of the assay. doi:10.1371/journal.pone.0070552.g007 Figure 6. Inhibition of IgG1 binding to HRV-C3 VP1 by HRV-A or HRV-B and HPV Sabin. Inhibition by lysate containing HRV-A, or a mixture of HRV-B and HPV Sabin in 30 subjects with high total IgG1 titres to HRV-C3. Percentage of cross-reactivity to the inhibiting species was calculated by (Titre after absorption/Total IgG1)6100. P,0.0001 between inhibition by HRV-A and inhibition by HRV-B and HPV Sabin. Three negative control sera and a titration of reference sera were included on every plate to assess reproducibility and quantitation of IgG1 binding. doi:10 1371/journal pone 0070552 g006 serum albumins and haemoglobins from different species can readily distinguish between antigens with 70% sequence identity [41] and the anti-Psp-A antibodies to Streptococcus pneumonia with 69% sequence identity or less, induce largely non-cross-reactive responses [32]. The house dust mite allergens Der p 5 and Blo t 5 with 38% identity show no cross-reaction despite both frequently eliciting large IgE antibody responses [42]. changes that alter neutralization [8], the antibodies against the infecting genotype might be difficult to measure, especially for HRV-A and HRV-B. The low prevalence of species-specific binding to HRV-C is unlikely to be due to the poor antigenicity of the recombinant construct since it bound strongly prior to absorption with HRV-A and the other species, and could inhibit binding to itself with the same dose-response characteristics of HRV-A. The CD measure- ments also did not indicate a deficient structure or instability. Another anomaly is that, HRV-C infection is detectable at almost the same prevalence as HRV-A and twice that of HRV-B [36] for which species-specific antibody binding was far more prevalent. The cross-reactivity and sequence identity of HRV-C with HRV- A was greater than that of HRV-B but this does not account for the high prevalence of HRV-A specific binding after absorption with HRV-C. Discussion Three negative control sera and a titration of reference sera were included on every plate to assess reproducibility and quantitation of IgG1 binding. doi:10.1371/journal.pone.0070552.g006 Figure 7. Species-specific IgG1 antibody binding (ng/ml) to HRV and HPV Sabin VP1 antigens in 63 healthy adults. Each sera was absorbed with lysates containing the VP1 of the other HRV species, as well as HPV Sabin VP1. IgG1 binding to HRV-C (HRV-C3 and HRV-C5) antigens were significantly lower than HRV-A, HRV-B and HPV Sabin titres (P,0.0001) as indicated by . Human myeloma IgG1 was used as a negative control to determine non-specific binding and identification of negative control sera. Three negative control sera for each antigen and a titration of reference sera were included on every plate for the quantitation of IgG1 binding and to assess reproducibility of the assay. doi:10.1371/journal.pone.0070552.g007 Figure 7. Species-specific IgG1 antibody binding (ng/ml) to HRV and HPV Sabin VP1 antigens in 63 healthy adults. Each sera was absorbed with lysates containing the VP1 of the other HRV species, as well as HPV Sabin VP1. IgG1 binding to HRV-C (HRV-C3 and HRV-C5) antigens were significantly lower than HRV-A, HRV-B and HPV Sabin titres (P,0.0001) as indicated by . Human myeloma IgG1 was used as a negative control to determine non-specific binding and identification of negative control sera. Three negative control sera for each antigen and a titration of reference sera were included on every plate for the quantitation of IgG1 binding and to assess reproducibility of the assay. doi:10.1371/journal.pone.0070552.g007 Figure 7. Species-specific IgG1 antibody binding (ng/ml) to HRV and HPV Sabin VP1 antigens in 63 healthy adults. Each sera was absorbed with lysates containing the VP1 of the other HRV species, as well as HPV Sabin VP1. IgG1 binding to HRV-C (HRV-C3 and HRV-C5) antigens were significantly lower than HRV-A, HRV-B and HPV Sabin titres (P,0.0001) as indicated by . Human myeloma IgG1 was used as a negative control to determine non-specific binding and identification of negative control sera. Three negative control sera for each antigen and a titration of reference sera were included on every plate for the quantitation of IgG1 binding and to assess reproducibility of the assay. doi:10.1371/journal.pone.0070552.g007 Figure 7. Species-specific IgG1 antibody binding (ng/ml) to HRV and HPV Sabin VP1 antigens in 63 healthy adults. Each sera was absorbed with lysates containing the VP1 of the other HRV species, as well as HPV Sabin VP1. Discussion It is possible that for most subjects there is the phenomenon of ‘‘original antigenic sin’’ operating, where prior responses to HRV-A dictate the specificities induced by the related HRV-C and as described for influenza, can compromise the induction of protective responses [37]. HRV-C has also been reported to have greater genotypic diversity than HRV-A and HRV-B [38], although here the diverse genotypes correlated strongly. Speculatively, it might be due to the different biology perhaps with reduced growth due to its different receptor and unknown target cell and temperature requirements [39,40]. A priority would be to determine if HRV-C infection does elicit HRV-C specific-antibodies and this would, given the low prevalence, might be easy to detect serology to monitor defined infectious episodes. The HRV-C construct made with the N-terminal truncation only detected IgG1 binding in the sera of subjects that retained anti-HRV-C antibodies when the cross-reactivity to HRV-A was removed by absorption. This could accordingly make a convenient construct for screening anti-HRV-C responses as the main cross- reactivity was with HRV-A, and minimal cross-reactivity with HRV-B or HPV Sabin was found. The truncation was originally studied because it was proposed that removing the surface inaccessible alpha helical region of the VP1 might prevent intermolecular interactions of the VP1 and improve the yields in recombinant expression. The same pentameric-sized complex was, however, produced and it retained similar structure to full-length HRV-C3 VP1. The N-terminal region has separately been of interest since it becomes exposed when the VP4 protein dissociates from the capsid in the acid environment of the lysosome [43]. Furthermore, a high frequency of IgG binding to a synthetic peptide representing this region has been detected [44]. There is sequence conservation between HRV-A and HRV-C within the truncated region that might explain the phenomenon but the truncation might also influence the conformation of other parts of VP1. The study here describes recombinant constructs that can be produced in E.coli as structurally characterized, defined molecules to measure species-specific antibodies produced to HRV-A, -B and -C. The IgG binding studied to date shows evidence for a high degree of antibody production and for a high degree of cross- reactivity between the VP1 of different HRV species. August 2013 \| Volume 8 \| Issue 8 \| e70552 Discussion The VP1 antigen constructs of human rhinovirus species A, B and C all bound high titres of IgG1 antibody with negligible binding to myeloma controls. The total IgG1 binding by the HRV-A and -C antigens varied over a 2-log range but at high titre; the highest titres probably reflect antibodies induced by multiple infections with related viruses and recent infection causing transient elevations. The range of binding of specific titres at 250 - 16107 ng/ml is similar to values obtained for other microbial and vaccine antigens [24,31–34]. All the antibody binding could be competitively inhibited with low concentrations of the homologous antigen. The recombinant glutathione-S- transferase VP1 fusion constructs purified by size exclusion chromatography were discrete, reproducible reagents that had a molecular mass equivalent to five VP1-GST dimers, which could reflect the pentameric interactions between the VP1 proteins in the apical pentameric subunits of the HRV particle [8,35]. CD analysis of the HRV species showed that they had similar secondary structures to the natural protein. The competitive inhibition performed with a sample of sera showed a sometimes high and variable degree of cross-reactivity between the different species including the HPV Sabin VP1, August 2013 \| Volume 8 \| Issue 8 \| e70552 August 2013 \| Volume 8 \| Issue 8 \| e70552 7 PLOS ONE \| www.plosone.org Antibody Binding to Human Rhinovirus Antigens Figure 6. Inhibition of IgG1 binding to HRV-C3 VP1 by HRV-A or HRV-B and HPV Sabin. Inhibition by lysate containing HRV-A, or a mixture of HRV-B and HPV Sabin in 30 subjects with high total IgG1 titres to HRV-C3. Percentage of cross-reactivity to the inhibiting species was calculated by (Titre after absorption/Total IgG1)6100. P,0.0001 between inhibition by HRV-A and inhibition by HRV-B and HPV Sabin. Three negative control sera and a titration of reference sera were included on every plate to assess reproducibility and quantitation of IgG1 binding. doi:10.1371/journal.pone.0070552.g006 serum albumins and haemoglobins from different species can readily distinguish between antigens with 70% sequence identity Figure 6. Inhibition of IgG1 binding to HRV-C3 VP1 by HRV-A or HRV-B and HPV Sabin. Inhibition by lysate containing HRV-A, or a mixture of HRV-B and HPV Sabin in 30 subjects with high total IgG1 titres to HRV-C3. Percentage of cross-reactivity to the inhibiting species was calculated by (Titre after absorption/Total IgG1)6100. P,0.0001 between inhibition by HRV-A and inhibition by HRV-B and HPV Sabin. Discussion Most notably, while some people have high titres of species-specific antibodies to HRV-C, most do not, even though they have high The degree of cross-reactivity between the HRV-A and HRV-C is not only unusual because of its unidirectional bias but is different in magnitude to that observed for many antigens with a similar degree of sequence disparity. Hyperimmune sera produced to August 2013 \| Volume 8 \| Issue 8 \| e70552 August 2013 \| Volume 8 \| Issue 8 \| e70552 PLOS ONE \| www.plosone.org 8 Figure 9. Antibody responses to HRV-C VP1 with an N-terminal truncation in 30 adult subjects. (A) IgG1 binding (ng/ml) to truncated HRV-C3 VP1 expressed from residue 14 to 275, in comparison to full-length VP1. P,0.0001 between full-length and truncated HRV-C3 VP1. (B) Correlation of IgG1 binding (ng/ml) between full-length HRV-C3 VP1 pre-absorbed with HRV-A to remove cross-reactivity to HRV-A, and truncated HRV-C3 VP1. A reference sera and three negative sera that did not have IgG1 binding to HRV-C3, as determined by the immunoassays and immunoabsorptions, were included on every plate for quantitation of binding. doi:10.1371/journal.pone.0070552.g009 Antibody Binding to Human Rhinovirus Antigens Antibody Binding to Human Rhinovirus Antigens component of early immune response to HRV immunity and to be influenced by prior infection by all species. Supporting Information Figure S1 Example of a standard curve used for Figure 8. Intra-species correlation of IgG1 responses in 63 adult subjects. Log correlation of IgG1 binding (ng/ml) between two genotypes of (A) HRV-A (B) HRV-B and (C) HRV-C. doi:10.1371/journal.pone.0070552.g008 Figure 9. Antibody responses to HRV-C VP1 with an N-terminal truncation in 30 adult subjects. (A) IgG1 binding (ng/ml) to truncated HRV-C3 VP1 expressed from residue 14 to 275, in comparison to full-length VP1. P,0.0001 between full-length and truncated HRV-C3 VP1. (B) Correlation of IgG1 binding (ng/ml) between full-length HRV-C3 VP1 pre-absorbed with HRV-A to remove cross-reactivity to HRV-A, and truncated HRV-C3 VP1. A reference sera and three negative sera that did not have IgG1 binding to HRV-C3, as determined by the immunoassays and immunoabsorptions, were included on every plate for quantitation of binding. doi:10.1371/journal.pone.0070552.g009 y g g Figure 9. Antibody responses to HRV-C VP1 with an N-terminal truncation in 30 adult subjects. (A) IgG1 binding (ng/ml) to truncated HRV-C3 VP1 expressed from residue 14 to 275, in comparison to full-length VP1. P,0.0001 between full-length and truncated HRV-C3 VP1. Discussion (B) Correlation of IgG1 binding (ng/ml) between full-length HRV-C3 VP1 pre-absorbed with HRV-A to remove cross-reactivity to HRV-A, and truncated HRV-C3 VP1. A reference sera and three negative sera that did not have IgG1 binding to HRV-C3, as determined by the immunoassays and immunoabsorptions, were included on every plate for quantitation of binding. doi:10.1371/journal.pone.0070552.g009 component of early immune response to HRV immunity and to be influenced by prior infection by all species. component of early immune response to HRV immunity and to be influenced by prior infection by all species. Figure 8. Intra-species correlation of IgG1 responses in 63 adult subjects. Log correlation of IgG1 binding (ng/ml) between two genotypes of (A) HRV-A (B) HRV-B and (C) HRV-C. doi:10.1371/journal.pone.0070552.g008 References p 6. Hao W, Bernard K, Patel N, Ulbrandt N, Feng H, et al. (2012) Infection and Propagation of Human Rhinovirus C in Human Airway Epithelial Cells. J Virol 86: 13524–13532. 25. Hales BJ, Bosco A, Mills KL, Hazell LA, Loh R, et al. 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Barclay WS, Al-Nakib W, Higgins PG, Tyrrell DAJ (1989) The time course of the humoral immune response to rhinovirus infection. Epidemiol Infect 103: 659–669. 28. Brockwell D, Yu L, Cooper S, Mccleland S, Cooper A, et al. (2001) Physicochemical consequences of the perdeuteriation of glutathione S- transferase from S. japonicum. Protein Sci 10: 572–580. 10. Butler WT, Waldmann TA, Rossen RD, Douglas Jr RG, Couch RB (1970) Changes in IgA and IgG concentrations in nasal secretions prior to the appearance of antibody during viral respiratory infection in man. J Immunol 105: 584–591. 29. Kim S, Smith TJ, Chapman MS, Rossmann MG, Pevear DC, et al. (1989) Crystal structure of human rhinovirus serotype 1A (HRV1A). J Mol Biol 210: 91–111. 30. Zhang Y, Simpson AA, Ledford RM, Bator CM, Chakravarty S, et al. (2004) Structural and virological studies of the stages of virus replication that are affected by antirhinovirus compounds. J Virol 78: 11061–11069. 11. Van Kempen M, Bachert C, Van Cauwenberge P (1999) An update on the pathophysiology of rhinovirus upper respiratory tract infections. Rhinology 37: 97–103. 31. Aarntzen EH, de Vries IJM, Go¨ertz JH, Beldhuis-Valkis M, Brouwers HM, et al. References 1. Ma¨kela¨ MJ, Puhakka T, Ruuskanen O, Leinonen M, Saikku P, et al. (1998) Viruses and bacteria in the etiology of the common cold. J Clin Virol 36: 539– 542. 1. Ma¨kela¨ MJ, Puhakka T, Ruuskanen O, Leinonen M, Saikku P, et al. (1998) Viruses and bacteria in the etiology of the common cold. J Clin Virol 36: 539– 542. 19. Ledford RM, Patel NR, Demenczuk TM, Watanyar A, Herbertz T, et al. (2004) VP1 sequencing of all human rhinovirus serotypes: insights into genus phylogeny and susceptibility to antiviral capsid-binding compounds. J Virol 78: 3663–3674. 20. McErlean P, Shackelton LA, Lambert SB, Nissen MD, Sloots TP, et al. (2007) Characterisation of a newly identified human rhinovirus, HRV-QPM, discovered in infants with bronchiolitis. J Clin Virol 39: 67–75. 2. Lau SKP, Yip CCY, Tsoi H, Lee RA, So L, et al. (2007) Clinical Features and Complete Genome Characterization of a Distinct Human Rhinovirus (HRV) Genetic Cluster, Probably Representing a Previously Undetected HRV Species, HRV-C, Associated with Acute Respiratory Illness in Children. J Clin Microbiol 45: 3655–3664. 21. Hales BJ, Elliot CE, Chai LY, Pearce LJ, Tipayanon T, et al. (2013) Quantitation of IgE Binding to the Chitinase and Chitinase-Like House Dust Mite Allergens Der p 15 and Der p 18 Compared to the Major and Mid-Range Allergens. Int Arch Allergy Immunol 160: 233–240. 3. Fry AM, Lu X, Olsen SJ, Chittaganpitch M, Sawatwong P, et al. (2011) Human rhinovirus infections in rural Thailand: epidemiological evidence for rhinovirus as both pathogen and bystander. PLoS One 6: e17780. 22. Whitmore L, Wallace BA (2008) Protein secondary structure analyses from circular dichroism spectroscopy: Methods and reference databases. Biopolymers 89: 392–400. 4. Ferguson PE, Gilroy NM, Faux CE, Mackay IM, Sloots TP, et al. (2013) Human rhinovirus C in adult haematopoietic stem cell transplant recipients with respiratory illness. J Clin Virol 56: 339–343. 23. Lees JG, Miles AJ, Wien F, Wallace BA (2006) A reference database for circular dichroism spectroscopy covering fold and secondary structure space. Bioinfor- matics 22: 1955–1962. 5. Bizzintino J, Lee W, Laing I, Vang F, Pappas T, et al. (2011) Association between human rhinovirus C and severity of acute asthma in children. Eur Respir J 37: 1037–1042. 24. Hales BJ, Chai LY, Elliot CE, Pearce LJ, Zhang G, et al. (2012) Antibacterial antibody responses associated with the development of asthma in house dust mite-sensitised and non-sensitised children. Thorax 67: 321–327. References (2012) Humoral anti-KLH responses in cancer patients treated with dendritic cell-based immunotherapy are dictated by different vaccination parameters. Cancer Immunol Immunother 61: 2003–2011. 12. Stanekova´ Z, Vareckova E (2010) Conserved epitopes of influenza A virus inducing protective immunity and their prospects for universal vaccine development. Virol J 7: 351. 13. Wrammert J, Koutsonanos D, Li GM, Edupuganti S, Sui J, et al. (2011) Broadly cross-reactive antibodies dominate the human B cell response against 2009 pandemic H1N1 influenza virus infection. J Exp Med 208: 181–193. 32. Nabors GS, Braun PA, Herrmann DJ, Heise ML, Pyle DJ, et al. (2000) Immunization of healthy adults with a single recombinant pneumococcal surface protein A (PspA) variant stimulates broadly cross-reactive antibodies to heterologous PspA molecules. Vaccine 18: 1743–1754. 14. Weinfurter JT, Brunner K, Capuano SV, Li C, Broman KW, et al. (2011) Cross- reactive T cells are involved in rapid clearance of 2009 pandemic H1N1 influenza virus in nonhuman primates. PLoS Path 7: e1002381. 33. Vance E, George S, Guinan E, Wheeler C, Antin J, et al. (1998) Comparison of multiple immunization schedules for Haemophilus influenzae type b-conjugate and tetanus toxoid vaccines following bone marrow transplantation. Bone Marrow Transplant 22: 735–741. p 15. Kennedy JL, Turner RB, Braciale T, Heymann PW, Borish L (2012) Pathogenesis of rhinovirus infection. Curr Opin Virol 2: 287–293. 34. Semenova V, Steward-Clark E, Stamey K, Taylor T, Schmidt D, et al. (2004) Mass value assignment of total and subclass immunoglobulin G in a human standard anthrax reference serum. Clin Diagn Lab Immunol 11: 919–923. 16. Laza-Stanca V, Stanciu LA, Message SD, Edwards MR, Gern JE, et al. (2006) Rhinovirus replication in human macrophages induces NF-kappaB-dependent tumor necrosis factor alpha production. J Virol 80: 8248–8258. 35. McErlean P, Shackelton LA, Andrews E, Webster DR, Lambert SB, et al. (2008) Distinguishing molecular features and clinical characteristics of a putative new rhinovirus species, human rhinovirus C (HRV C). PLoS One 3: e1847. 17. Gern JE, Brooks GD, Meyer P, Chang A, Shen K, et al. (2006) Bidirectional interactions between viral respiratory illnesses and cytokine responses in the first year of life. J Allergy Clin Immunol 117: 72–78. 36. Lau SKP, Yip CCY, Lin AWC, Lee RA, So LY, et al. (2009) Clinical and molecular epidemiology of human rhinovirus C in children and adults in Hong Kong reveals a possible distinct human rhinovirus C subgroup. J Infect Dis 200: 1096–1103. 18. Acknowledgments We would like to acknowledge the participants who took part in the study, and L.A. Hazell and T.L.Y. Chai for assistance with protein antigen preparation. Figure S2 Circular dichroism (CD) analysis of recom- binant HRV-C VP1 protein. (A) CD spectrum of GST-fusion HRV-C3 VP1. (B) CD spectrum of HRV-C3 VP1 following subtractive CD analysis in which the GST control was subtracted from the fusion protein. The diagrams represent the ultraviolet spectra of the purified recombinant proteins analysed using CD spectroscopy in the range 260–190 nm. Structural analysis of the data was performed using DiChroWeb Server: CDSSTR algorithm, reference set 4. Author Contributions Conceived and designed the experiments: JI WS SRS WRT BJH. Performed the experiments: JI SRS. Analyzed the data: JI SRS WRT BJH. Conceived and designed the experiments: JI WS SRS WRT BJH. Performed the experiments: JI SRS. Analyzed the data: JI SRS WRT BJH. Contributed reagents/materials/analysis tools: WRT SRS. Wrote the paper: JI WS SRS WRT BJH. Contributed reagents/materials/analysis tools: WRT SRS. Wrote the paper: JI WS SRS WRT BJH. Supporting Information Figure S1 Example of a standard curve used for quantitation of IgG1 binding. Each assay was calibrated by interpolating the results from a titration curve constructed with recombinant GST-fusion Der p 2 and a standardised humanised anti-Der p 2 chimeric IgG1. The linear section of the Der p 2 chimeric curve used for analysis had a slope = 5.0060.07 and is indicated in red. Equivalent concentrations of recombinant VP1 antigens was used to coat wells and bound with a titration of reference sera (example of HRV-A34 shown). The 2-fold dilutions used are indicated. The linear section of the reference titration curve (indicated in red) had a slope = 4.9560.69, comparable to the Der p 2 chimeric curve. A titration curve of reference sera was included on every plate to construct a standard curve, which was then used to convert europium counts to absolute IgG1 binding titres of cross-reactive antibodies mainly to HRV-A, that bind the HRV-C. Further investigation of these responses with clinically defined subjects and paediatric populations in conjunction with virus detection is thus warranted. The antibodies might be used as a marker of CD4 T-cell responses and be extended in the examination of other antibody isotypes. They might also have critical functions in the immunopathology produced by these viruses where for example they could increase the involvement of cytokine-producing monocytes and other inflammatory cells, as well as antigen presentation. As shown for other virus infections [12–14], non-neutralising antibodies also play a key role in immunity and they would be expected to be quantitatively a large PLOS ONE \| www.plosone.org August 2013 \| Volume 8 \| Issue 8 \| e70552 PLOS ONE \| www.plosone.org 9 Antibody Binding to Human Rhinovirus Antigens (ng/ml). The mean europium count of the negative control, human myeloma IgG1, is indicated. For HRV-A34, the mean and standard deviation (SD) europium count for human myeloma IgG1 was 25726795 (n = 3), which was calculated to be 499 ng/ ml. Negative sera was determined using mean +3SD of human myeloma IgG1 binding for each antigen. (TIF) (TIF) Table S1 Nucleotide and protein sequences of HRV and HPV Sabin VP1 used in this study. (DOCX) 42. Weghofer M, Dall’Antonia Y, Grote M, Sto¨cklinger A, Kneidinger M, et al. (2008) Characterization of Der p 21, a new important allergen derived from the gut of house dust mites. Allergy 63: 758–767. 41. Ro¨hm C, Zhou N, Su¨ss J, Mackenzie J, Webster R (1996) Characterization of a novel influenza hemagglutinin, H15: criteria for determination of influenza A subtypes. Virology 217: 508–516. References Rossmann MG, Arnold E, Erickson JW, Frankenberger EA, Griffith JP, et al. (1985) Structure of a human common cold virus and functional relationship to other picornaviruses. Nature 317: 145–153. August 2013 \| Volume 8 \| Issue 8 \| e70552 August 2013 \| Volume 8 \| Issue 8 \| e70552 PLOS ONE \| www.plosone.org 10 Antibody Binding to Human Rhinovirus Antigens Antibody Binding to Human Rhinovirus Antigens Antibody Binding to Human Rhinovirus Antigens 37. Kim JH, Skountzou I, Compans R, Jacob J (2009) Original antigenic sin responses to influenza viruses. J Immunol 183: 3294–3301. 38. McIntyre CL, McWilliam Leitch EC, Savolainen-Kopra C, Hovi T, Simmonds P (2010) Analysis of genetic diversity and sites of recombination in human rhinovirus species C. J Virol 84: 10297–10310. yp gy 42. Weghofer M, Dall’Antonia Y, Grote M, Sto¨cklinger A, Kneidinger M, et al. (2008) Characterization of Der p 21, a new important allergen derived from the gut of house dust mites. Allergy 63: 758–767. p J 39. Bochkov YA, Palmenberg AC, Lee WM, Rathe JA, Amineva SP, et al. (2011) Molecular modeling, organ culture and reverse genetics for a newly identified human rhinovirus C. Nat Med 17: 627–632. g gy 43. Giranda VL, Heinz BA, Oliveira MA, Minor I, Kim KH, et al. (1992) Acid- induced structural changes in human rhinovirus 14: possible role in uncoating. Proc Natl Acad Sci U S A 89: 10213–10217. 40. Ashraf S, Brockman-Schneider R, Bochkov YA, Pasic TR, Gern JE (2012) Biological characteristics and propagation of human rhinovirus-C in differen- tiated sinus epithelial cells. Virology 436: 143–149. 44. Niespodziana K, Napora K, Cabauatan C, Focke-Tejkl M, Keller W, et al. (2012) Misdirected antibody responses against an N-terminal epitope on human rhinovirus VP1 as explanation for recurrent RV infections. FASEB J 26: 1001– 1008. PLOS ONE \| www.plosone.org August 2013 \| Volume 8 \| Issue 8 \| e70552 PLOS ONE \| www.plosone.org 11 11
https://openalex.org/W2924659556	https://www.nature.com/articles/s41598-019-41460-2.pdf	English	null	Targeting hormone refractory prostate cancer by in vivo selected DNA libraries in an orthotopic xenograft mouse model	Scientific reports	2,019	cc-by	15,043	Targeting hormone refractory prostate cancer by in vivo selected DNA libraries in an orthotopic xenograft mouse model Received: 15 October 2018 Accepted: 8 March 2019 Published: xx xx xxxx Laia Civit1, Ioanna Theodorou2, Franziska Frey1, Holger Weber3,10, Andreas Lingnau3,11, Carsten Gröber4, Michael Blank4, Chloé Dambrune2, James Stunden5, Marc Beyer6,7,8, Joachim Schultze 6,7, Eicke Latz 5, Frédéric Ducongé2, Michael H. G. Kubbutat3,10 & Günter Mayer1,9 The targeting of specific tissue is a major challenge for the effective use of therapeutics and agents mediating this targeting are strongly demanded. We report here on an in vivo selection technology that enables the de novo identification of pegylated DNA aptamers pursuing tissue sites harbouring a hormone refractory prostate tumour. To this end, two libraries, one of which bearing an 11 kDa polyethylene glycol (PEG) modification, were used in an orthotopic xenograft prostate tumour mouse model for the selection process. Next-generation sequencing revealed an in vivo enriched pegylated but not a naïve DNA aptamer recognising prostate cancer tissue implanted either subcutaneous or orthotopically in mice. This aptamer represents a valuable and cost-effective tool for the development of targeted therapies for prostate cancer. The described selection strategy and its analysis is not limited to prostate cancer but will be adaptable to various tissues, tumours, and metastases. This opens the path towards DNA aptamers being experimentally and clinically engaged as molecules for developing targeted therapy strategies. Prostate cancer is the most common cancer among men, with approximately 1.1 million new cases diagnosed per year worldwide1. It accounts for the second most incidence of cancer-related death although the 5-year survival rate is over 80%. Especially, hormone refractory prostate cancer is not curable and targeted therapies are yet not available. Hormone refractory prostate tumour cells likely metastasise to distal sites accounting for poor progno- sis and survival of patients2. For prostate tumours not responding to initial hormone therapy, chemotherapy with docetaxel (taxotere) results in an improved survival of patients3. Alternative therapies are immunotherapy with sipuleucel-T, an autologous dendritic cell- based cancer vaccine, agents interfering with androgen signalling, i.e. abiraterone or enzalutamide, and radiopharmaceutical therapy for bone metastases4–7. p py Tomasetti and Vogelstein recently described occurrence of random mutations during DNA replication in can- cer stem cells playing an important role in the development of certain types of tumours8. This was associated with the development of cancer in which environmental or hereditable factors have a low impact on tumour onset and 1Chemical Biology and Chemical Genetics, Life and Medical Sciences (LIMES) Institute, University of Bonn, Gerhard- Domagk-Str. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports/ progression9, e.g. in prostate cancer. Developing new therapeutic strategies with high specificity for the malignant tissue is therefore of strong interest but challenging once prostate cancer progresses to an androgen-independent, hence hormone refractory state. progression9, e.g. in prostate cancer. Developing new therapeutic strategies with high specificity for the malignant tissue is therefore of strong interest but challenging once prostate cancer progresses to an androgen-independent, hence hormone refractory state. y Aptamers are an emerging class of molecules for developing targeted therapy approaches10,11. They are single chained nucleic acids, folding into well-defined three-dimensional shapes based on which they recognise target structures with high affinity and specificity12. Aptamers targeting tumour cells are commonly identified by an in vitro selection process using cultured cell lines or isolated membrane proteins. However, only a few examples are described in which these aptamers are also capable of recognising the respective target or cells in the related in vivo microenvironment13–19. Recently, a prostate cancer targeting RNA aptamer selected in an internalisation cell-SELEX procedure was successfully used, in combination with two highly toxic drugs, for the inhibition of tumour growth in vivo20.t g In contrast, selection procedures conducted solely in vivo, e.g. using xenograft tumour model systems are sup- posedly suitable for the de novo identification of effective tumour recognising aptamers. Here, an in vivo selection approach is described, employing DNA libraries for the identification of aptamers targeting androgen independ- ent prostate tumours in an orthotopic xenograft mouse model. This approach resulted in the identification of a series of DNA aptamers that show tumour targeting properties in orthotopic and subcutaneous xenograft mouse models. Among them, one representative aptamer (D3P-21) was further characterised and found to reproducibly recognise prostate tumours in vivo. The interaction properties of D3P-21 were found to depend strictly upon the presence of a 5′-polyethylen glycol (PEG) moiety already implemented during the in vivo selection procedure. Spectroscopic data indicate that the conformation of the DNA aptamer is not impaired by the PEG moiety, hence it might directly interact with the target structures, explaining D3P-21’s PEG-dependent interaction properties.h g y g p g p p p The study opens the path towards in vivo selection procedures using DNA libraries in suitable model systems, an endeavour which previously has been supposed to be non-effective due to low stability and the rapid clearance of DNA in vivo. Results Th As further control, we prepared tumours from mice injected with PBS only and subjected these tumours to the same recovery procedure and PCR protocol (Suppl. Fig. S2a,b). In Supplementary Table S1, the weights of the resected tumours (between 28 and 388 mg five weeks after implantation) and kidneys from each selection cycle are given. After ten in vivo selection cycles (the conditions of which are summarised in Suppl. Table S2), we analysed each of the obtained DNA libraries by next-generation sequencing (NGS)30. Between 1.5·105 and 1.2·107 sequences per selection cycle were analysed from both selection procedures (Suppl. Table S3). The number of unique sequences was found to decrease significantly in later cycles, indicating both libraries being enriched (Fig. 2a). The PEG-modified DNA library D3P showed a steady decrease of unique sequences over the monitored DNA populations obtained from the different selection cycles. In contrast, the number of unique sequences in the naïve DNA library D3 was strongly decreased from the selection cycle 7 to 8 (Fig. 2a). This behaviour was also eminent from the distribution of the four nucleotides (dA, dG, dT, and dC) throughout the initial random region, which was similar up to selection cycle 4 and 7 of the libraries D3P or D3, respectively (Fig. 2b,c and Suppl. Fig. S3a,b). In turn, the nucleotide distribution was clearly altered when analysing the DNA populations obtained after ten selection cycles, which already became evident in selection cycles 6 (D3P) and 8 i Briefly, we established two in vivo selection protocols using the DNA library D3. The first selection protocol employed D3 in its naïve variant, whereas the second protocol made use of a 5′–polyethylene glycol (PEG, 11 kDa) modified version, named D3P. Besides the nature of the DNA library used, both protocols differed in the work up procedure of the DNA molecules after tumour and kidney resection and homogenisation (for details please refer to the methods section). The PEG moiety was chosen as it has been shown to increase the half-life of DNA mol- ecules in the blood circulation28,29. Prior to injection, the DNA libraries were prepared as a solution in PBS. After 20 minutes of circulation, the mice were perfused with PBS and sacrificed by cervical dislocation. Subsequently, the prostate tumour was removed, snap frozen, and stored at −80 °C until further processing. www.nature.com/scientificreports/ The data not only provide evidence for DNA aptamers being suitable for this approach but also that DNA aptamers are less immunogenic than previously anticipated21. Based on the study results, DNA aptam- ers may be revived as a less costly and alternative class of aptamers for basic research in vivo and therapeutic use. To overcome the reluctance of using DNA aptamers for in vivo purposes, more studies are required showing proof-of-concept and superior performance in validation studies. A systematic analysis of the potential of DNA aptamers to induce innate immune responses will be necessary, to address the probability of these compounds in a broad manner. Moreover, DNA is more robust and chemically stable than RNA and accepts chemical modifica- tions as well as non-canonical nucleobases, which can be used for extending the genetic alphabet22–25. Results Th The general outline of the in vivo selection scheme using orthotopic xenograft prostate tumour models is depicted in Fig. 1. The prostate cancer cell line PC-3 was implanted into the prostate of male NMRI nude mice. PC-3 is a human hormone insensitive prostate tumour cell line, which represents a well-accepted tumour model for castra- tion resistant prostate cancer26 with respect to its sensitivity to current treatments, i.e. exhibiting tumour growth inhibitory effects after treatment with docetaxel27 and its metastatic profile. The used cell line bears an intrinsic luciferase reporter protein enabling post-surgery monitoring of the tumour growth (Suppl. Fig. S1a). Approx. five weeks after implantation, the respective DNA library or phosphate buffered saline (PBS) was injected into the mice’s tail vein (Suppl. Fig. S1b). Tumours from mice injected with PBS were used as negative tumours during the library extraction and amplification steps to discard contamination of the samples (Supp. Fig. S2a,b).lhi i Briefly, we established two in vivo selection protocols using the DNA library D3. The first selection protocol employed D3 in its naïve variant, whereas the second protocol made use of a 5′–polyethylene glycol (PEG, 11 kDa) modified version, named D3P. Besides the nature of the DNA library used, both protocols differed in the work up procedure of the DNA molecules after tumour and kidney resection and homogenisation (for details please refer to the methods section). The PEG moiety was chosen as it has been shown to increase the half-life of DNA mol- ecules in the blood circulation28,29. Prior to injection, the DNA libraries were prepared as a solution in PBS. After 20 minutes of circulation, the mice were perfused with PBS and sacrificed by cervical dislocation. Subsequently, the prostate tumour was removed, snap frozen, and stored at −80 °C until further processing. After thawing, the tissues of three mice were homogenised as one sample and the nucleic acid library recovered either by silica col- umn purification (D3P) or phenol/chloroform extraction followed by ethanol precipitation (D3). After recovery, the library was amplified by PCR, subjected to single-strand generation and used for the next selection cycle. In each selection cycle, the DNA molecules associated with the kidneys were also recovered and subjected to PCR, which allowed to control the general workflow as the kidneys represent the major clearance pathway of DNA aptamers in vivo10. Targeting hormone refractory prostate cancer by in vivo selected DNA libraries in an orthotopic xenograft mouse model 1, 53121, Bonn, Germany. 2CEA, DRT, Institut de biologie François-Jacob, Molecular Imaging Research Center (MIRCen), UMR CNRS 9199, 18 Route du Panorama, 92260, Roses, France. 3KTB Tumorforschungsgesellschaft mbH, Research Division ProQinase, Breisacher Str. 117, 79106, Freiburg, Germany. 4AptaIT GmbH, Am Klopferspitz 19a, 82152, Planegg, Martinsried, Germany. 5Institute of Innate Immunity, University Hospital Bonn, Sigmund- Freud-Str. 25, 53127, Bonn, Germany. 6Genomics and Immunoregulation, Life and Medical Sciences (LIMES) Institute, University of Bonn, Carl-Troll-Straße 31, 53115, Bonn, Germany. 7Platform for Single Cell Genomics and Epigenomics at the DZNE and the University of Bonn, Sigmund-Freud-Str. 27, 53127, Bonn, Germany. 8Molecular Immunology in Neurodegeneration, German Center for Neurodegenerative Diseases (DZNE), Sigmund-Freud-Str. 27, 53127, Bonn, Germany. 9Center of Aptamer Research and Development (CARD), University of Bonn, Gerhard- Domagk Str. 1, 53121, Bonn, Germany. 10Present address: ProQinase GmbH, Breisacher Straße 117, 79106, Freiburg, Germany. 11Present address: Genmab B.V., Yalelaan 60, 3584 CM, Utrecht, The Netherlands. Correspondence and requests for materials should be addressed to G.M. (email: gmayer@uni-bonn.de) Scientific Reports \| (2019) 9:4976 \| https://doi.org/10.1038/s41598-019-41460-2 Scientific Reports \| (2019) 9:4976 \| https://doi.org/10.1038/s41598-019-41460-2 1 www.nature.com/scientificreports/ Results Th After thawing, the tissues of three mice were homogenised as one sample and the nucleic acid library recovered either by silica col- umn purification (D3P) or phenol/chloroform extraction followed by ethanol precipitation (D3). After recovery, the library was amplified by PCR, subjected to single-strand generation and used for the next selection cycle. In each selection cycle, the DNA molecules associated with the kidneys were also recovered and subjected to PCR, which allowed to control the general workflow as the kidneys represent the major clearance pathway of DNA aptamers in vivo10. As further control, we prepared tumours from mice injected with PBS only and subjected these tumours to the same recovery procedure and PCR protocol (Suppl. Fig. S2a,b). In Supplementary Table S1, the weights of the resected tumours (between 28 and 388 mg five weeks after implantation) and kidneys from each selection cycle are given. After ten in vivo selection cycles (the conditions of which are summarised in Suppl. Table S2), we analysed each of the obtained DNA libraries by next-generation sequencing (NGS)30. Between 1.5·105 and 1.2·107 sequences per selection cycle were analysed from both selection procedures (Suppl. Table S3). Th b f f d d fi l l l d b h l b The number of unique sequences was found to decrease significantly in later cycles, indicating both libraries being enriched (Fig. 2a). The PEG-modified DNA library D3P showed a steady decrease of unique sequences over the monitored DNA populations obtained from the different selection cycles. In contrast, the number of unique sequences in the naïve DNA library D3 was strongly decreased from the selection cycle 7 to 8 (Fig. 2a). This behaviour was also eminent from the distribution of the four nucleotides (dA, dG, dT, and dC) throughout the initial random region, which was similar up to selection cycle 4 and 7 of the libraries D3P or D3, respectively (Fig. 2b,c and Suppl. Fig. S3a,b). In turn, the nucleotide distribution was clearly altered when analysing the DNA populations obtained after ten selection cycles, which already became evident in selection cycles 6 (D3P) and 8 Scientific Reports \| (2019) 9:4976 \| https://doi.org/10.1038/s41598-019-41460-2 2 www.nature.com/scientificreports/ Figure 1. In vivo selection process using orthotopically xenograft prostate cancer models. PC-3 cells expressing luciferase were implanted to the prostate of nude mice and the tumour growth was monitored by bioluminescence imaging during 5 weeks until complete growth of the tumours. Results Th Pegylated (D3P) or naive DNA libraries (D3) were injected via the tail vein. After 20 min, mice were perfused and tumours and kidneys extracted and snap frozen. Homogenisation of the tumours and control organ, kidney, for extraction of bound oligonucleotides was then performed by means of mechanical and chemical homogenisation followed by phenol/chloroform purification and ethanol precipitation for D3 library and silica columns purification for D3P library. Extracted oligonucleotides were amplified and ssDNA was generated for the next SELEX cycle. In total, 10 selection cycles were performed for both libraries. NGS analysis of the in vivo SELEX was performed and selected identified sequences were tested by an in vivo screening assay in orthotopic and subcutaneous mouse models. Figure 1. In vivo selection process using orthotopically xenograft prostate cancer models. PC-3 cells expressing luciferase were implanted to the prostate of nude mice and the tumour growth was monitored by bioluminescence imaging during 5 weeks until complete growth of the tumours. Pegylated (D3P) or naive DNA libraries (D3) were injected via the tail vein. After 20 min, mice were perfused and tumours and kidneys extracted and snap frozen. Homogenisation of the tumours and control organ, kidney, for extraction of bound oligonucleotides was then performed by means of mechanical and chemical homogenisation followed by phenol/chloroform purification and ethanol precipitation for D3 library and silica columns purification for D3P library. Extracted oligonucleotides were amplified and ssDNA was generated for the next SELEX cycle. In total, 10 selection cycles were performed for both libraries. NGS analysis of the in vivo SELEX was performed and selected identified sequences were tested by an in vivo screening assay in orthotopic and subcutaneous mouse models. (D3) (Suppl. Fig. S3a,b). In the following and if not otherwise stated, we mainly describe the DNA populations of both libraries obtained after 10 selection cycles. Both libraries still contained unique sequences (11% (D3) and 9% (D3P)), which might be due to the high diversity of targets and the complex nature of the in vivo selection approach. Of note, 0.27% of the sequences were found to be present in the DNA populations of both libraries from the 10th selection cycle, which can be explained due to i) common unspecific targeting, ii) higher resistance to nucleases or iii) a more favourable amplification by PCR. The sequences were grouped in different populations according to their copy numbers in each selection cycle, i.e. Results Th ≤10, 11–100, 101–1000, 1001–10000, and >10000 and normalised with respect to the total number of copies in each selection cycle (Fig. 3a,b). This grouping reveals that sequences with >10 copy numbers are increasingly observed from selection cycle 5 and 3 for D3 and D3P, respectively (Fig. 3a). However, the vast majority of sequences within the DNA population from these selection cycles have copy numbers of 10 or less (98.3% in D3 and 95.6% in D3P). In contrast at cycle 10, 48.5% of the sequences from the D3 library and 36.3% of those found in the D3P library correspond to few strongly enriched sequences (among them 25 (D3) and 7 (D3P) sequences) with >10000 copies (Fig. 3b).i g g Next, individual sequences were chosen for further testing based on their enrichment profiles. In particular, collection was defined by i) the frequency of an individual sequence in a DNA population and ii) amplification fold, i.e. change of copy numbers from one selection cycle to another (Suppl. Fig. S4a,b). Based on these criteria 46 sequences (22 from D3 and 24 from D3P) were selected for further assessment, among them 17 sequences with low copy numbers, i.e. frequency <0.5% but enrichment profiles similar to those found for the most abun- dant sequences (Suppl. Tables S4 and S5 and Suppl. Fig. S4a,b). The other 29 sequences were the most abundant sequences found with frequencies >0.5%, whereas one sequence, named either D3-0 (24.23%) or D3P-7 (5.63%), was found in both libraries. Of note is the frequency of each sequence found in the kidney. In contrast to the selec- tion done with D3, where lower copy numbers of each individual sequence were found in the kidney compared to the tumour tissue, the selection using D3P revealed sequences with higher copy numbers associated with the kidney compared to the tumour (Supp. Table S4 and S5). This observation might be due to a more rapid renal clearance of the non-pegylated (D3) sequences (MW ~25 kDa, which is below the cut-off threshold of the renal glomerulus31) in comparison to the pegylated sequences (D3P, MW ~36 kDa). We subjected all 46 sequences to an initial in vivo screening procedure using variants of the sequences bearing a near-infrared fluorescent dye (Alexa Fluor 680) at their 3′-ends. Sequences obtained from the library D3P were additionally equipped with a 5′–11 kDa PEG moiety. Scientific Reports \| (2019) 9:4976 \| https://doi.org/10.1038/s41598-019-41460-2 Results Th The sequences were evaluated using fluorescence reflectance imaging (FRI) of mice that bear orthotopic and subcutaneous xenograft tumours. The latter was necessary since the renal clearance of oligonucleotides hampers differentiation of sequences enriched in the orthotopic prostate tumours from those eliminated through the bladder. Two nanomole of the individual sequences were injected in the tail vein of anesthetised mice and whole-body FRI of the dorsal side view was performed before, 5, 60, and 180 minutes post injection. Subsequently, all animals were euthanised and several organs, including the ortho- topic and the subcutaneous tumours, were harvested and analysed by ex vivo FRI. An initial screening using a sin- gle mouse per individual sequence was performed. Each sequence was evaluated for tumour targeting comparing Scientific Reports \| (2019) 9:4976 \| https://doi.org/10.1038/s41598-019-41460-2 3 www.nature.com/scientificreports/ Figure 2. Next-generation sequence analysis of the DNA libraries D3 and D3P obtained by in vivo selection. (a) Analysis of the number of unique sequences within the obtained libraries from the in vivo selection using D3 (squares) and D3P (dots). The distribution of nucleotides over the 43 nucleobases of the initial random region of the starting library and the DNA libraries form the selection cycles 7 and 10 of the in vivo selection using D3 (b) and the distribution of nucleotides over the 43 nucleobases of the initial random region of the starting library and the DNA libraries from selection cycles 4 and 10 of the in vivo selection using D3P (c). Orange: dA, green: dC, blue: dG, and red: dT. Figure 2. Next-generation sequence analysis of the DNA libraries D3 and D3P obtained by in vivo selection Figure 2. Next-generation sequence analysis of the DNA libraries D3 and D3P obtained by in vivo selection. (a) Analysis of the number of unique sequences within the obtained libraries from the in vivo selection using D3 (squares) and D3P (dots). The distribution of nucleotides over the 43 nucleobases of the initial random region of the starting library and the DNA libraries form the selection cycles 7 and 10 of the in vivo selection using D3 (b) and the distribution of nucleotides over the 43 nucleobases of the initial random region of the starting library and the DNA libraries from selection cycles 4 and 10 of the in vivo selection using D3P (c). Orange: dA, green: dC, blue: dG, and red: dT. Results Th DNA libraries D3 and D3P obtained by in vivo selection Figure 2. Next-generation sequence analysis of the DNA libraries D3 and D3P obtained by in vivo selection. (a) Analysis of the number of unique sequences within the obtained libraries from the in vivo selection using D3 (squares) and D3P (dots). The distribution of nucleotides over the 43 nucleobases of the initial random region of the starting library and the DNA libraries form the selection cycles 7 and 10 of the in vivo selection using D3 (b) and the distribution of nucleotides over the 43 nucleobases of the initial random region of the starting library and the DNA libraries from selection cycles 4 and 10 of the in vivo selection using D3P (c). Orange: dA, green: dC, blue: dG, and red: dT. the mean fluorescence inside the subcutaneous tumour to the mean fluorescence measured in a healthy zone adjacent to the tumour (Suppl. Fig. S5a). No clear differences were measured at 5 and 60 minutes after injection, due to a high background fluorescence before clearance of non-bound sequences, but several sequences provided a two-fold higher fluorescence signal in the tumour compared to the healthy zone after 180 minutes (Fig. 4a,b and Suppl. Fig. S5b). These sequences were subjected to further testing, which included the sequences D3P-16, –6, and –7 that revealed a lower fluorescence ratio of the tumour vs. the normal tissue and considered as non- or weak-targeting sequences (Fig. 4b and Suppl. Fig. S5b). Of note, none of the sequences derived from the library D3 were found to target efficiently the subcutaneous tumours (Suppl. Figs S5c and S6).h gfi y ( pp g ) Furthermore, the calculated ratios obtained by FRI were ≤2 for most of the sequences (Suppl. Fig. S6). The repetition of the screening experiments with the sequences D3-39, -21, -34, and -27 revealed a high variation of obtained ratios and in some cases (D3-39 and D3-21) a strong fluorescence signal detectable throughout the whole mouse. Due to these findings, the D3-related sequences were decided not being further evaluated. Instead we focussed on the characterisation of the sequences obtained from the selection experiments using D3P. The majority of re-screened sequences obtained from the library D3P also showed a high variation and difference in their biodistribution, e.g. D3P-1, -4, -6, -7, -11, -16, -18, -19, -24, and -44 (Fig. 4b and Suppl. Fig. S5b). Results Th Orthotopic tumour targeting was also assessed by ex vivo fluorescence measurements comparing the fluorescence of prostatic tumours vs. muscle. However, this analysis was limited since half of the mice used during our screening did not Scientific Reports \| (2019) 9:4976 \| https://doi.org/10.1038/s41598-019-41460-2 4 www.nature.com/scientificreports/ Figure 3. Next-generation sequence analysis of the DNA population obtained from sequencing the individual DNA libraries of the indicated in vivo selection cycles. (a) Individual sequences were grouped according to their frequency in the libraries derived from each selection cycle. Upper panel refers to the libraries obtained from the in vivo selection using D3; lower panel refers to the libraries obtained from the in vivo selection using D3P. (b) The proportion of those sequences in the total population. Upper panel refers to the libraries obtained from the in vivo selection using D3; lower panel refers to the libraries obtained from the in vivo selection using D3P. Figure 3. Next-generation sequence analysis of the DNA population obtained from sequencing the individual DNA libraries of the indicated in vivo selection cycles. (a) Individual sequences were grouped according to their frequency in the libraries derived from each selection cycle. Upper panel refers to the libraries obtained from the in vivo selection using D3; lower panel refers to the libraries obtained from the in vivo selection using D3P. (b) The proportion of those sequences in the total population. Upper panel refers to the libraries obtained from the in vivo selection using D3; lower panel refers to the libraries obtained from the in vivo selection using D3P. Figure 4. Planar imaging of pegylated sequences. (a) Imaging of mice injected with 2 nmol of the D3P-library, D3P-44, and D3P-21 at different time points post injection (0, 5, 60, and 180 min). Dorsal view; exposure time 1000 ms. Arrows depicted in mice images at 180 min point the subcutaneous tumour. (b) Ratio of subcutaneous fluorescence signal obtained from the tumour tissue compared to surrounding tissue (please see also Suppl. Fig. S5a, left panel) of all indicated sequences 180 min post injection. (c) Ratio of the fluorescence signal of the orthotopic tumour ex vivo compared to muscle fluorescence (please see also Suppl. Fig. S5a, right panel) of all indicated sequences at 180 min post injection. Five mice did not bear an orthotopic tumour, wherefore no values are given in (c) for the sequences D3P-15, D3P-26, D3P-44, and D3P-library. Figure 4. Results Th The values obtained from the starting library (D3P-lib) (n = 3), and the aptamers D3P-10 (n = 5), D3P-20 (n = 6), as well as D3P- 21 (n = 5) were compared to all other sequences tested and pooled as “others” (n = 31). The aptamers D3P-21, D3P-P20 and D3P-P10 have a statistically significantly higher tumour to healthy tissue ratios (2.76 ± 0.09, 2.75 ± 0.49, and 2.48 ± 0.34, respectively) compared to the mice injected with the other aptamers (1.75 ± 0.09). In contrast, the difference obtained with the naïve library (D3P-lib) (2.400 ± 0.55) was not significant. Statistical significance was calculated using Graphpad Prism 6 using an unpaired t test model assuming that all data have the same standard deviation (SD). P < 0.01 and P < 0.001. develop orthotopic tumours in the prostate. Nevertheless, the fluorescence ratio between prostate tumour and muscle were ≥2 for most of the sequences tested and even higher ratios were obtained by D3P-10 and D3P-21 (Fig. 4c). Together with D3P-20, these two sequences revealed superior subcutaneous tumour targeting as well (Fig. 4b). Further testing of these sequences using additional subcutaneous mouse model samples revealed a more reproducible tumour targeting when compared to the starting library and all other analysed sequences (Fig. 5). Among them, D3P-21 was the most promising sequence as it showed an average fluorescence ratio of tumour to healthy tissue of 2.76 ± 0.09, which is higher and more reproducible compared to the average ratio obtained by all other sequences (1.75 ± 0.09).i q For further validation, the amount of aptamer localised to the tumour tissue was quantified by quantitative PCR (qPCR). Both orthotopic and subcutaneous tumours from mice treated with D3P-21 or the D3P-library were homogenised and the DNA extracted. The obtained DNA was then subjected to qPCR (Suppl. Fig. S7a). This analysis revealed a higher amount of D3P-21 recovered from subcutaneous tumours compared to the D3P-library (Suppl. Fig. S7a left). Interestingly, qPCR data also reveal that more copies of D3P-21 could be recovered from the orthotopic compared to the subcutaneous prostate tumour tissue (Suppl. Fig. S7a, right). The heterogeneity of the recovered DNA amounts might be explained by the fact that the samples of the test set were non-perfused (in contrast to the samples directly obtained from the selection procedures and which were used as sources to generate the results shown in in Figs 2, 3 and Supp. Results Th Planar imaging of pegylated sequences. (a) Imaging of mice injected with 2 nmol of the D3P-library, D3P-44, and D3P-21 at different time points post injection (0, 5, 60, and 180 min). Dorsal view; exposure time 1000 ms. Arrows depicted in mice images at 180 min point the subcutaneous tumour. (b) Ratio of subcutaneous fluorescence signal obtained from the tumour tissue compared to surrounding tissue (please see also Suppl. Fig. S5a, left panel) of all indicated sequences 180 min post injection. (c) Ratio of the fluorescence signal of the orthotopic tumour ex vivo compared to muscle fluorescence (please see also Suppl. Fig. S5a, right panel) of all indicated sequences at 180 min post injection. Five mice did not bear an orthotopic tumour, wherefore no values are given in (c) for the sequences D3P-15, D3P-26, D3P-44, and D3P-library. Scientific Reports \| (2019) 9:4976 \| https://doi.org/10.1038/s41598-019-41460-2 5 www.nature.com/scientificreports/ www.nature.com/scientificreports tificreports/ Figure 5. Validation of aptamers evolved from the DNA library D3P by planar imaging. The ratio of subcutaneous fluorescence signal obtained from the tumour tissue compared to surrounding tissue (please see also Suppl. Fig. S5a, left panel) of all indicated sequences 180 min post injection is given. The values obtained from the starting library (D3P-lib) (n = 3), and the aptamers D3P-10 (n = 5), D3P-20 (n = 6), as well as D3P- 21 (n = 5) were compared to all other sequences tested and pooled as “others” (n = 31). The aptamers D3P-21, D3P-P20 and D3P-P10 have a statistically significantly higher tumour to healthy tissue ratios (2.76 ± 0.09, 2.75 ± 0.49, and 2.48 ± 0.34, respectively) compared to the mice injected with the other aptamers (1.75 ± 0.09). In contrast, the difference obtained with the naïve library (D3P-lib) (2.400 ± 0.55) was not significant. Statistical significance was calculated using Graphpad Prism 6 using an unpaired t test model assuming that all data have the same standard deviation (SD). P < 0.01 and **P < 0.001. Figure 5. Validation of aptamers evolved from the DNA library D3P by planar imaging. The ratio of subcutaneous fluorescence signal obtained from the tumour tissue compared to surrounding tissue (please see also Suppl. Fig. S5a, left panel) of all indicated sequences 180 min post injection is given. Results Th (a) Flow cytometry analysis of the interaction of aptamer D3P-21 and the DNA library D3P (D3P-lib) with prostate cancer PC-3 cells, murine splenocytes, and murine peripheral blood mononuclear cells (PBMC) and other cancer cell lines (MCF7, H460, A549, and Ramos). Depicted is the ratio of binding of D3P-21 compared to the D3P-library (left panel) and the total percentage of cells bound by D3P-21 in (right panel). Cells were incubated with 200 nM of the D3P-21 or D3P-lib labelled at the 3′-end with Alexa Fluor 680. (b) Flow cytometry analysis of the interaction of D3P-21 and D3P-lib with the prostate cancer PC-3 and LNCaP cells. Shown is the ratio of binding of D3P-21 compared to D3P-lib (left l) d th t t l t f ll b d b D3P 21 ( i ht l) C ll i b t d ith 100 M f Figure 6. Evaluation of D3P-21 properties in vitro. (a) Flow cytometry analysis of the interaction of aptamer D3P-21 and the DNA library D3P (D3P-lib) with prostate cancer PC-3 cells, murine splenocytes, and murine peripheral blood mononuclear cells (PBMC) and other cancer cell lines (MCF7, H460, A549, and Ramos). Depicted is the ratio of binding of D3P-21 compared to the D3P-library (left panel) and the total percentage of cells bound by D3P-21 in (right panel). Cells were incubated with 200 nM of the D3P-21 or D3P-lib labelled at the 3′-end with Alexa Fluor 680. (b) Flow cytometry analysis of the interaction of D3P-21 and D3P-lib with the prostate cancer PC-3 and LNCaP cells. Shown is the ratio of binding of D3P-21 compared to D3P-lib (left panel) and the total percentage of cells bound by D3P-21 (right panel). Cells were incubated with 100 nM of the aptamer or D3P-lib labelled with Atto647N at the 3′-end. (c) Flow cytometry analysis of the impact of the PEG moiety of D3P-21 on its interaction properties. PC-3 cells were incubated with pegylated (D3P-21), non- pegylated D3P-21 (D3P-21non-PEG), or the non-pegylated D3P-library (D3P-lib non-PEG) labelled with Atto647N at the 3′-end and the ratio of binding is shown compared to D3P-lib. (d) Analysis of the impact of the PEG moiety on D3P-21 on PC-3 cell interaction using non-labelled DNA and qPCR for quantification. Shown is the ratio of recovered fmoles of the indicated oligodeoxynucelotide compared to D3P-lib. Represented as mean ± SD (n = 4). Results Th Figs S2–S4), resulting in residual amounts of blood remaining in the tissue that interfere in the analysis. y A pre-requisite for the potential therapeutic application of aptamers in vivo is their long-term stability in mammalian serum. As D3P-21 was selected in vivo, this inherently indicates that the sequence has certain nuclease resistance. To further explore its stability, its degradation in serum (Suppl. Fig. S7b, quadrants) and in Dulbeccos’s phosphate buffer saline (DPBS) (Suppl. Fig. S7b, circles) as a control were tested. These experiments revealed that after 1 h of incubation at 37 °C, 92.8% of the aptamer D3P-21 remain full length (Suppl. Fig. S7b, left panel) in serum, which is ~20% more compared to its non-pegylated variant (73.1%) (Suppl. Fig. S7b, right panel). This difference becomes less significant after 3 hours of incubation, after which 61.1% (D3P-21) and 50.8% (non-pegylated D3P-21) of the respective full-length aptamers were detected. These data are in line with the qPCR results that also project towards a good stability of D3P-21 in vivo. Of note, while the PEG moiety seems to increase the endurance of D3P-21 towards nucleases up to 3 h, extended incubation times revealed even more degradation of the pegylated variant compared to the non-pegylated aptamer. When incubated in DPBS, no deg- radation of the aptamer variants was observed.ti p After having identified the aptamer D3P-21 and validated its performance in vivo, the aptamer’s characteristics and properties in vitro were analysed. To characterise the binding properties of D3P-21 in vitro, flow cytometry studies were performed using PC-3 cells. In these experiments, the aptamer D3P-21 revealed a two-fold increase in binding to cultured PC-3 cells compared to the naïve D3P library (Fig. 6a and Suppl. Fig. S8). Subsequently, the interaction of D3P-21 with other cancer cell lines, i.e. A459, H460, MCF-7, Ramos, and the androgen-dependent prostate cancer cell line LNCaP was tested. Furthermore, peripheral blood mononuclear cell (PMBC) and sple- nocytes from mice were also investigated. Of note, for technical reasons oligonucleotides (ODN, aptamer and controls) labelled with Atto647N fluorophore were used in the flow cytometry assay with LNCaP cells, while Scientific Reports \| (2019) 9:4976 \| https://doi.org/10.1038/s41598-019-41460-2 6 www.nature.com/scientificreports/ www.nature.com/scientificreports /scientificreports/ Figure 6. Evaluation of D3P-21 properties in vitro. www.nature.com/scientificreports/ Next, the impact of the PEG moiety on the binding characteristics of D3P-21 was analysed. Therefore, the interaction of pegylated or non-pegylated D3P-21 with PC-3 cells was measured by flow cytometry (Fig. 6c and Suppl. Fig. S12) and qPCR (Fig. 6d) and compared to the D3 and D3P libraries, respectively. These experiments demonstrated a loss of binding performance of D3P-21 in the absence of the PEG moiety. Conversely, the binding of the D3 library to PC-3 cells was found to be independent of its pegylation status (Fig. 6c,d). y p p gy ( g , ) D3P-21 is a G-rich sequence (33.75%, Suppl. Table S5) and, thus, might be capable of folding into G-quadruplex structures32. We therefore analysed the dependence of cell binding of the aptamer on the presence of potassium ions as these are key to G-quadruplex structure stabilisation we analysed the dependence of cell binding of the aptamer on the presence of potassium ions as these are key to G-quadruplex structure stabili- sation33. The obtained results show that the interaction of D3-P21 with PC-3 cells depends on the presence of potassium ions in the binding buffer (Fig. 7a and Supp. Fig. 13). In order to elucidate whether D3P-21 forms a G-quadruplex structure and to analyse the impact of the PEG moiety on the conformation of D3P-21, circular dichroism (CD) spectroscopy experiments were performed. CD spectra of the aptamer were characterised by a positive peak at 273 nm (D3P-21) and 270 nm (non-pegylated D3P-21) and a negative peak at 244 nm (Fig. 7b, upper left (D3P-21) and right (D3P-21 non-PEG) panels). These typical CD profiles indicate that D3P-21 most likely contains a B-form conformation34. Notably, the absence or presence of potassium ions did not have an impact on the CD spectra, indicating that the conformation of D3P-21 is K+-independent. In contrast, the CD spectra of C10.36, a previously described parallel G-quadruplex containing aptamer35, were found to be strongly affected by the absence or presence of K+ (Fig. 7b, lower left panel). Minor differences were observed in the CD spectra of the pegylated vs. the non-pegylated aptamer revealing that the 11 kDa PEG moiety has little or no effect on the aptamer’s conformation (Fig. 7b, lower right panel). These data also indicate, that D3P-21 most likely does not fold into a common G-quadruplex structure. Discussion l d Nuclease-mediated degradation and rapid renal filtration of nucleic acids categorises the in vivo selection of aptamers highly challenging. To date, a few examples of in vivo selected aptamers are described employing 2′-deoxy-2′-fluoro pyrimidine (2′-fluoro)-modified RNA molecules38–40 and more recently, with a phosphoro- thioate containing DNA library41. 2′-fluoro RNA is considered to be more resistant toward serum nucleases and less prone to elicit innate immune responses31. Likewise, the majority of aptamers investigated for therapeutic approaches are built from 2′-fluoro RNA42–44. Exceptions are thrombin inhibiting aptamers and AS1411, all of which were shown to fold into highly stable G-quadruplex structures. These G-quadruplexes are investigated in vivo for the treatment of coronary artery syndrome or acute myeloid leukaemia45–47. In comparison to DNA molecules, 2′-fluoro RNA holds a series of disadvantages, i.e. very cost demanding and, thus, more difficult to engineer and optimise. In addition, 2′-fluoro RNA aptamers extending 50 nucleotides in length are still difficult to synthesise chemically and in larger quantities, as required for therapeutic approaches10. Thus, a minimisation of the active aptamer might be required before subjection to further therapeutic development. These characteristics make 2′-fluoro RNA challenging to be employed for basic research and drug development. Alternatively, DNA is affordable but branded as unstable and to evoke immune responses, e.g. mediated by the Toll-like receptor superfamily21. To this end, neither a systemic investigation of the inherent capability of DNA aptamers to activate the innate immune system nor their eligibility for in vivo selection approaches has been studied comprehensively.i y g y pp p y Herein, an in vivo selection procedure is described that enabled the identification of prostate tumour recog- nising DNA aptamers. An orthotopic xenograft mouse model was applied during the selection and validation procedure to secure tissue penetration and recognition of the relevant microenvironment of the tumour by the resultant aptamers. Orthotopically implanted xenograft tumour models are considered to represent one of the most accurate and available mimics of the tumour microenvironment in regard to a patient’s clinical situation. The androgen-independent PC-3 cell line was used for implantation, which is hormone independent and PSA negative. Hormone refractory prostate tumours are considered being non-curable yet26.h The overall results of the in vivo selection experiments and the fact that the best performing aptamer, D3P-21, was enriched from a pegylated DNA library indicate that such libraries reveal superior performance compared to a naïve DNA library. www.nature.com/scientificreports/ q p Finally, the impact of D3P-21 on the innate immune system was evaluated since previous studies suggest that DNA aptamers activate the immune system, mainly mediated by the Toll-like receptor (TLR) superfamily21. To determine D3P-21’s potential in this regard, the secretion of TNFα by immortalised murine embryonic stem cell-derived macrophages upon aptamer treatment was measured36. The results demonstrated a very low immu- nogenicity by D3P-21 and its non-pegylated variant at concentrations up to 3 µM (Fig. 8a,b). In contrast, the D3P-library induced TNFα secretion at concentrations above 0.375 µM. This finding is not surprising as the initial library contains ~1015 sequences, some of which most likely sharing structural motifs capable of TLR rec- ognition. However, the activation of the innate immune response by the DNA library was found to be >3 orders of magnitude less pronounced compared to controls, i.e. CpG and LPS37. These data indicate that D3P-21 does not activate the innate immune system. Results Th ODNs (aptamer and controls) labelled with Alexa Fluor 680 were employed for the other cell lines. These exper- iments revealed that D3P-21 interacts with the lung cancer cell lines A549 and H460 (lung carcinoma), while no binding to splenocytes, PMBCs, Ramos (Burkitt’s lymphoma), and MCF7 (breast cancer) cells was observed (Fig. 6a and Suppl. Fig. S8). Notably, binding to androgen-dependent prostate cancer LNCaP cells was also not observed (Fig. 6b and Suppl. Fig. S9). Other aptamer candidates tested (D3P-20, D3P-10, D3-28, D3-39, and D3-N1) showed no binding to PC-3 cells in vitro (Suppl. Figs S10a,b, S11a,b). Scientific Reports \| (2019) 9:4976 \| https://doi.org/10.1038/s41598-019-41460-2 7 www.nature.com/scientificreports/ Discussion l d The tested sequences obtained from the selection using the naïve DNA library did not reveal a consistently performing aptamer yet. However, it cannot be excluded that a well-performing aptamer could still be identified from this library as the enriched diverse population may contain these sequences albeit at very low abundance and the tools yet applied to analyze the library are not sufficient to reveal them. The continuation of the in vivo selection process might further enrich targeting sequences from this library as well. Likewise, D3P-21 was ranked on position 17 of all identified sequences regarding copy numbers and frequency within the enriched pegylated DNA library. Noteworthy, the most abundant sequences were not found to be the best targeting aptam- ers. This observation can be explained by a higher circulation half-life, targeting of non-tumour related structures, or by being more prone to PCR replication and thereby biasing the sequence populations48. Scientific Reports \| (2019) 9:4976 \| https://doi.org/10.1038/s41598-019-41460-2 8 www.nature.com/scientificreports/ / p p / Figure 7. Impact of potassium ions on the interaction D3P-21 with PC-3 cells. (a) Flow cytometry analysis to monitor the influence of potassium in the binding of aptamer D3P-21 to PC-3 cells. Shown is the ratio of binding of D3P-21 compared to D3P-lib (left panel) and the total percent of cells bound by D3P-21 (right panel). The cells were incubated with 100 nM of the aptamer labelled with Atto647N at the 3′-end. Represented as mean ± SD (n = 4). (b) CD spectroscopy analysis of D3P-21. CD spectra of D3P-21 (upper left panel), non-pegylated D3P-21 (D3P-21 non-PEG) (upper right panel), and C10.36 (lower left panel) under different conditions, i.e. H2O, PBS w/o potassium ions (pH 7.4), and PBS with increasing concentrations of potassium ions as indicated. Comparison of the obtained CD spectra of different aptamers and variants at a concentration of 4 mM potassium ions (lower right panel). Figure 7. Impact of potassium ions on the interaction D3P-21 with PC-3 cells. (a) Flow cytometry analysis to monitor the influence of potassium in the binding of aptamer D3P-21 to PC-3 cells. Shown is the ratio of binding of D3P-21 compared to D3P-lib (left panel) and the total percent of cells bound by D3P-21 (right panel). The cells were incubated with 100 nM of the aptamer labelled with Atto647N at the 3′-end. Represented as mean ± SD (n = 4). (b) CD spectroscopy analysis of D3P-21. Discussion l d CD spectra of D3P-21 (upper left panel), non-pegylated D3P-21 (D3P-21 non-PEG) (upper right panel), and C10.36 (lower left panel) under different conditions, i.e. H2O, PBS w/o potassium ions (pH 7.4), and PBS with increasing concentrations of potassium ions as indicated. Comparison of the obtained CD spectra of different aptamers and variants at a concentration of 4 mM potassium ions (lower right panel). A rapid renal clearance of the naïve DNA library (D3) compared to a PEG-modified variant might also account for unsuccessful use during the in vivo selection approach. Renal clearance is due to the average molecu- lar weight of D3 of ~25 kDa, which is below the cut-off threshold of the renal glomerulus31. In turn, an increased circulation time of the PEG-modified library might enhance the exposure time of candidate sequences to the relevant tumour tissue. Thus, under this scenario the enrichment of specifically associated aptamers is favoured compared to enriching for those sequences with simply higher resistance towards nuclease degradation, e.g. because of appropriate structure formation. This effect was intended by the addition of the 11 kDa PEG moiety to the D3P-library, thereby increasing the library’s molecular weight to ~ 36 kDa, which is in the range of the renal glomerulus threshold31. It has also been demonstrated that pegylation could induce steric repulsion of plasma Scientific Reports \| (2019) 9:4976 \| https://doi.org/10.1038/s41598-019-41460-2 9 www.nature.com/scientificreports/ Figure 8. Evaluation of aptamer immunogenicity. (a) Increasing concentrations of D3P-library (grey line), D3P-21 (blue), D3P-21 non-pegylated (green), CpG ODN 1826 type B (dark grey) and LPS (black) were incubated with immortalized murine embryonic stem cell-derived macrophages for 24 h and concentration of TNF-alpha in the supernatant was determined by HTRF assay. Results without CpG ODN and LPS are depicted in (b). Represented as mean ± SEM (n = 5). Figure 8. Evaluation of aptamer immunogenicity. (a) Increasing concentrations of D3P-library (grey line), D3P-21 (blue), D3P-21 non-pegylated (green), CpG ODN 1826 type B (dark grey) and LPS (black) were incubated with immortalized murine embryonic stem cell-derived macrophages for 24 h and concentration of TNF-alpha in the supernatant was determined by HTRF assay. Results without CpG ODN and LPS are depicted in (b). Represented as mean ± SEM (n = 5). proteins and decrease phagocytosis by the reticuloendothelial system49. It is unknown whether even larger PEG moieties would improve the selection outcome, but as PEG has recently been linked with side effects, i.e. Discussion l d allergy responses, anaphylactic reactions, and hypersensitivity in humans50–52, alternative tags such as lipids or protein carriers might also be worthwhile to investigate in the context of in vivo selection experiments53,54.t g g p D3P-21 showed consistent tumour targeting in the employed orthotopic and subcutaneous xenograft mouse models. The aptamer followed a constant enrichment profile, with steadily increasing frequency up to 0.94% in the DNA population obtained after 10 selection cycles. The maximal fold of amplification was seen from selec- tion cycle 3 to 4, while it was predominantly from cycle 2 to 3 for most of the highly abundant sequences. This finding underlines the importance of considering less frequent sequences for further characterisation instead of focussing only on the most abundant ones, as similarly described in previous literature reports55,56. Sophisticated population analysis procedures are of particular importance for aptamer selection approaches using complex target structures, e.g. cells, exosomes57, and tissues58, albeit still in its infancy but thought to develop rapidly alongside the availability and analysis of next-generation sequencing data59,60. The NGS-analysis further revealed that the sequence population from the last two selection cycles did not change dramatically, e.g. no new enriched sequences were detected within these populations. The data shown here underline NGS being an indispensable tool to track the evolution of DNA populations during in vivo selection procedures and for the identification of potential candidate aptamers. It might be supportive in detecting aptamers already in early selection cycles, thus reducing the entire number of selection cycles, which is of great importance when costly and time-consuming experiments as well as living animals are required. This study provides a comprehensive and unprecedented pop- ulation analysis of the progression of an in vivo selection process of DNA aptamers by NGS. In contrast, the few reported in vivo selection procedures relied on standard cloning and Sanger sequencing38,40 of ~50 sequences for identifying aptamer candidates and only one study performed Sanger sequencing in comparison with deep sequencing39. However, owing to the nature of Sanger sequencing this procedure most probably reveals the most abundant sequences, which as implied by this study might limit the success rate.f Strong heterogeneity between different mice treated with the same candidate aptamer was observed for many of the tested sequences. This reflects the challenge of an in vivo screening approach, which was manifested also by different degrees of metastasis, i.e. Scientific Reports \| (2019) 9:4976 \| https://doi.org/10.1038/s41598-019-41460-2 Discussion l d to improve the pharmacokinetic properties of aptamers42,63,64. The introduction of a PEG moiety in these aptam- ers did not affect their affinity to its target molecules significantly, however minor differences in binding were observed depending on the nature (linear or branched) and size of the PEG moiety65. p g y Targeting of tumour tissue in its native environment is a major challenge in cancer therapy in general and in particular for respective organs that are difficult to treat, e.g. prostate and pancreas. Aptamers represent an excellent compound class for achieving this. They can be selected in vivo, thereby optimally adapted towards their desired application38–40. Likewise, aptamers can also be selected in vitro, e.g. targeting cultured tumour cells or homogenously purified extracellular domains of proteins normally found on tumour cell surfaces66. The latter is greatly represented by a 2′-fluoro RNA aptamer that binds to prostate-specific membrane antigen (PSMA) and which has been shown to be valuable for targeting therapies in mice63,67–70. Which of the strategies of aptamer generation, i.e. in vivo and/or in vitro will be more straight forward in identifying aptamers with efficient targeting properties in a clinical setting though remains elusive yet.t p p g g y In conclusion, the data in this study show that murine orthotopic xenograft models are compatible with in vivo selection experiments. As these model systems reflect the natural microenvironment of tumours in cancer biology very well, the resultant aptamers may bear great potential to be further developed as targeted therapeutic regimen. This finding also opens the path towards many more applicable orthotopic model systems, e.g. mela- noma, breast cancer, colon cancer, pancreatic cancer, non-small cell lung cancer, and even metastasis model sys- tems71–74, with which the developed procedure is compatible. The obtained data essentially illustrate the benefit of combining in vivo selection with high-throughput sequencing and thorough data analysis. The more sequencing data on further in vivo selection studies will be available the more knowledge on the evolution of sequences will be gained, which will support to make more direct hits on aptamers from these endeavours. We performed a comparative in vivo study, head-to-head evaluating pegylated vs. non-pegylated DNA libraries and conclude that the presence of PEG entities enhances selection success. Material and Methods Ethics Statement. All animal use procedures for subcutaneous PC-3 tumour models and in vivo fluores- cence imaging were in strict accordance with the recommendations of the European Community (86/609/CEE) and the French National Committee (décret 87/848) for the care and use of laboratory animals. Ethics committee of CETEA – CEA DSV (Comité d’Ethique en Expérimentation Animale (CETEA), de la Direction des Sciences du Vivant (DSV) du Commissariat à l’Energie Atomique et aux énergies alternatives (CEA)) approved the study (ref: 12-093). Experimental protocols for performing in vivo orthotopic PC-3 models and Aptamer in vivo selection studies had been approved by the Ethics Committee for Animal Experimentation, and were registered by the Regierungspräsidium Freiburg (G-12/62), Germany. The animal experimentation is in accordance with the European Community guideline 2010/63/EU. Orthotopic PC-3 tumour model. On Day 0.3·106 PC-3 tumour cells expressing luciferase in 15 µl PBS were implanted orthotopically into 20 male NMRI nude mice. To prevent pain, Meloxicam (Metacam, 1 mg/kg, s.c.) was applied 1 h prior to surgery and 24 h post implantation. Male NMRI nude mice were anesthetised in a separate box using 1.5–2 Vol% Isoflurane with an oxygen flow of 0.6 l/min. The mice were positioned on a heated operating table with the left side upwards. The skin was cleaned, shaved and sterilised. An incision of approx. 1 cm was made in order to display the seminal vesicle and the prostate. A cell suspension of 3·106 PC-3Luc cells in 15 µl PBS was injected orthotopically into the prostate using a 29 G needle syringe. The seminal vesicle and the prostate were carefully pushed back into the visceral cavity and the abdominal wall closed by suturation. Thereafter the mouse was warmed in a separate box while recovering from anaesthesia. In the following, animal weights were measured three times weekly (Monday, Wednesday and Friday). Subcutaneous PC-3 tumour model. For in vivo screening experiments, mice were subcutaneously injected between shoulder blades with 3·106 PC-3 cells in a volume of 200 ml of Matrigel (BD Bioscience, Le Pont de Claix, France) and phosphate- buffered saline (PBS) (50:50). Tumours were then allowed to grow for 3–5 weeks until a size around 300 mm3 before in vivo imaging experiments. During each injection and imaging experiments, mice were anesthetised with isoflurane–1.25% in a 1:3 mixture of O2 and air. Discussion l d in the bones, pancreas, liver, or abdomen between mice bearing orthotopic xenograft prostate tumours potentially affecting the biodistribution of aptamers. Nevertheless, aptamer D3P-21 revealed very low heterogeneity in vivo, when targeting the subcutaneous and orthotopic tumours. D3P-21 also interacts with cultured PC-3 cells in vitro, which in turn demonstrates the capacity of this aptamer to recognise a target that is present on the tumour cells in culture and in the tumour microenvironment in vivo. Besides PC-3 cells, D3P-21 also recognises lung cancer cell lines, e.g. A549 and H460, whereas the breast cancer cell line MCF-7 and the androgen receptor and PSA positive prostate cancer cell line LNCaP were not bound. This finding is in line with recent data on mRNA expression profiles of various cell lines that reveal A459 cells clustering with PC-3 but not MCF-761. This behaviour of D3P-21 is in accordance with previous studies that show aptamers selected by cell-SELEX recognising other cell lines than the one employed for the selection itself18 and specificity for a defined tumour tissue was not part of the in vivo selection regimen. No interaction of D3P-21 with murine splenocytes, PBMC’s, and human blood cancer cell lines, e.g. Ramos (Burkitt’s lymphoma), was detected. These data are in accordance with the in vivo selection setting, during which the DNA libraries when injected into the tail vein were ‘co-incubated’ with PBMCs, which most likely served as a parallel negative selection as only those DNA sequences associated with the tumour tissue were processed further. Together, these data indicate that D3P-21 is a tumour targeting aptamer with good specificity.h g g p g pi y The aptamer’s cell binding properties depend on the presence of the PEG moiety and potassium ions. CD spec- troscopy indicated that D3P-21 partially folds into a B-form helical structure but does not reveal a G-quadruplex. Scientific Reports \| (2019) 9:4976 \| https://doi.org/10.1038/s41598-019-41460-2 10 www.nature.com/scientificreports/ As the PEG moiety seems not to have a great impact on the aptamer’s folding it most likely directly interacts with the target structure of the cell surface. To the best of our knowledge, this is the first study demonstrating the successful selection of aptamers from a pegylated DNA library. The introduction of flanking moieties during selection has been proven successful compared to post-selection incorporation to avoid aptamer folding and binding issues62. Several literature examples are known in which the PEG was introduced post-selection, e.g. Discussion l d Finally, the study establishes DNA molecules and librar- ies for in vivo selection procedures and might revive the application of DNA aptamers, besides 2′–fluoro RNA aptamers and beyond highly stable G-quadruplex structures as applicable compound class for developing targeted therapies and/or for validating targeting strategies. The recently demonstrated therapeutic efficacy of chemically modified DNA aptamers, so-called SOMAmers75–77 and the lack of stimulating the innate immune system by D3P-21, a major drawback traditionally linked to DNA, further supports this notion. In particular, future work will be required to reveal the molecular target of D3P-21, e.g., using pull-down experiments from the PC3 cell line followed by LC-MS/MS analysis, the dependence on the PEG moiety, and whether the aptamer functions as vehicle for the specific delivery of cargo therapeutics, e.g. paclitaxel or antagomirs to prostate tumours. Material and Methods Subcutaneous injec- tion was performed 3 weeks after orthotopical implantation as previously described or in mice without orthotopic implantation. Scientific Reports \| (2019) 9:4976 \| https://doi.org/10.1038/s41598-019-41460-2 11 www.nature.com/scientificreports/ In vivo bioluminescence imaging. During the course of the study, tumour growth was monitored in vivo using bioluminescence imaging. For this purpose, 150 mg/kg D-Luciferin was injected intraperitoneally (i.p.) into the mice 7 min before anesthetisation. Light emission was measured 10 min post injection with a CCD-camera for 5 min using a NightOWL LB 981 bioluminescence imaging system (Berthold Technologies, Germany). In vivo bioluminescence imaging. During the course of the study, tumour growth was monitored in vivo using bioluminescence imaging. For this purpose, 150 mg/kg D-Luciferin was injected intraperitoneally (i.p.) into the mice 7 min before anesthetisation. Light emission was measured 10 min post injection with a CCD-camera for 5 min using a NightOWL LB 981 bioluminescence imaging system (Berthold Technologies, Germany). In vivo SELEX. All oligonucleotides, including DNA libraries and primers, were synthesised by Ella Biotech GmbH (Munich, Germany). Two separate 80-nt single-stranded DNA libraries, D3 and D3P, were used consisting in a 43-nt random region. D3P library contained an 11 kDa polyethylene glycol (PEG) moi- ety on the 5′-end. Both libraries were amplified by PCR using the following primers: forward primer (Fw) 5′-GCTGTGTGACTCCTGCAA-3′, with 5′–11kDa PEG moiety in the case of D3P library, and reverse primer (Rv-Pho) 5′-Phosphate-GGAGACAAGATACAGCTGC-3′). PCR reaction was performed by using GoTaq® G2 Flexi DNA Polymerase (Promega) and 1 µM of both Fw and Rv-Pho primers with the following cycling program (2 min 95 °C; 30 sec. 95 °C, 30 sec. 64 °C, 45 sec. 72 °C; hold 10 °C) in a Veriti 96 well thermal cycler (Applied Biosystems). In vivo selection was done using tumour-bearing animals. Mice were either treated intravenously with one of two aptamer libraries (D3 or D3P) or left untreated. Five nmol of libraries (consisting of one copy of each sequence) were injected in cycle 1 and the amount was reduced to 2, 1 and 0.5 nmol for cycles 2, 3 and 4 respec- tively. From cycle 5 to 10, 0.1 nmol of the libraries were injected. Prior injection, libraries were prepared in 110 µL of Dulbecco’s phosphate-buffered saline (DPBS) containing Ca++ and Mg++ (Gibco), denatured at 80 °C for 3 min and slowly cooled down to RT for proper folding. Material and Methods After 20 min, animals were perfused with DPBS, killed by cervi- cal dislocation and tumours and kidney were collected. Tumours and kidney were snap-frozen in liquid nitrogen and stored at −80 °C before recovering the nucleic acids from the tissues. Weight of the tumours and kidneys used during in vivo SELEX are summarised in Supp. Table S1. Extraction of the nucleic acids from 3 tumours and 3 kidneys from injected mice was performed by first homogenising the organs with a 7 mL dounce tissue grinder with large and small clearance pistils (Landgraf Laborsysteme HLL GmbH). To lyse the cells, TE-SDS Lysis buffer (0.1 M Tris-HCl pH 8, 1 mM EDTA pH 8, 0.5% SDS) supplemented with 0.5 µg/µL of Proteinase K (Roth) was used for D3 injected tumours or kidney, followed by 10 min incubation at 95 °C. For D3P library, buffers A1, A2 and A3 from NucleoSpin® Plasmid kit (Macherey-Nagel) were used for lysis of the cells followed by 10 min centrifugation at 4000 rcf to remove cellular debris. Purification of extracted oligonucleotides was performed by means of phenol/chloroform extraction and ethanol precipitation for D3 library, and by silica col- umns (DNA Clean & ConcentratorTM −500 (Zymo Research)) in the case of D3P library. A negative control was always included with tumours extracted from control mice (injected with DPBS) following the same procedure described above. Purified oligonucleotides were then re-dissolved in milliQ water and a first PCR amplification was performed. RNA digestion was performed before PCR amplification by using a 1:1 mixture of RNase T1 (Roche) and RNase A (Macharey-Nagel). Agarose gel (4%) purification was then performed in order to separate the library band from genomic DNA and primers with the NucleoSpin® Clean-Up kit. Further PCR amplifi- cation was then performed to reach the required amount of library the next cycle. All tissue homogenisations and PCR preparations were performed in two different PCR workstations (Peqlab) in order to avoid contami- nations. Single strand displacement of the purified PCR product was carried out by λ-exonuclease digestion in 1 × λ-exonuclease reaction buffer and 5000 U/mL of λ-exonuclease (Thermo Scientific). After 30 min incubation at 37 °C, λ-exonuclease was inactivated at 80 °C for 10 min. Subsequently the samples were purified with the NucleoSpin® Clean-Up kit using the NTC buffer and resulting DNA libraries were freeze dried and frozen prior usage for next selection cycle. Material and Methods Detailed selection conditions are summarised in Supp. Table S2. Next Generation Sequencing. After 10 selection cycles, samples from all cycles for both tumour and kidney of the two DNA libraries were prepared for next generation sequencing (NGS) analysis on Illumina HiSeq1500 platform following the protocol from Tolle et al.78. Shortly, a first PCR with index containing primers was performed. Those indexes allow the analysis of 12 different samples on the same round. After purification of the PCR product as described above, up to 12 different samples with different indexes were mixed with equal amounts of DNA, to a final amount of 2 µg DNA. Then, addition of adapter sequences by enzymatic ligation was performed according to the manufacturer by using TruSeq DNA PCR-Free Sample Preparation Kit LT (Illumina), following the steps “End Repair”, “Adenylation” and “Adapter Ligation”. Samples were then purified via agarose gel (2%) and silica based spin-columns, and eluted in resuspension buffer. Quantitative PCR was performed for library validation with the KAPA library quantification kit (Sigma-Aldrich) prior sequencing. Seventy-five base pair single end sequencing was carried out. Raw NGS data was analysed using the COMPAS (COMmonPAtternS) software. In vivo Planar NIR fluorescence imaging of candidate aptamers. Mice were housed under standard conditions with food and water ad libitum but using chlorophyll free diet 15 days before imaging in order to reduce autofluorescence signal of the animals. Imaging experiments and analysis were performed using a fluores- cence Diffuse Optical Tomography (fDOT) imaging system as previously described79. Basically, the acquisition of Fluorescence reflectance imaging (FRI) is based on the excitation of fluorophores by the LEDs (emitting light between 650 and 670 nm) placed above the animal and on the reception of the fluorescence signal using the CCD camera and a band-pass filter (730 ± 15 nm). The CCD camera is focused at the top surface of the animal. Prior to intravenous injection and imaging of the aptamers, solutions of all sequences containing 2 nmol were prepared in DPBS with calcium and magnesium. All sequences were then heated for 3 minutes at 80 °C, spin down, let to cool in ice for 3 minutes and store at room temperature. Prior to imaging, mice were anesthetised with 4% isoflurane gas. Afterwards the level of isoflurane concentration was lowered down to 2–2.5%. Material and Methods The natural auto-fluorescence of the mice was recorded just before injection and was further subtracted in order to obtain the Scientific Reports \| (2019) 9:4976 \| https://doi.org/10.1038/s41598-019-41460-2 12 www.nature.com/scientificreports/ accurate fluorescence signal from the injected fluorescent probes. Then, the fluorescent aptamers were injected in the tail vein using a 29 G (insulin-type) syringe in a volume of 100 µL. Fluorescence images of dorsal side view were acquired 5 min, 90 min and 180 min post injection. From experience, good contrast is obtained after exposition times of a few milli-seconds. Since aptamers are rapidly eliminated by the urinary pathway, the bio- distribution of aptamers in prostate tumours could not be measured by in vivo imaging. Therefore, animals were euthanised 3 h after injection and organ resection permitted ex vivo fluorescence analysis of tumours and muscles. Planar Image analysis. For the semi-quantitative analysis of fluorescence planar images, the ImageJ soft- ware (http://rsbweb.nih.gov/ij/) was used. The first step was to subtract the intrinsic background noise of the camera from each image acquired. Second step was to normalise the images to the same exposure time. An ROI was manually drawn, to delineate the tumour, based on the white images (photographs) that are always acquired before initialising the experiments. The mean of intensity in this region was subtracted from the mean of inten- sity in the same area before injection, which corresponds to the auto-fluorescence of the animal at time t0. Using normalised images as well, a ROI is manually drawn for each time to delineate a reference healthy area close to the tumour tissue. The tumour targeting of aptamers was evaluated by dividing the mean fluorescence from the tumour by the mean fluorescence from the healthy zone. For ex vivo analysis, the same protocol was used and the tumour/muscle ratios were calculated. qPCR of tissue. Orthotopic and xenograft tumours from mouse injected with Alexa Fluor 680 labeled D3P- 21 and control sequences D3P-library, D3P-4 and D3P-16 from the in vivo screening were homogenised and purified as described above for D3P library. The amount of extracted DNA was quantified with NanoDrop 2000C (Thermo Scientific) and for qPCR quantification samples were normalised to a same OD. Material and Methods Two µL sample of the extracted DNA were added to 18 µL of a PCR master mix, containing 1X GoTaq colorless buffer, 2 mM MgCl2, 0.2 mM dNTPs, 300 nM of non-modified reverse and forward primers, 1X SYBR Green I (Sigma Aldrich) and 2.5 U GoTaq polymerase. Thermal conditions were optimised to 10 min 95 °C followed by 40 cycles of 30 s at 95 °C, 30 s at 64 °C and 45 s at 72 °C. Thermal cycling was performed in an iCycler Thermal Cycler upgraded with the iQ5 real-time PCR detection system (Bio-Rad, Germany). DNA standards were included; 20–0.002 fmol in 1 to 10 dilution. Each sample and standard were run in duplicates. Cell culture. For the in vitro evaluation of D3P-21 aptamer, different cell lines were used. The tumour cell line PC-3 was obtained from ProQinase, Ramos (Burkitt’s lymphoma), A549 (human non-small cell lung cancer) and H460 (large cell lung cancer) were obtained from ATCC (American Type Culture Collection). MCF7 cells (breast cancer) were purchased from CLS (Cell lines service). Splenocytes and PMBC’s were obtained from the spleen and the blood, respectively, of C57/BL6J mouse strain (kindly provided by Dr. Sven Burgdorf from the LIMES Institute in Bonn). Ramos, MCF7, H460 and LNCaP cells were cultured in RPMI 1640 medium while PC-3 and A549 cells were cultured in DMEM, high glucose, GlutaMAXTM supplemented (ThermoFisher) both with 10% fetal bovine serum (Sigma) at 37 °C in humidified air containing 5% CO2, and maintained by routine passage every 2-3 days. Prior usage, cells were counted with a hemacytometer. Suspension cells were centrifuged 5 min at 200 rcf and the pellet was suspended in fresh medium to obtain a cell suspension with the desired densities. For adherent cells, 100000 cells/well in appropriate medium were seeded in 24 well plates 24 hours prior the assay and proper amount of LNCaP cells were seeded in T12.5 cell culture flasks 48 hours prior the assay. Binding assays. Flow cytometry assays were performed in a BD FACSCanto cytometer and qPCR assays with an iCycler Thermal Cycler upgraded with the iQ5 real-time PCR detection system (Bio-Rad, Germany). Flow cytometry. Material and Methods The spectra were recorded with 100 nm min-1 scanning speed and 4 accumulations. Na2HPO4·H2O and 3 mM NaH2PO4·2H2O, pH 7.4), and increasing concentrations of KCl (0.1, 1, 4 and 10 mM). The spectra were recorded with 100 nm min-1 scanning speed and 4 accumulations. TNF-α HTRF assay. The TNF-α homogeneous time-resolved fluorescence (HTRF) assay was performed in accordance with the manufacturer guidelines (Cisbio). Briefly, immortalised murine embryonic stem cell-derived macrophages in 96-well plates were treated with increasing concentrations of D3P-library and aptamers D3P-21 and D3P-20, both containing or lacking of the 5′–11 kDa PEG moiety, for 24 hours. CpG oligonucleotide and LPS were used as positive controls. The cell supernatants were collected and stained with anti-TNF-α antibodies conjugated to FRET molecules. Changes in the fluorescence emission spectrum were proportional to the TNF-α concentration. TNF-α HTRF assay. The TNF-α homogeneous time-resolved fluorescence (HTRF) assay was performed in accordance with the manufacturer guidelines (Cisbio). Briefly, immortalised murine embryonic stem cell-derived macrophages in 96-well plates were treated with increasing concentrations of D3P-library and aptamers D3P-21 and D3P-20, both containing or lacking of the 5′–11 kDa PEG moiety, for 24 hours. CpG oligonucleotide and LPS were used as positive controls. The cell supernatants were collected and stained with anti-TNF-α antibodies conjugated to FRET molecules. Changes in the fluorescence emission spectrum were proportional to the TNF-α concentration. References Barciszewski) (Springer, Berlin, Heidelberg, 2014).h p g g 12. Mayer, G. The Chemical Biology of Aptamers. Angewandte Chemie International Edition 48, 2672–2689 (2009). h 13. Alibolandi, M. et al. Smart AS1411-aptamer conjugated pegylated PAMAM dendrimer for the superior delivery of camptothec colon adenocarcinoma in vitro and in vivo. 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Scientific Reports \| (2019) 9:4976 \| https://doi.org/10.1038/s41598-019-41460-2 References J Biol Methods 2 (2015).fi 27. Fizazi, K. et al. High efficacy of docetaxel with and without androgen deprivation and estramustine in preclinical models of a prostate cancer. Anticancer Res 24, 2897–2903 (2004). p 28. Healy, J. M. et al. Pharmacokinetics and biodistribution of novel aptamer compositions. Pharm Res 21, 2234–2246 (2004).h 29. Drolet, D. W., Green, L. S., Gold, L. & Janjic, N. Fit for the Eye: A 29. Drolet, D. W., Green, L. S., Gold, L. & Janjic, N. Fit for the Eye: Aptamers in Ocular Disorders. Nucleic Acid Ther 26, 127 146 (2016). 30. Blank, M. Next-Generation Analysis of Deep Sequencing Data: Bringing Light into the Black Box of SELEX Experiments. Methods Mol Biol 1380, 85–95 (2016). Mol Biol 1380, 85 95 (2016). 1. Zhou, J. & Rossi, J. Aptamers as targeted therapeutics: current potential and challenges. Nat Rev Drug Discov 16, 181–202 (2017). 31. Zhou, J. & Rossi, J. 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Recognition of nucleic acid and nucleic acid analogs by Toll-like receptors 7, 8 and 9. Immunobiology 213, 315–328 (2008). p gy ( ) 38. Mi, J. et al. In vivo selection of tumor-targeting RNA motifs. Nature Chemical Biology 6, 22–24 (2010).i 39. Cheng, C., Chen, Y. H., Lennox, K. A., Behlke, M. A. & Davidson, B. L. In vivo SELEX for Identification of Brain-penetrating Aptamers. Molecular Therapy - Nucleic Acids 2, e67 (2013).tih ph py 0. Mi, J. et al. Material and Methods For studying the interaction of D3P-21 aptamer to different cancer cell lines, 3′-Alexa Fluor 680 labeled D3P-21 and control library D3P-library were used whereas 3′-Atto647N labeled oligonucleotides were used to study the 11-kDa PEG moiety influence in the aptamer binding and the interaction of D3P-21 aptamer to both prostate cancer cell lines PC-3 and LNCaP. Cells were incubated with 100 or 200 nM of D3P-21 aptamer or D3P-library control in 200 µL of binding buffer or 750 µL for LNCaP cells (DPBS with 0.49 mM MgCl2, 0.9 mM CaCl2 and 0.5 mg/mL salmon sperm) for 30 minutes at 37 °C and 5% CO2. Then, cells were washed 3 times with washing buffer (DPBS with 0.49 mM MgCl2, 0.9 mM CaCl2) via centrifugation at 200 g for 5 minutes at room temperature for suspension cells, and with scraping of the adherent cells in the last washing step followed by centrifugation for volume reduction. For LNCaP cells, CaCl2 was removed from the last washing step in order to prevent clumping of the cells. For each measurement, 10000 cells were analysed in the flow cytometer. The data was analysed using FlowJo software. qPCR. The same incubation protocol as used for flow cytometry analysis for the individual sequences/library was followed. After 3 washing steps, cold ddH20 was added and cells were incubated at 4 °C for 30 min. Cells were then recovered from the well plate, heated at 95 °C for 5 min, and diluted to 5 cells/µL for analysis via qPCR. qPCR protocol was identical to the one described above (qPCR of tissue section). Stability of D3P-21 sequence. Two µM D3P-21 aptamer either bearing or lacking the 11-kDa PEG moiety in the 5′-end were incubated in human serum and DPBS containing calcium and magnesium at 37 °C. Samples were collected at different time points (0, 1, 3 and 18 hours) and intact DNA was quantified with qPCR as described above. Human serum was kindly provided by Dr. Jens Müller from the University Hospital Bonn. CD Spectroscopy studies. CD spectra were recorded at 20 °C with a Jasco J-810 spectrophotometer. The measurements were performed with 8 µM of DNA oligos in water, PBS without potassium (130 mM NaCl, 7 mM Scientific Reports \| (2019) 9:4976 \| https://doi.org/10.1038/s41598-019-41460-2 13 www.nature.com/scientificreports/ Na2HPO4·H2O and 3 mM NaH2PO4·2H2O, pH 7.4), and increasing concentrations of KCl (0.1, 1, 4 and 10 mM). References Chemically Modified Interleukin-6 Aptamer Inhibits Development of Collagen-Induced Arthritis in Cynomolgus Monkeys. Nucleic Acid Ther 26, 10–19 (2016).l h 7. Mor-Vaknin, N. et al. DEK-targeting DNA aptamers as therapeutics for inflammatory arthritis. Nat Commun 8, 14252 (2017). 8 Tolle F & Mayer G In Nucleic Acid Aptamers: Selection Characterization and Application (ed Günter Mayer) 77 84 (Springer Ne 77. Mor-Vaknin, N. et al. DEK-targeting DNA aptamers as therapeutics for inflammatory arthritis. Nat Commun 8, 14252 (2017). 78. Tolle, F. & Mayer, G. 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Polyethylene Glycol (PEG)-Induced Anaphylactic Reaction During Bowel Preparation. ACG Case Rep J 2, 216 (2015). 51. Wenande, E. C., Skov, P. S., Mosbech, H., Poulsen, L. K. & Garvey, L. H. References Optimizing aptamer activity for gene therapy applications using expression cassette SELEX. Mol Ther 6, 30–34 (2002). h 3. Dassie, J. P. et al. Systemic administration of optimized aptamer-siRNA chimeras promotes regression of PSMA-expressing tumors Nat Biotechnol 27, 839–849 (2009). 4. Diener, J. L. et al. Inhibition of von Willebrand factor-mediated platelet activation and thrombosis by the anti-von Willebrand facto A1-domain aptamer ARC1779. J Thromb Haemost 7, 1155–1162 (2009).i ph 5. Da Pieve, C., Williams, P., Haddleton, D. M., Palmer, R. M. & Missailidis, S. Modification of thiol functionalized aptamers by conjugation of synthetic polymers. Bioconjug Chem 21, 169–174 (2010).fi j g y y j g 6. Lupold, S. E., Hicke, B. J., Lin, Y. & Coffey, D. S. 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FASTAptamer: A Bioinformatic Toolkit for High-throughput Sequence Analys Combinatorial Selections Mol Ther Nucleic Acids 4 e230 (2015) 60. Alam, K. K., Chang, J. L. & Burke, D. H. FASTAptamer: A Bioinformatic Toolkit for High-throughput Sequence Analysis of Combinatorial Selections. Mol Ther Nucleic Acids 4, e230 (2015).h Combinatorial Selections. Mol Ther Nucleic Acids 4, e230 (2015).h h 61. Thul, P. J. et al. A subcellular map of the human proteome. Science (2017). h 61. Thul, P. J. et al. A subcellular map of the human proteome. Science (2017) h p p 62. Martell, R. E., Nevins, J. R. & Sullenger, B. A. Acknowledgementsh g This work has been made possible by funds from ERA-NET ‘Euronanomed’ to M.K., F.D. and G.M. (Grant acronym: META, Grant number: 13N12249). The authors also thank the European Commission for a Marie Curie Intra European fellowship to L.C. and BMBF (13N12247), J.L.S. and M.B. are members of the Excellence Cluster ImmunoSensation. J.L.S. was funded by the DFG (SFB704). We thank Silvana Hassel for critical reading the manuscript. Scientific Reports \| (2019) 9:4976 \| https://doi.org/10.1038/s41598-019-41460-2 15 www.nature.com/scientificreports/ Author Contributions G.M. designed the study, evaluated and discussed experiments and experimental results. L.C. and G.M. wrote the manuscript. L.C. and F.F. performed the in vivo SELEX steps were no animals were involved, the NGS analysis and the selection of sequences for in vivo screening as well as all in vitro studies. M.B. and J.L.S. performed sequencing, discussed experiments and experimental results and helped with writing the manuscript. C.G. and M.Bl. performed the data analysis of the NGS. I.T. and C.D. performed the in vivo screening of aptamers using fluorescence imaging. I.T. and F.D. designed the study on in vivo screening, evaluated and discussed experiments and experimental results. M.K. contributed to design of the in vivo selection studies and evaluated and discussed experiments and results. A.L. and H.W. performed the in vivo screening of aptamer libraries and discussed experiments and results. J.S. and E.L. evaluated the impact of the aptamer on the innate immune system. G.M. designed the study, evaluated and discussed experiments and experimental results. L.C. and G.M. wrote the manuscript. L.C. and F.F. performed the in vivo SELEX steps were no animals were involved, the NGS analysis and the selection of sequences for in vivo screening as well as all in vitro studies. M.B. and J.L.S. performed sequencing, discussed experiments and experimental results and helped with writing the manuscript. C.G. and M.Bl. performed the data analysis of the NGS. I.T. and C.D. performed the in vivo screening of aptamers using fluorescence imaging. I.T. and F.D. designed the study on in vivo screening, evaluated and discussed experiments and experimental results. M.K. contributed to design of the in vivo selection studies and evaluated and discussed experiments and results. A.L. and H.W. performed the in vivo screening of aptamer libraries and discussed experiments and results. J.S. and E.L. evaluated the impact of the aptamer on the innate immune system. Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-019-41460-2 Competing Interests: The authors declare no competing interests. Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps an 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) 2019 Scientific Reports \| (2019) 9:4976 \| https://doi.org/10.1038/s41598-019-41460-2 16
https://openalex.org/W4224241326	https://www.nature.com/articles/s41598-022-10454-y.pdf	English	null	Comparison of sequential CT arterioportography-arteriosplenography with standard cross-sectional imaging and endoscopy in children with portal hypertension	Scientific reports	2,022	cc-by	6,577	Simone Hammer1, Florian Zeman2, Hans Jürgen Schlitt3, Christian Stroszczynski1, Barbara Greiner1, Michael Christian Doppler4 & Wibke Uller4* In this study the diagnostic capability and additional value of sequential CT arterioportography– arteriosplenography (CT AP–AS) in comparison to standard cross-sectional imaging and upper gastrointestinal endoscopy (UGE) in pediatric portal hypertension (PH) was analyzed. Patients with clinical signs of PH who underwent CT AP–AS in combination with additional contrast-enhanced magnetic resonance imaging (CE-MR) and/or contrast-enhanced computed tomography (CE-CT) were included. Two radiologists reviewed independently imaging regarding the capability to prove patency of (1) extrahepatic and intrahepatic main stem portal vein (PV), (2) intrahepatic PV system and (3) splenomesenteric venous axis. Imaging was reviewed for detection of abdominal varices and results were compared to UGE. Main venous supply of varices (PV and/or splenic vein system) and splenorenal shunting were evaluated. 47 imaging studies (20 CT AP-AS, 16 CE-MR, 11 CE-CT) and 12 UGE records of 20 patients were analyzed. CT AP–AS detected significantly more splenorenal shunts (p = 0.008) and allowed more confident characterization of the extra-/intrahepatic PV-system and splenomesenteric veins in comparison to CE-MR (p < 0.001). Extra- and intrahepatic PV-system were significantly more confidently assessed in CT AP–AS than in CE-CT (p = 0.008 and < 0.001 respectively). CT AP–AS was the only modality that detected supply of varices and additional gastric/duodenal varices. In this retrospective study CT AP–AS was superior to standard cross-sectional imaging concerning confident assessment of the venous portosplenomesenteric axis in pediatric patients. CT AP–AS detected additional varices, splenorenal shunting and supply of varices. Biliary atresia and extrahepatic portal vein obstruction (EHPVO) with development of cavernous transforma- tion are the most common causes of PH in children1–3. The impaired hepatopetal blood flow from the susperior mesenteric vein (SMV) and splenic vein (SV) leads to congestion of the portomesenteric venous system with development of collateral pathways resulting in varices and portosystemic shunts. Splenomegaly and upper gastrointestinal variceal hemorrhage are the most common initial clinical manifestations of pediatric PH4,5. Management consists of preventing and controlling variceal hemorrhage by pharmacological and endoscopic treatment. Radiologic-interventional (transjugular intrahepatic portosystemic shunting) and surgical techniques (portosystemic shunting, liver transplantation) are alternative therapy options in case of endoscopic or phar- macologic treatment failure6. 1Department of Radiology, University Hospital Regensburg, Franz‑Josef‑Strauss‑Allee 11, 93053 Regensburg, Germany. 2Center for Clinical Trials, University Hospital Regensburg, Franz‑Josef‑Strauß‑Allee 11, 93053 Regensburg, Germany. 3Department of Surgery, University Hospital Regensburg, Franz‑Josef‑Strauss‑Allee 11, 93053 Regensburg, Germany. www.nature.com/scientificreports www.nature.com/scientificreports Comparison of sequential CT a rterioportography‑arteriosp lenography with standard cross‑sectional imaging and endoscopy in children with portal hypertension OPEN Simone Hammer1, Florian Zeman2, Hans Jürgen Schlitt3, Christian Stroszczynski1, Barbara Greiner1, Michael Christian Doppler4 & Wibke Uller4* Simone Hammer1, Florian Zeman2, Hans Jürgen Schlitt3, Christian Stroszczynski1, Barbara Greiner1, Michael Christian Doppler4 & Wibke Uller4* 4Department of Diagnostic and Interventional Radiology, Medical Center‑University of Freiburg, Faculty of Medicine, University of Freiburg, Hugstetter Straße 55, 79106 Freiburg, Germany. *email: wibke.uller@uniklinik-freiburg.de \| https://doi.org/10.1038/s41598-022-10454-y Scientific Reports \| (2022) 12:6554 www.nature.com/scientificreports/ Figure 1. Flowchart of the study cohort. Figure 1. Flowchart of the study cohort. Figure 1. Flowchart of the study cohort. Figure 1. Flowchart of the study cohort. In patients with recurrent gastrointestinal bleeding despite medical and endoscopic therapy7, cross-sectional imaging is essential for assessing the whole portomesenteric venous system as well as the SV for choosing adequate therapeutic management. The extent of PV thrombosis to the splanchnic veins and intrahepatic PV as well as patency of the SV, SMV, recessus of Rex and the intrahepatic branches of the PV are crucial to decide on the best therapy options1. py p Sequential CT AP–AS is based on sequential contrast application into the splenic artery (SA) and the supe- rior mesenteric artery (SMA) during CT and proved to evaluate individual venous hemodynamic changes in children with symptomatic PH8. This technique enables maximal contrast filling of the targeted abdominal veins, differentiation of the main supply of varices and determination of blood flow direction.h f pp yl The aim of this study was to assess the diagnostic capability of CT AP–AS regarding the patency of the portal and splenomesenteric veins in comparison to standard cross-sectional imaging (CE-CT and CE-MR). Moreover the present study was designed to analyze delineation of varices in CT AP–AS in comparison to standard cross- sectional imaging and upper gastrointestinal endoscopy (UGE). Results S d Study cohort. Between November 2011 and October 2020 38 CT AP–AS were conducted in 33 pediatric patients (flowchart of the study cohort, Fig. 1). 5 follow-up CT AP–AS were excluded. For 8 patients standard cross-sectional imaging was not available or standard-cross sectional imaging was outside the defined time- interval (18 months before or after CT AP-AS). Another 4 patients were excluded as they underwent surgical or radiological-interventional treatment between standard cross-sectional imaging and CT AP-AS. Severe artifacts in standard cross-sectional imaging led to exclusion of 1 patient. Cause of PH in the 13 excluded patients (n = 6 female; median age 8.5 years, range 2.5–15.7 years) was prehepatic in 10 cases and intrahepatic in 3 cases. 20 https://doi.org/10.1038/s41598-022-10454-y https://doi.org/10.1038/s41598-022-10454-y CT AP–AS performed in 20 patients were included in this study (n = 9 female; median age 13.6 years, range 1.7–18.4 years; median body weight 37.2 kg; range 14.1–80.6 kg). Etiology of PH was prehepatic in 15 cases and intrahepatic in 5 cases. 11 patients underwent CE-CT and 16 patients CE-MR (7 patients underwent both, CE-CT and CE-MR). Median time interval between CT AP–AS and standard cross-sectional imaging was −2.4 months (range −16.3–14.8 months). Medical records of UGE were available in 12 patients. Median time interval between CT AP–AS and UEG was −0.4 months (range −3.9–7.1 months). CT AP–AS performed in 20 patients were included in this study (n = 9 female; median age 13.6 years, range 1.7–18.4 years; median body weight 37.2 kg; range 14.1–80.6 kg). Etiology of PH was prehepatic in 15 cases and intrahepatic in 5 cases. 11 patients underwent CE-CT and 16 patients CE-MR (7 patients underwent both, CE-CT and CE-MR). Median time interval between CT AP–AS and standard cross-sectional imaging was −2.4 months (range −16.3–14.8 months). Medical records of UGE were available in 12 patients. Median time interval between CT AP–AS and UEG was −0.4 months (range −3.9–7.1 months). Comparison CT AP–AS and standard cross‑sectional imaging. Evaluation portosplenomesenteric veins. Results for diagnostic capability are shown in Table 1. CT AP–AS allowed confident characterization of the extra- and intrahepatic main stem PV (region 1) in all cases. Status of extrahepatic and intrahepatic main stem PV was significantly better characterized with CT AP–AS compared to CE-CT (p = 0.008) and CE-MR (p < 0.001).hi p The intrahepatic PV system (region 2) was confidently assessed in CT AP-AS, only in 1 patient the right peripheral intraheptic PV was assigned to the category "not reliably assessable" (52/53, 98.1%). Confident cat- egorization in CE-CT and CE-MR for the intrahepatic PV system (region 2) was possible for 25/53 intrahepatic portal veins (48.4%) and 41/72 intrahepatic portal veins (56.9%) respectively (p < 0.001 and < 0.001). Ability to determine status of the left central intrahepatic PV was significantly better at CT AP–AS compared to CE-CT (p = 0.041) and CE-MR (p = 0.023). Capability to diagnose status of the right central intrahepatic vein increased with use of CT AS-AP compared to CE-CT (p = 0.07) and CE-MR (p = 0.07). For determination of the left peripheral branches differences between CT AS-AP and CE-MR were significant (p = 0.023). https://doi.org/10.1038/s41598-022-10454-y Vessel CT AP-AS CE-CT p values CT AP-AS CE-MR p values (n = 11) (n = 16) Region 1 33/33 (100) 24/33 (72.7) 0.008 48/48 (100) 33/48 (68.8) < 0.001 Extrahepatic PV 11/11 (100) 8/11 (72.7) 0.24 16/16 (100) 12/16 (75.0) 0.13 Main stem intrahepatic PV 11/11 (100) 6/11 (54.5) 0.07 16/16 (100) 11/16 (68.8) 0.07 Cavernous transformation main stem PV 11/11 (100) 10/11(90.9) 1.00 16/16 (100) 10/16 (62.5) 0.041 Region 2 52/53 (98.1) 25/53 (48.4) < 0.001 72/72 (100) 41/72 (56.9) < 0.001 Left central intrahepatic PVa 11/11 (100) 5/11 (45.5) 0.041 16/16 (100) 9/16 (56.3) 0.023 Left peripheral intrahepatic PVb 11/11 (100) 7/11 (63.6) 0.13 16/16 (100) 9/16 (56.3) 0.023 Right central intrahepatic PVa, c 10/10 (100) 5/10 (50.0) 0.07 12/12 (100) 7/12 (58.3) 0.07 Right peripheral intrahepatic PVb, c 9/10 (90.0) 2/10 (20.0) 0.023 12/12 (100) 5/12 (41.7) 0.023 Cavernous transformation intrahepatic PV 11/11 (100) 6/11 (54.5) 0.07 16/16 (100) 10/16 (62.5) 0.041 Region 3 32/33 (97.0) 26/33 (78.8) 0.08 45/48 (93.8) 31/48 (64.6) 0.001 SMV 11/11 (100) 9/11 (81.8) 0.48 15/16 (93.8) 11/16 (68.8) 0.22 SV 11/11 (100) 9/11 (81.8) 0.48 16/16 (100) 13/16 (81.3) 0.25 SMV/SV confluence 10/11 (90.9) 8/11 (72.7) 0.63 14/16 (87.5) 7/16 (43.8) 0.023 Table 1. Diagnostic capability CT AP–AS versus CE-CT and CT AP–AS versus CR-MR (frequency of i Table 1. Diagnostic capability CT AP–AS versus CE-CT and CT AP–AS versus CR-MR (frequency of confident characterization per imaging modality and vessel). Values are number of studies, numbers in parentheses are percentages. p values are written in bold when statistically significant. a contrast enhancement within the right/left PV to first branching. b contrast enhancement within the expected course of the first order intrahepatic left/rigth PV extending peripherally. c n = 1 and n = 4 patients after split-liver transplantation in the CE-CT group and CE-MR group respectively. g p y q y confident characterization per imaging modality and vessel). Values are number of studies, numbers in parentheses are percentages. p values are written in bold when statistically significant. a contrast enhancement within the right/left PV to first branching. b contrast enhancement within the expected course of the first order intrahepatic left/rigth PV extending peripherally. c n = 1 and n = 4 patients after split-liver transplantation in the CE-CT group and CE-MR group respectively. https://doi.org/10.1038/s41598-022-10454-y Scientific Reports \| (2022) 12:6554 \| www.nature.com/scientificreports/ Vessel CT AP-AS CE-CT p values CT AP-AS CE-MR p values (n = 11) (n = 16) Region 1 33/33 (100) 24/33 (72.7) 0.008 48/48 (100) 33/48 (68.8) < 0.001 Extrahepatic PV 11/11 (100) 8/11 (72.7) 0.24 16/16 (100) 12/16 (75.0) 0.13 Main stem intrahepatic PV 11/11 (100) 6/11 (54.5) 0.07 16/16 (100) 11/16 (68.8) 0.07 Cavernous transformation main stem PV 11/11 (100) 10/11(90.9) 1.00 16/16 (100) 10/16 (62.5) 0.041 Region 2 52/53 (98.1) 25/53 (48.4) < 0.001 72/72 (100) 41/72 (56.9) < 0.001 Left central intrahepatic PVa 11/11 (100) 5/11 (45.5) 0.041 16/16 (100) 9/16 (56.3) 0.023 Left peripheral intrahepatic PVb 11/11 (100) 7/11 (63.6) 0.13 16/16 (100) 9/16 (56.3) 0.023 Right central intrahepatic PVa, c 10/10 (100) 5/10 (50.0) 0.07 12/12 (100) 7/12 (58.3) 0.07 Right peripheral intrahepatic PVb, c 9/10 (90.0) 2/10 (20.0) 0.023 12/12 (100) 5/12 (41.7) 0.023 Cavernous transformation intrahepatic PV 11/11 (100) 6/11 (54.5) 0.07 16/16 (100) 10/16 (62.5) 0.041 Region 3 32/33 (97.0) 26/33 (78.8) 0.08 45/48 (93.8) 31/48 (64.6) 0.001 SMV 11/11 (100) 9/11 (81.8) 0.48 15/16 (93.8) 11/16 (68.8) 0.22 SV 11/11 (100) 9/11 (81.8) 0.48 16/16 (100) 13/16 (81.3) 0.25 SMV/SV confluence 10/11 (90.9) 8/11 (72.7) 0.63 14/16 (87.5) 7/16 (43.8) 0.023 Table 1. Diagnostic capability CT AP–AS versus CE-CT and CT AP–AS versus CR-MR (frequency of confident characterization per imaging modality and vessel). Values are number of studies, numbers in parentheses are percentages. p values are written in bold when statistically significant. a contrast enhancement within the right/left PV to first branching. b contrast enhancement within the expected course of the first order intrahepatic left/rigth PV extending peripherally. c n = 1 and n = 4 patients after split-liver transplantation in the CE-CT group and CE-MR group respectively. https://doi.org/10.1038/s41598-022-10454-y Concerning the right peripheral PV, CT AS-AP provided the highest diagnostic capability (p = 0.023) in comparison with CE- CT and CE-MR (Figs. 2 and 3).fi Differences between CT AP–AS and CE-MR were significant concerning the diagnostic capability of extra- and intrahepatic cavernous transformation (p = 0.041). Cavernous transformation of the PV was equally diag- nosed at CE-CT and CT AP–AS with a trend towards higher diagnostic capability at CT AP–AS concerning intrahepatic cavernous transformation (p = 0.07).i p p CT AP–AS allowed confident assessment of the splenomesenteric veins (region 3) for 32/33 (97%) vessels in the CT AP–AS vs. CE-CT group and for 45/48 (93.8%) vessels in the CT AP–AS vs. CE-MR group. For region 3 confident diagnosis at CE-CT was possible for 26/33 (78.8%) veins and at CE-MR for 31/48 (64.6%) veins (p = 0.08 and 0.001 respectively). With respect to the SMV and SV CT AP-AS, CE-CT and CE-MR performed equally. Concerning the con- fluence, CT AP–AS and CE-CT also performed equally whereas CE-MR differed significantly from CT AP–AS results (p = 0.63 and 0.023 respectively). Evaluation varices and splenorenal shunting. CT AP–AS delineated significantly more varices compared to CE-CT: stomach (p = 0.041), duodenum (p = 0.041) and small intestinum (p = 0.023).i p p p In comparison to CE-MR CT AP–AS shows significantly more gastric (p = 0.002), duodenal (p = 0.013), small intestinal (p = 0.004) and colonic (p = 0.041) varices. There was no significant difference between CT AP–AS and https://doi.org/10.1038/s41598-022-10454-y Scientific Reports \| (2022) 12:6554 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 2. A 8-year-old female patient with PH. Coronal and axial images of CT-AP (a, b) and CE-CT (c, d). The left central and peripheral intrahepatic PV (arrows) were categorized "not reliably assessable" in CE-CT (c, d). CT-AP delineates the small, but clearly patent left intrahepatic PV. Figure 2. A 8-year-old female patient with PH. Coronal and axial images of CT-AP (a, b) and CE-CT (c, d). The left central and peripheral intrahepatic PV (arrows) were categorized "not reliably assessable" in CE-CT (c, d). CT-AP delineates the small, but clearly patent left intrahepatic PV. Figure 2. A 8-year-old female patient with PH. Coronal and axial images of CT-AP (a, b) and CE-CT (c, d). The left central and peripheral intrahepatic PV (arrows) were categorized "not reliably assessable" in CE-CT (c, d). CT-AP delineates the small, but clearly patent left intrahepatic PV. Figure 3. https://doi.org/10.1038/s41598-022-10454-y A 6-year-old male patient with EHPVO. Axial images of CT-AP (a, b) and CE-MR (c, d). The left central (arrows) and peripheral PV (arrowheads) were not confidently assessed in CE-MR, especially for the peripheral left PV CE-MR was non-diagnostic (circle in d). Figure 3. A 6-year-old male patient with EHPVO. Axial images of CT-AP (a, b) and CE-MR (c, d). The left central (arrows) and peripheral PV (arrowheads) were not confidently assessed in CE-MR, especially for the peripheral left PV CE-MR was non-diagnostic (circle in d). Figure 3. A 6-year-old male patient with EHPVO. Axial images of CT-AP (a, b) and CE-MR (c, d). The left central (arrows) and peripheral PV (arrowheads) were not confidently assessed in CE-MR, especially for the peripheral left PV CE-MR was non-diagnostic (circle in d). Scientific Reports \| (2022) 12:6554 \| https://doi.org/10.1038/s41598-022-10454-y www.nature.com/scientificreports/ Figure 4. Axial and coronal images of CT-AP of two 16-year-old male patients with congenital hepatic fibrosis (a, b) and hepatoportal sclerosis (c, d). CT-AP detected small duodenal varices not described in UEG (arrow) and small intestinal varices (arrowheads) not detectable in CE-CT. Figure 4. Axial and coronal images of CT-AP of two 16-year-old male patients with congenital hepatic fibrosis (a, b) and hepatoportal sclerosis (c, d). CT-AP detected small duodenal varices not described in UEG (arrow) and small intestinal varices (arrowheads) not detectable in CE-CT. standard cross-sectional imaging for esophageal/paraesophageal varices. Figure 4 illustrates small intestinal varices only detectable using CT AP-AS. CT AP–AS delineated main venous supply of varices (SMV versus SV system): esophagus (2/15 SMV, 8/15 SV, 5/15 both), paraesophageal space (3/14 SMV, 7/14 SV, 4/14 both), stomach (12/18 SV, 6/18 both), duodenum (9/10 SMV, 1/10 both), small intestinum (13/13 SMV), colon (9/9 SMV), rectum (3/8 SMV, 4/8 SV, 1/8 both). Significantly more spontaneous splenorenal shunts were detected in CT AP–AS compared to CE-MR (p = 0.008). CT AP–AS delineated main venous supply of varices (SMV versus SV system): esophagus (2/15 SMV SV, 5/15 both), paraesophageal space (3/14 SMV, 7/14 SV, 4/14 both), stomach (12/18 SV, 6/18 both), duo (9/10 SMV, 1/10 both), small intestinum (13/13 SMV), colon (9/9 SMV), rectum (3/8 SMV, 4/8 SV, 1/8 b Significantly more spontaneous splenorenal shunts were detected in CT AP–AS compared to C (p = 0.008). Comparison CT AP–AS and UEG. CT AP–AS identified 11/12 endoscopic described esophageal, 12/12 gastric and 2/2 duodenal varices. https://doi.org/10.1038/s41598-022-10454-y In 6/12 cases CT AP–AS showed additional varices in the fundus and corpus of the stomach (Fig. 5). Furthermore CT AP–AS detected in 2 patients small duodenal varices not described in UGE (Fig. 4). Comparison CT AP–AS and UEG. CT AP–AS identified 11/12 endoscopic described esophageal, 12/12 gastric and 2/2 duodenal varices. In 6/12 cases CT AP–AS showed additional varices in the fundus and corpus of the stomach (Fig. 5). Furthermore CT AP–AS detected in 2 patients small duodenal varices not described in UGE (Fig. 4). Discussion Enhancement of varices of the gastric cardia (arrowheads) and esophagus (white arrows) predmominates in CT-AS (d–f) in comparison to CT-AP (a–c). Enhancement of paraesophageal varices (open arrows) in both CT-AP (c) and CT-AS (f). In UEG varices at the gastric cardia were described, CT-AS (d, e) delineates additional small fundic varices (black arrows). Figure 5. A 15-year-old male patient with PH. Coronal and axial images of CT-AP (a–c) and CT-AS (d–f). Enhancement of varices of the gastric cardia (arrowheads) and esophagus (white arrows) predmominates in CT-AS (d–f) in comparison to CT-AP (a–c). Enhancement of paraesophageal varices (open arrows) in both CT-AP (c) and CT-AS (f). In UEG varices at the gastric cardia were described, CT-AS (d, e) delineates additional small fundic varices (black arrows). CE-CT in portalvenous phase depicts also the SV and renal veins. This is important when key criteria for the meso-Rex bypass procedure are not fulfilled and distal splenorenal shunting is an alternative. If CE-CT is not conclusive, CT-AP should be performed9. Shneider et al.6 proposed transjugular retrograde or percutaneous transhepatic portal venography, if CE-CT or CE-MR are non-diagnostic for the intrahepatic PV system. How- ever, this invasive technique lacks the possibility for postprocessing to generate 2D and 3D reconstructions and determination of the exact size of intrahepatic portal vein branches10. Parekh et al.11 showed that 4D flow MR imaging allows 3D visualization of the PV and quantification of PV hemodynamics in a small pediatric study cohort. However, since PV system was not assessed in detail and results were not compared to any other imaging modalities, the value of this technique in the workup of pediatric PH remains unclear.i CT AP–AS delineated significantly more varices of the stomach, duodenum and small intestinum compared to CE-CT and CT-MR despite standard cross-sectional imaging is suitable to depict upper gastrointestinal varices in adults and children with PH12–14. CT AP–AS displays excellent venous contrast due to the direct contrast injection into the SA and the SMA. In contrast to peripheral intravenous contrast injection dilution effects do not occur and accordingly, higher detection rate of varices results. The excellent delineation of abdominal varices and moreover, direction of flow is essential for surgical treatment planning. l g p g In contrast to standard cross-sectional imaging CT AP–AS revealed small varices in more uncommon loca- tions like the duodenum, small intestinum and colon. Discussion In comparison to standard cross-sectional imaging CT AP–AS allowed significantly more confident assessment of the extra- and intrahepatic PV system. Especially the left central and peripheral intrahepatic PV were more confidently characterized in CT AP–AS compared to CE-MR. In contrast to CE-CT CT AP–AS facilitates sig- nificantly more confident assessment of the intrahepatic portalvenous system as a whole (98.1% for CT AP–AS versus 48.4% for CE-CT) and in particular of the left central PV (100% for CT AP–AS versus 45.5% for CE-CT). The low performance of CE-CT regarding the intrahepatic portalvenous system is remarkable as, in our experi- ence, CE-CT is very reliable for the evaluation of the portalvenous system in the adult population. In pediatric patients, however the contrast-to-noise ratio (most probable due to insufficient contrast-medium timing) is often not sufficient for confident diagnosis. Furthermore, perfect timing in pediatric patients is very individual and volume of contrast is restricted to the body weight of the child. Our results regarding the assessment of the intrahepatic portalvenous system meet the findings of Kwan et al. They compared the diagnostic capability of different invasive/non-invasive imaging modalities (CE-CT, CE-MR, ultrasound, transsplenic portography, transhepatic portography, transarterial portography and CT-AP) for preoperative meso-Rex bypass planning in pediatric patients. CT-AP was available for only 6 cases, but interestingly, in contrast to all other imaging modalities CT-AP allowed confident assessment of the crucial structures for the meso-Rex bypass procedure (left intrahepatic PV, PV contiguity and SMV) in all cases. All but one of these patients (5/6) underwent non- diagnostic imaging studies prior to CT-AP. Therefore Kwan et al. concluded, that CT-AP should be the first or second line imaging modality for preoperative planning. They proposed CE-CT for first assessment, because https://doi.org/10.1038/s41598-022-10454-y Scientific Reports \| (2022) 12:6554 \| www.nature.com/scientificreports/ Figure 5. A 15-year-old male patient with PH. Coronal and axial images of CT-AP (a–c) and CT-AS (d–f). Enhancement of varices of the gastric cardia (arrowheads) and esophagus (white arrows) predmominates in CT-AS (d–f) in comparison to CT-AP (a–c). Enhancement of paraesophageal varices (open arrows) in both CT-AP (c) and CT-AS (f). In UEG varices at the gastric cardia were described, CT-AS (d, e) delineates additional small fundic varices (black arrows). Figure 5. A 15-year-old male patient with PH. Coronal and axial images of CT-AP (a–c) and CT-AS (d–f). www.nature.com/scientificreports/ CT AP–AS detected significantly more spontaneous splenorenal shunts compared to CE-MR. Three-dimen- sional CE-MR is a standard imaging technique suitable for assessment of portosystemic collaterals including spontaneous splenorenal shunts in children18,19. CT imaging like CT AP–AS however has higher spatial resolution and is less prone to motion artifacts which may explain the higher detection rate of splenorenal shunts in CT AP-AS. Additionally due to the direct contrast medium injection into the SA the renal veins are not opacified in CT AP-AS, consequently even very small splenorenal shunts are detectable. Spontaneous splenorenal shunt- ing is of utmost interest for interventional access routes and planning pediatric liver transplantation, as (large) splenorenal shunts can cause postoperative PV flow steal syndrome which may require ligation of the splenorenal shunt20. Recent studies evaluated the clinical relevance of spontaneous portosystemic shunts including spleno- renal shunting on CT imaging in adult liver cirrhosis and found an association of spontaneous portosystemic shunts with higher mortality and higher complication rate for PV thrombosis, hepatic encephalopathy and gas- trointestinal hemorrhage21,22. Equivalent studies for pediatric PH do not yet exist, but these findings underline the importance of a comprehensive imaging evaluation of patients with PH.hh p p g g p The major limitations of this study are its retrospective design and the small study cohort. There might be a selection bias because only complex patients underwent CT AS-AP and 13 patients were excluded owing to a lack of CE-CT or CE-MR (within the defined period). The defined time interval between standard cross-sectional imaging and CT AP–AS (18 months before or after CT AP-AS) was chosen arbitrarily and might seem rather long. Only complicated patients in need for surgical or radiologic-interventional treatment underwent CT AP–AS due to its invasive nature. In many cases CE-CT or CE-MR was done for initial diagnostics and CT AP–AS later in case of deterioration. As CT AP–AS delivers all therapy-relevant information, standard cross-sectional imaging was often not repeated. Patency of the portalvenous system and the development of portosystemic collaterals are dynamic, to overcome this bias CE-CT/CE-MR conducted before AND after CT AP–AS were analyzed. UGE was the only reference standard regarding upper gastrointestinal varices. Analysis of CT AP–AS concerning the patency of portosplenomesenteric veins was restricted to diagnostic capability because comprehensive reference standards like intraoperative findings do not exist in every patient. Methods Th l This single center study was conducted according to the principles expressed in the Declaration of Helsinki. The ethics committee of the University of Regensburg approved this retrospective study (approval no. 20-2123-104) and waived the requirement for informed consent. Pediatric patients (18 years old or younger) with clinical signs of PH who underwent CT AP–AS at our tertiary referral university hospital center with available standard contrast-enhanced cross-sectional imaging (CE-CT in portalvenous phase and/or CE-MR with axial T1-weighted dynamic three-dimensional gradient-echo sequences) within 18 months before or after CT AP–AS were included in this study. Follow-up CT AP–AS and standard cross-sectional imaging studies with severe artifacts were excluded. Medical records of UGE were reviewed when available. Imaging. CT AP–AS was conducted following a prospectively determined protocol as described in detail before8: (1) 4-F sheaths and 4-F catheters were used to catheterize the SMA and SA selectively by transfemoral approaches. Subsequently the patient was transferred to the CT suite with the two catheters secured at the groin. g (2) CT-AP (contrast injection through the catheter placed in the SMA) and CT-AS (contrast injection through the catheter placed in the SA) were conducted at a helical 256 (2 × 128)-slice dual source CT scanner (Somatom Flash scanner; Siemens, Forchheim, Germany) with automatic tube voltage selection (80 kV or 100 kV) and automatic tube current modulation. 0.4 mL nonionic contrast agent per kilogram bodyweight diluted with the same volume of NaCl was injected at a rate of 2.5 mL/s. (2) CT-AP (contrast injection through the catheter placed in the SMA) and CT-AS (contrast injection through the catheter placed in the SA) were conducted at a helical 256 (2 × 128)-slice dual source CT scanner (Somatom Flash scanner; Siemens, Forchheim, Germany) with automatic tube voltage selection (80 kV or 100 kV) and automatic tube current modulation. 0.4 mL nonionic contrast agent per kilogram bodyweight diluted with the same volume of NaCl was injected at a rate of 2.5 mL/s. Portalvenous phase of contrast-enhanced CT and dynamic T1-weighted three-dimensional gradient-echo sequences of CE-MR after peripheral intravenous contrast injection were evaluated. Imaging analysis. Two radiologists (12 and 14 years of experience in the interpretation of vascular imaging studies) reviewed independently imaging using a dedicated PACS viewer (Syngo; Siemens, Forchheim, Ger- many) in a random order. Time interval between the readout sessions were at least 4 weeks. Readers used a structured template. www.nature.com/scientificreports/ pi g y p In conclusion, CT AP–AS with direct contrast-application in the SA and SMA via arterial transfemormal catheterization is a novel, invasive technique that guides evaluation of complex anatomic and hemodynamic changes in complicated pediatric PH more confident in comparison to standard cross-sectional imaging and delineates variceal formation. Discussion Varices in these locations are of interest because they can be origin of occult gastrointestinal hemorrhage. Though systematic data regarding these varices are scarce and restricted to endoscopy. A recent study based on esophageal capsule endosopy conducted for screening and surveillance purposes showed a prevalence of 0.07% for duodenal varices15. Kumar et al.16 found colonic varices in 3.7% of colonoscopies in pediatric patients with EHPVO. The prevalence of duodenal and colonic varices using CT AP–AS in this series was much more higher (10/20 and 9/20 patients respectively). CT AP–AS detected endoscopic described esophageal varices in all but one case but additional gastric varices in 6/12 cases and additional duodenal varices in 2/12 cases. Gulati et al. compared CE-CT imaging of the esophageal and gastric vasculature in pediatric EHPVO with UGE. In their study, CE-CT showed a false positivity of 20% for gastric varices compared to UEG. Interestingly, endoscopy detected varices in all false positive cases during follow up. Since endoscopic evaluation is restricted to the inner surface of the gastric wall, veins within the gastric wall or smaller varices may be missed due to air insufflations during endoscopy17. CE-CT and CT AP–AS probably pick up changes in the gastric vasculature earlier because this technique visualizes both the superficial and the intramural veins. The even higher percentage of false positive findings for gastric varices in our study may be explained by the higher venous contrast in CT AP–AS and the better image quality in comparison to the CE-CT imaging studies of Gulati et al.f Moreover, combination of CT-AP and CT-AS differentiates the main venous supply of varices (SMV versus SV system)8. To our knowledge no other image modality provides this hemodynamic information. According to the experience of our interdisciplinary team of interventional radiologists, surgeons and gastroenterologists main venous supply of varices is important for planning of successful therapeutic interventions in order to decompress the SMV or SV system. However further systematic evaluation of the relevance of these findings for the therapeutic outcome is necessary. https://doi.org/10.1038/s41598-022-10454-y Scientific Reports \| (2022) 12:6554 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ occluded) the overall assessment of the vessel was defined as “confident characterization”. If two readers assigned different categories the overall assessment of the regarding vessel was "not reliably assessable". occluded) the overall assessment of the vessel was defined as “confident characterization”. If two readers assigned different categories the overall assessment of the regarding vessel was "not reliably assessable". Evaluation varices and splenorenal shunting. Varices (paraesophageal space, esophagus, stomach, duodenum, small intestinum, colon, rectum) and splenorenal shunting were reviewed in consensus. Medical records of UGE were reviewed concerning presence and localization of varices (esophagus, cardia/fundus/cor- pus of the stomach, duodenum).f p Enhancement patterns of CT-AP and CT-AS were compared in consensus to differentiate variceal origin main supply (SV system or SMV system). Statistical analysis. Data are presented as absolute and relative frequencies. CT AP–AS was compared to standard cross-sectional imaging by using the McNemar test. A p-value < 0.05 was considered statistically significant. All analyses were performed using R version 4.1.0 (The R Foundation for Statistical Computing). Data availability The data that support the findings of this study are available within the article. The data that support the findings of this study are available within the article. Received: 7 January 2022; Accepted: 29 March 2022 Author contributions S.H. and W.U. wrote the manuscript. B.G. and M.C.D. collected data. C.S. and H.J.S. performed literature review. S.H. and W.U. performed the reading of MRI and CT images. S.H. and W.U. prepared figures. F.Z. carried out statistical evaluation. All authors reviewed the manuscript. References Eur. Radiol. Exp. 4, 65 (2020). p yp p ( ) 9. Kwan, S. W., Fidelman, N., Durack, J. C., Roberts, J. P. & Kerlan, R. K. Rex shunt preoperative imaging: diagnostic capability of imaging modalities. PLoS ONE 6, e22222. https://doi.org/10.1371/journal.pone.0022222 (2011). p yp p 9. Kwan, S. W., Fidelman, N., Durack, J. C., Roberts, J. P. & Kerlan, R. K. Rex shunt preoperative imaging: diagnostic capabili imaging modalities PLoS ONE 6 e22222 https://doi org/10 1371/journal pone 0022222 (2011) p yp p 9. Kwan, S. W., Fidelman, N., Durack, J. C., Roberts, J. P. & Kerlan, R. K. Rex shunt preoperative imaging: diagnostic capability of imaging modalities. PLoS ONE 6, e22222. https://doi.org/10.1371/journal.pone.0022222 (2011). g g , p g j p ( ) 10. Chaves, I. J. et al. Pre- and postoperative imaging and interventions for the meso-Rex bypass in children and young adults. Pediat. Radiol. 42, 220–232 (2012).l g g p g j p 10. Chaves, I. J. et al. Pre- and postoperative imaging and interventions for the meso-Rex bypass in children and young adults. Pediat. Radiol. 42, 220–232 (2012).l 1. Parekh, K. et al. 4D flow MR imaging of the portal venous system: a feasibility study in children. Eur. Radiol. 27, 832–840 (2017) l g g p y y y 12. Kim, M., Mitchell, D. G. & Ito, K. Portosystemic collaterals of the upper abdomen: review of anatomy and demonstration on MR imaging. Abdom. Imag. 25, 462–470 (2000). g g g 3. Saddik, D., Frazer, C., Robins, P., Reed, W. & Davis, S. Gadolinium-enhanced three-dimensional MR portal venography. AJR. Am J. Roentgenol. 172, 413–417 (1999).h g 14. Kang, H. K. et al. Three-dimensional multi-detector row CT portal venography in the evaluation of portosystemic collateral vessels in liver cirrhosis. Radiogr. Rev. Publ. Radiol. Soc. N. Am. 22, 1053–1061 (2002). g 15. Pai, A. K., Jonas, M. M. & Fox, V. L. Esophageal capsule endoscopy in children and young adults with portal hypertension. J. Pediat. Gastroenterol. Nutrit. 69, 641–664 (2019). 6. Kumar, N. et al. Prevalence, nature, and predictors of colonic changes in children with extrahepatic portal vein obstruction Gastrointest. Endosc. 91, 849–858 (2020). ( ) 7. Gulati, M. S., Paul, S. B., Arora, N. K., Mathur, P. & Berry, M. Esophageal and gastric vasculature in children with extrahepatic portal hypertension: evaluation by intravenous CT portography. Clin. Imag. 24, 351–356 (2000). 18. Albuquerque, P. A. B., Morales Ramos, D. A. References 1. Carollo, V. et al. Multimodality imaging of the Meso-Rex bypass. Abdom. Radiol. (N. Y.) 44, 1379–1394 (2019). K A F l N l f d l h H l C ( ) 1. Carollo, V. et al. Multimodality imaging of the Meso-Rex bypass. Abdom. Radiol. (N. Y.) 44, 1379–1394 (2019) 2 K A F t l N l f di i l h i H t l C 4 1346 1352 (2020) 2. 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Magnetic resonance imaging of the liver and biliary tree in children. Curr. Prob. Diagn. Radiol. 38, 126–134 (2009). g 19. Teo, E. L., Strouse, P. J. & Prince, M. R. Applications of magnetic resonance imaging and magnetic resonance angiograph evaluate the hepatic vasculature in the pediatric patient. Pediat. Radiol. 29, 238–243 (1999). 20. Uchida, H. et al. A central approach to splenorenal shunt in pediatric living donor liver transplantation. Pediat. Transp. 19, E142– E145. https://doi.org/10.1111/petr.12543 (2015). p g p 1. Nardelli, S. et al. Relevance of spontaneous portosystemic shunts detected with CT in patients with cirrhosis. Radiology 299 133–140 (2021). g 21. Nardelli, S. et al. Relevance of spontaneous portosystemic shunts detected with CT in patients with cirrhosis. Radiology 299, 133–140 (2021). 21. Nardelli, S. et al. Relevance of spontaneous portosystemic shunts detected with CT in patients with cirrhosis. Radiology 299, 133–140 (2021). 22 Si ó T l M t l A i ti b t t t i h t d i d li ti d t lit i ti t ith 133–140 (2021). 22. Simón-Talero, M. et al. Association between portosystemic shunts and increased complications and mortality in patients with 133–140 (2021). 22. Simón-Talero, M. et al. Association between portosystemic shunts and increased complications and mortality in patients with cirrhosis Gastroenterology 154 1694 1705 (2018) 2. Simón-Talero, M. et al. Association between portosystemic shunts and increased complications and mortality in patients with cirrhosis. Gastroenterology 154, 1694–1705 (2018). Methods Th l They were blinded to clinical data and results of imaging. Evaluation portosplenomesenteric veins. Patency of the extrahepatic/intrahepatic PV system, SMV, SV and SMV/SV confluence was evaluated. These vessels were summarized in 3 vessel regions. Region 1: extra- hepatic and intrahepatic main stem PV. Region 2: intrahepatic PV system with left/right central intrahepatic PV (defined as the section between PV main trunk bifurcation and first branching of the intrahepatic PV) and left/right peripheral intrahepatic PV branches (defined as evident contrast enhancement of PV branches in the peripheral liver parenchyma). Region 3: SMV, SV, SMV/SV confluence. Status of the vessels was classified: (1) patent, (2) occluded, (3) not reliably assessable. If the readers assigned the same classification-type (patent or https://doi.org/10.1038/s41598-022-10454-y Scientific Reports \| (2022) 12:6554 \| www.nature.com/scientificreports/ Funding g Open Access funding enabled and organized by Projekt DEAL. g Open Access funding enabled and organized by Projekt DEAL. Competing interests The authors declare no competing interests. Competing interests The authors declare no competing interests. Competing interests The authors declare no competing interests. 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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) 2022 Additional information Correspondence and requests for materials should be addressed to W.U. Correspondence and requests for materials should be addressed to W.U. Reprints and permissions information is available at www.nature.com/reprints. © The Author(s) 2022 Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and nstitutional 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 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) 2022 https://doi.org/10.1038/s41598-022-10454-y Scientific Reports \| (2022) 12:6554 \|
https://openalex.org/W4387545623	https://eumj.med.sumdu.edu.ua/index.php/journal/article/download/362/249	Ukrainian	null	COMPLICATIONS AFTER PENIS ENHANCEMENT WITH HYALURONIC ACID: A LITERATURE REVIEW	Shìdnoukraïnsʹkij medičnij žurnal	2,023	cc-by	5,100	© 2023 by the author(s). © 2023 by the author(s). This work is licensed under Creative Commons Attribution 4.0 International License https://creativecommons.org/licenses/by/4.0/ DOI: https://doi.org/10.21272/eumj.2023;11(3):232-240 How to сite / Як цитувати статтю: Lytvak O, Shaprynskyi V, Markova M, Zasieda Yu. [Complications after penis enhancement with hyaluronic acid: a literature review]. East Ukr Med J. 2023;11(3):232-240 ABSTRACT Due to the growing demand for these procedures, the issue of their safety is 232 Lytvak O, Shaprynskyi V, Markova M, Zasieda Yu East. Ukr. Med. J. 2023;11(3):232-240 relevant. According to the available statistics, the overall incidence of complications after HA injections is spontaneous without further side effects. If complications are detected, the prognosis after treatment is satisfactory. As a result, there are no standardized surgical protocols and recommendations for penile enhancement with hyaluronic acid in the world. Keywords: hyaluronic acid, girth enlargement, filler, penis, phalloplasty. Corresponding author: Olena Lytvak, State Institution of Science «Research and Practical Centre of Preventive and Clinical Medicine» State Administrative Department, Kyiv, Ukraine e-mail: 0506902242@ukr.net Corresponding author: Olena Lytvak, State Institution of Science «Research and Practical Centre of Preventive and Clinical Medicine» State Administrative Department, Kyiv, Ukraine e-mail: 0506902242@ukr.net ABSTRACT Olena Lytvak https://orcid.org/0000-0001-5362-670X State Institution of Science «Research and Practical Centre of Preventive and Clinical Medicine» State Administrative Department, Kyiv, Ukraine Introduction. The penis is a symbol of the primal sense of male fertility and sexual activity, which affects the sense of male personality and the ability to satisfy a partner. Some patients experience discomfort, even if their penis is within the normal size range from an anatomical point of view. The reason for the spread of penis enlargement procedures is related to the feeling of masculinity that the appearance of the genitals can give a man, as well as a changed perception of his body. Penis enlargement procedures are becoming more and more common among men who seek to achieve a more desirable aesthetic of their male organ. In addition, penis enlargement methods remain a controversial issue, as they do not have clear indications and can cause complications. Vasyl Shaprynskyi https://orcid.org/0000-0002-1437-7410 State Institution of Science «Research and Practical Centre of Preventive and Clinical Medicine» State Administrative Department, Kyiv, Ukraine Materials and methods. This study reviewed the literature on complications after penile enhancement with hyaluronic acid (HA). To achieve this goal, we conducted a systematic search for scientific medical information in English-language databases such as MEDLINE, Embase, AMED (Allied and Complementary Medicine), and HMIC (Health Management Information Consortium). We analyzed all articles that met the specified search parameters. In addition, we manually searched for relevant references in the found texts. Marianna Markova https://orcid.org/0000-0003-0726-4925 Kharkiv Medical Academy of Postgraduate Education, Kharkiv, Ukraine Marianna Markova https://orcid.org/0000-0003-0726-4925 Kharkiv Medical Academy of Postgraduate Education, Kharkiv, Ukraine Yuriy Zasieda https://orcid.org/0000-0001-7307-3591 Zaporizhzhya State Medical University, Ministry of Health of Ukraine, Zaporizhzhya, Ukraine Yuriy Zasieda https://orcid.org/0000-0001-7307-3591 Zaporizhzhya State Medical University, Ministry of Health of Ukraine, Zaporizhzhya, Ukraine Results. The use of HA injections for penis enlargement in men shows a low rate of complications both in the early- and long-term period after the procedure. In a small percentage of patients, early and expected side effects included swelling, bruising, subcutaneous nodules, and infections. In this work, radical ways of their correction are proposed for all types of complications, which makes the use of HA safe and effective for penis enlargement. Conclusions. Nowadays, there is a lack of high-level evidence- based information on complications after HA injections. Corresponding author: Olena Lytvak, State Institution of Science «Research and Practical Centre of Preventive and Clinical Medicine» State Administrative Department, Kyiv, Ukraine e-mail: 0506902242@ukr.net РЕЗЮМЕ Олена Литвак https://orcid.org/0000-0001-5362-670X Державна наукова установа «Нау- ково-практичний центр профілак- тичної та клінічної медицини» Дер- жавного управління справами, м. Київ, Україна Олена Литвак https://orcid.org/0000-0001-5362-670X Державна наукова установа «Нау- ково-практичний центр профілак- тичної та клінічної медицини» Дер- жавного управління справами, м. Київ, Україна Вступ. Статевий член є символом первісного почуття чоло- вічої родючості та сексуальної активності, що впливає на від- чуття чоловічої особистості та здатності задовольняти партне- рку. Але деякі пацієнти відчувають дискомфорт, навіть якщо з анатомічного погляду їх статевий орган за розміром входить у межі норми. Причина поширення процедур зі збільшення стате- вого члена пов’язана із відчуттям мужності, яку може додати чоловікові зовнішній вигляд його статевих органів, а також змі- нене сприйняття свого тіла. Процедури збільшення статевого члена стають все більш поширеними серед чоловіків, які праг- нуть досягти бажанішої естетики цього чоловічого органа. Крім того, методи збільшення статевий член залишаються супереч- ливим питанням, оскільки не мають чітких показань та можуть викликати ускладнення. Василь Шапринський https://orcid.org/0000-0002-1437-7410 Державна наукова установа «Нау- ково-практичний центр профілак- тичної та клінічної медицини» Дер- жавного управління справами, м. Київ, Україна INTRODUCTION / ВСТУП що обхват статевого члена вважається важливі- шим за довжину [10,11]. У зв’язку із вище зазна- ченими причинами все більше чоловіків консуль- туються щодо збільшення статевого члена і шука- ють способи підвищити сексуальний потяг і задо- волення. Статевий член є символом первісного почуття чоловічої родючості та сексуальної активності, що впливає на відчуття чоловічої особистості та здатності задовольняти партнерку [1, 2]. Але деякі пацієнти відчувають дискомфорт, навіть якщо з анатомічного погляду їх статевий орган за розмі- ром входить у межі норми [3]. Причина поши- рення процедур зі збільшення статевого члена пов’язана із відчуттям мужності, яку може додати чоловікові зовнішній вигляд його статевих орга- нів, а також змінене сприйняття свого тіла [1]. Найчастіше пацієнти чоловічої статті зверта- ються за медичною допомогою з питань збіль- шення обхвату статевого члена, передчасної еяку- ляції, причому кількість таких звернень останнім часом зростає [12, 13]. Сьогодні все більше звертають увагу на вико- ристання у процедурах збільшення розмірів ста- тевого члена мінімально інвазивних процедур. Серед таких процедур зросла популярність вико- ристання наповнювачів м’яких тканин із віднов- ленням інтересу до їх андрологічного застосу- вання [14]. Велике дослідження у 25 594 здорових чолові- ків показало, що 45% хотіли б мати більший пенiс [4]. Іноді, за бажанням збільшення пенiса, хова- ється бажання доставити більше задоволення пар- тнерці, хоча власне для самих чоловіків розмір пенiса має більше значення, ніж для жінок [5]. На- справді, сексуальне задоволення жінки може фізи- чно створюватися достатнім розширенням слизо- вої оболонки піхви, особливо дистальної третини передньої стінки піхви, де знаходиться значна гу- стина нервів i судин [6]. Тому, косметична хірур- гія статевого члена повинна враховувати потреби пацієнтів та бути безпечною. Хоча всебічно досліджуються різні наповню- вачі для використання в інших частинах тіла, проте їхнє застосування шляхом ін’єкції в пеніс ві- дрізняється через іншу анатомію та у зв’язку із за- стосуванням більшої кількості наповнювача для пеніса, що вимагає спеціальних клінічних дослі- джень [15]. Для збільшення розмірів статевого члена сьогодні використовують різні наповню- вачі, серед яких на особливу увагу заслуговує гіа- луронова кислота [16]. Проте, високоякісні докази ефективності ГК та безпеки є обмеженими, оскі- льки бракує доказів та рекомендацій щодо її вико- ристання в клінічній практиці. Процедури збільшення статевого члена стають все більш поширеними серед чоловіків, які праг- нуть досягти бажанішої естетики цього чолові- чого органу [7]. Розмір пеніса є важливим показ- ником для вимірювання статевого розвитку в чо- ловіків. Окрім того, розміри статевого члена сприймаються як символ здоров'я, сексуальної ак- тивності та мужності. Василь Шапринський https://orcid.org/0000-0002-1437-7410 Маріанна Маркова https://orcid.org/0000-0003-0726-4925 Харківська медична академія після- дипломної освіти, м. Харків, Україна Матеріали та методи. У науковому дослідженні проведе- ний огляд літературних джерел щодо ускладнень після контур- ної пластики статевого члена гіалуроновою кислотою (ГК). Для досягнення цієї мети, ми здійснили системний пошук наукової медичної інформації в англомовних базах даних, таких як MEDLINE, Embase, AMED (Allied and Complementary Medicine) і HMIC (Health Management Information Consortium). Ми аналі- зували всі статті, що відповідали заданим пошуковим парамет- рам. Крім того, був проведений ручний пошук відповідних по- силань у знайдених текстах. Харківська медична академія після- дипломної освіти, м. Харків, Україна Юрій Засєда https://orcid.org/0000-0001-7307-3591 Запорізький державний медичний університет, м. Запоріжжя, Укра- їна Юрій Засєда https://orcid.org/0000-0001-7307-3591 Запорізький державний медичний університет, м. Запоріжжя, Укра- їна Результати. Застосування ін’єкцій ГК для збільшення стате- вого органу в чоловіків показує низький відсоток ускладнень як на ранніх, так і віддалених термінах після процедури. У незнач- ного відсотка пацієнтів ранні та очікувані побічні ефекти вклю- чали набряк, синці, підшкірні вузлики та інфекції. У роботі для всіх видів ускладнень запропоновані радикальні шляхи їх коре- кції, що робить застосування ГК безпечним та ефективним для збільшення розмірів статевого члена. Висновки. На сьогоднішній день є нестача інформації з ви- соким рівнем доказовості стосовно ускладнень після ін’єкцій ГК. У зв’язку зі зростаючим попитом на дані процедури, пи- тання їх безпечності є актуальним. Згідно з існуючою 233 Lytvak O, Shaprynskyi V, Markova M, Zasieda Yu East. Ukr. Med. J. 2023;11(3):232-240 статистикою, загальна частота ускладнень після ін’єкцій ГК є спонтанною без подальших побічних ефектів. При виявленні ускладнень прогноз після лікування задовільний. Зважаючи на це, стандартизовані хірургічні протоколи та рекомендації щодо контурної пластики статевого члена гіалуроновою кислотою у світі відсутні. Ключові слова: гіалуронова кислота, збільшення товщини, наповнювач, статевий член, фалопластика. Автор, відповідальний за листування: Олена Литвак, Державна наукова установа «Науково-практич- ний центр профілактичної та клінічної медицини» Державного управління справами, м. Київ, Україна e-mail: 0506902242@ukr.net Автор, відповідальний за листування: Олена Литвак, Державна наукова установа «Науково-практич- ний центр профілактичної та клінічної медицини» Державного управління справами, м. Київ, Україна e-mail: 0506902242@ukr.net INTRODUCTION / ВСТУП Деякі дослідження [8,9] вка- зують, що розмір чоловічого статевого органу пов’язаний із самооцінкою. Так, чоловіки, які не- дооцінюють свій розмір, можуть перебільшувати фізіологічні дефекти, що буде виражено психоло- гічним стресом. Такі чоловіки більше схильні до депресії, що викликає занепокоєння та сексуальне незадоволення [9]. Багато досліджень вказують, У деяких дослідженнях [8, 16, 17] оцінено суб’єктивне задоволення під час сексуальної акти- вності після збільшення пеніса з використанням ін’єкційних наповнювачів, проте детальна інфор- мація щодо еякуляції досі невідома. Окрім того, необхідно визначити, чи наповнювачі ГК з різ- ними властивостями (концентрація ГК і ступінь зшивання) виявляють подібні ефекти. Невідо- мими залишаються питання безпеки раніше 234 Lytvak O, Shaprynskyi V, Markova M, Zasieda Yu East. Ukr. Med. J. 2023;11(3):232-240 досліджених наповнювачів ГК. Тому, одним із за- вдань залишається оцінка ефективності та безпеки збільшення пеніса з використанням наповнювача ГК та його клінічний вплив на еякуляцію. маючи нормальний розмір статевого члена, недо- оцінюють розмір власного члена і перебільшують середній розмір інших чоловіків [19, 21]. Згідно з Міжнародною класифікацією хвороб 10-го перегляду (МКХ-10) та Діагностичним і ста- тистичним керівництвом з психічних розладів 5- го перегляду (Diagnostic and Statistics Manual of mental disorders, DSM-5), пенільна дисморфофобія є одним з дисморфічних розладів тіла. Цей розлад є соматоформним і характеризується станом три- воги через уявний або незначний дефект у фізич- ному вигляді, який викликає проблеми у різних сферах життя [21]. Основним симптомом є пове- дінка перед дзеркалом, коли пацієнт довго ди- виться на свій орган з різних ракурсів. Якщо лю- дина занепокоєна розміром свого члена і приділяє цій проблемі принаймні 1 годину на день, що при- зводить до повторюваної поведінки і видимих не- зручностей, це можна вважати пенільною дисмо- рфофобією [18, 26, 27]. Люди з цим розладом на- магаються приховувати свій член, уникають зага- льних роздягалень, бояться фотографуватися і хо- чуть відволікти від себе увагу. Чоловіки з цим ро- зладом можуть зазнавати депресію та відчувати розлади у соціальній, професійній та сексуальній сферах, викликані занепокоєнням розміром пе- ніса. На відміну від чоловіків із СМСЧ, пацієнти з пенільною дисморфофобією зазвичай не отриму- ють полегшення стану від збільшення розміру, і можуть навіть спостерігатися випадки погір- шення симптомів [23, 24]. На даний час існує кі- лька клінічних досліджень, які показали позитив- ний ефект консервативної терапії дисморфічного розладу тіла [28–31]. Однак, наразі не проводи- лось жодного дослідження, яке б вивчало ефекти- вність консервативної терапії для пенільної дис- морфофобії. INTRODUCTION / ВСТУП Крім того, було запропоновано опи- тувальник для ідентифікації чоловіків із пеніль- ною дисморфофобією, який може стати корисним інструментом для хірургів [32]. Незважаючи на те, що ін’єкційне лікування ГК в андрології зростає, поточна критика його андро- логічного застосування є другорядною щодо від- сутності встановлених процедур, погано визначе- них показань і занепокоєння щодо надійності іс- нуючої наукової літератури в цій галузі. На даний момент у літературі відсутні рекомендації та ста- ндарти щодо проведення подібних процедур [1, 18]. У цій оглядовій статті ми проаналізували лі- тературні джерела щодо використання ГК в анд- рології, що у подальшому допоможе скерувати клініциста до відповідального консультування па- цієнтів, які звертаються за медичною допомогою з приводу збільшення статевого члена. Мета роботи – огляд наукової інформації сто- совно можливих ускладнень після збільшення ро- змірів статевого члена за допомогою наповнювача гіалуронової кислоти, оцінити безпеку процедури. Стани, при яких виконується збільшення члена Враховуючи вищесказане, слід пам’ятати, що не всім потрібно збільшувати статевий орган. Тому назвемо категорії чоловіків, для яких це має значення. Синдром малого статевого члена (СМСЧ). Ба- гато чоловіків, які хочуть збільшити статевого члена, мають нормальний розмір, який достатньо функціональний для належної сексуальної діяль- ності [19, 20]. Зазвичай таким пацієнтам здається, що їх фалос менший за середньостатистичний [21]. Особливим у цьому стані є те, що таким чо- ловікам розмір статевого члена не заважає в по- всякденному житті та сексуальних стосунках. Але, слід зазначити, що при нормальному лiбiдo вони отримують менше задоволення від інтимної близькості [20]. Прагнення збільшити свій стате- вий орган базується на бажанні покращити зовні- шній вигляд та досягти пропорційності. Наразі не існує єдиного підходу до лікування чоловіків зі СМСЧ. Деякі дослідники вважають, що такі чоло- віки мають спочатку пройти психологічне консу- льтування [20, 22]. Дослідження також показали, що збільшення довжини статевого члена допома- гає полегшити стан та підвищує психосемоційне задоволення у чоловіків зі СМСЧ [23, 24]. Вкорочення статевого члена. Зменшення ро- зміру чоловічого статевого члена може бути спри- чинене різними медичними та хірургічними ста- нами, такими як рак передміхурової залози з вико- наною радикальною простатектомією, хвороба Пейроні та вроджені аномалії. Крім того, еректи- льна дисфункція може спричиняти вкорочення статевого члена. кислотою Незважаючи на те, що побічні ефекти від ін’єкції ГК зазвичай легкі та рідкісні, вони все одно можуть викликати значне незадоволення у постраждалих пацієнтів. Ahn S.T. та ін. [33] вико- нали систематичний огляд літератури з метою опису можливих ризиків використання ГК для збі- льшення розмірів статевого члена. Було виявлено, що наповнювачі ГК потенційно викликають побі- чні ефекти відразу після процедури або навіть че- рез роки. Найпоширенішими побічними наслід- ками були незадоволення косметичним виглядом органа, надмірна корекція, нерівності поверхні статевого члена, утворення гранульом і можливі некротичні зміни внаслідок судинного ураження. У дослідженні Lee W.K. et al. [34], протягом усього 6-місячного спостереження 14,3% пацієн- тів мали ускладнення, такі як підшкірна кровотеча (4,3%), підшкірні вузлики (5,4 %) та інфекція (2,4%), тоді як набряк статевого члена був виявле- ний у 21 (9,1 %) з 230 пацієнтів, усі з яких не були обрізані до процедури. Автори визначили можли- вий причинно-наслідковий зв’язок між післяопе- раційним набряком статевого члена та надлишко- вою передопераційною крайньою плоттю, оскі- льки це, за гіпотезою, спричиняє дистальне нако- пичення введеного гелю ГК, що призводить до лі- мфатичної компресії. Цікаво, що в дослідженнях Abdallah H. та ін. [35], де всі пацієнти були обрі- зані перед ін’єкцією гелю ГК, не повідомлялося про набряк статевого члена. Боротися з деформа- ціями статевого члена, що залишилися після опе- рації або після ін’єкцій речовин, що не всмокту- ються, є проблемою. Незважаючи на те, що дока- зів у цьому відношенні небагато, наповнювачі ГК також можна використовувати із задовільними та відтворюваними результатами для корекції зали- шкових деформацій обхвату статевого члена після операції по збільшенню пеніса. У дослідженні Kim J. та ін. [36], ін’єкції ГК проводили шляхом канюляції голками 27G і 30G в головку, що дозво- ляло спонтанно закрити місця входу голки без на- кладання швів. Повідомлялося, що використання Найпоширенішим ускладненням ін’єкції ГК серед пацієнтів є підшкірний вузлик статевого члена (загальна частота 2,2 %), який зазвичай ви- никає через 2 тижні після операції. Описані вуз- лики пов’язані з накопиченням ГК в одній ділянці [38, 39]. Оскільки ГК вимагає певного періоду часу для перерозподілу та формування, то для рі- вномірного розподілу ГК можна використовувати еластичні стрічки та масаж статевого члена. Од- нак, коли ГК вводять занадто швидко, вона може не розподілятися рівномірно. Це може призвести до розвитку вузликів, стійких до терапії масажем статевого члена через 1–2 тижні. Хоча ці вузлики, як правило, безболісні, вони можуть зберігатися протягом тривалого часу, якщо їх не лікувати [38]. Матеріали та методи Пенільна дисморфофобія. На думку провідних вчених, лікарі мають відрізняти пенільну дисмо- рофобію та СМСЧ [23, 25], оскільки вони дуже схожі між собою: при обох цих розладах чоловіки, У науковому дослідженні проведений огляд літературних джерел щодо ускладнень після кон- турної пластики статевого члена гіалуроновою ки- слотою (ГК). Для досягнення цієї мети, ми 235 East. Ukr. Med. J. 2023;11(3):232-240 Lytvak O, Shaprynskyi V, Markova M, Zasieda Yu канюлі 18G полегшує розподіл наповнювача ГК, але місця ін’єкцій можуть не заживати спонтанно, і може знадобитися накладання швів на місце ін’єкції, щоб запобігти можливому зараженню ім- плантату бактеріальною флорою статевих органів [37]. Інфекція є рідкісним наслідком після ін’єкції ГК при збільшенні статевого члена, але може при- звести до шкідливих наслідків. У звіті про випа- док [37], у хворого віком 35 років виникла інфек- ція статевого члена з вираженим набряком та ери- темою. Протягом 24 год у хворого розвинувся се- птичний шок з анурією, що потребувало переве- дення у відділення інтенсивної терапії. Через мі- сяць після госпіталізації спостерігалася значна по- верхнева втрата шкіри як на вентральній, так і на бічній частинах пеніса. здійснили системний пошук наукової медичної ін- формації в англомовних базах даних, таких як MEDLINE, Embase, AMED (Allied and Complementary Medicine) і HMIC (Health Management Information Consortium). Ми аналізу- вали всі статті, що відповідали заданим пошуко- вим параметрам. Крім того, був проведений руч- ний пошук відповідних посилань у знайдених те- кстах. кислотою Крім того, через тонку шкіру статевого члена під- шкірні вузлики розміром більше 1 см зазвичай стають помітними і прощупуються. В результаті пацієнти можуть відчувати дискомфорт і депре- сію під час сексу. Крім того, ці вузлики не можуть розсмоктуватися спонтанно. Варіантами ліку- вання цих ускладнень є операція і терапія гіалуро- нідазою [40, 41]. Терапія гіалуронідазою здійсню- ється шляхом ін’єкції тонкою голкою відповідної кількості (зазвичай 30 одиниць) гіалуронідази за- лежно від розміру вузлика. Іншим ускладненням можуть бути підшкірні кровотечі, які виникають у 1,3 % усіх пацієнтів. В основному це може відбуватися через 2–5 днів пі- сля операції і досягає максимуму в перший тиж- день. Основна причина підшкірної кровотечі при ін’єкції ГК – пряма пункція судини. Хоча ін’єкція ГК є мікроінвазивною хірургією, використання антитромботичних препаратів перед лікуванням має бути припинено, щоб зменшити ризик крово- течі. Крім того, було показано, що використання канюль замість голки допомагає знизити ризик 236 Lytvak O, Shaprynskyi V, Markova M, Zasieda Yu East. Ukr. Med. J. 2023;11(3):232-240 відповідно, залежно від використовуваного гелю ГК. Крім того, ін’єкцію гелю ГК проводили в го- ловку статевого члена без накладання швів. За да- ними [43] показано, що ін’єкція кореня пеніса за допомогою канюлі 18G забезпечує легкість рівно- мірного розподілу ГК. Однак недоліком є те, що у деяких пацієнтів місця ін’єкцій не загоюються спонтанно, а це може бути місцем інфікування флорою лобкового волосся за відсутності підго- товки шкіри. У результаті було зроблено уши- вання місця ін'єкції. Щоб зменшити ймовірність інфікування, використовують монофазну та син- тетичну ГК. захворювання [33]. Використання товстіших гол- кових трубок для встановлення канюлі може зме- ншити частоту підшкірної кровотечі. Тому, доре- чно використовувати канюлю 18G для створення шкірного тунелю для ін’єкції ГК. Підшкірна кро- вотеча, якщо така є, може спонтанно зникнути в перехідний період. Інфікування при ін’єкціях ГК в м’які тканини пеніса низька, оскільки спостерігається у 1,5 % па- цієнтів [42]. У дослідженні [43] інфекційний про- цес розвивався лише у двох пацієнтів, причому обидва пацієнти були хворі на цукровий діабет і мали статевий акт через 1–2 місяці після операції. Існує чотири основні елементи, які можуть сприяти зараженню: 1) особливості пацієнта; 2) статевий акт; 3) фактори ін’єкції та 4) характерис- тики гелю ГК. Такі особливості пацієнта, як діабет та імунодефіцит, збільшують ризик розвитку ін- фекцій, особливо в області статевих органів. Ста- тевий акт також може сприяти зараженню. Підшкірна кровотеча Підшкірні вузлики Інфекція Набряк статевого члена AUTHOR CONTRIBUTIONS / ВКЛАД АВТОРІВ Усі автори зробили істотний внесок у розробку початкової та доопрацьованої версій цієї статті. Вони несуть повну відповідальність за всі аспекти роботи і вирішення питань, пов'язаних з точністю або цілісністю наведеної інформації. кислотою Бакте- рії, які у великій кількості присутні у піхві та во- лоссі на лобку, можуть потрапити в незагоєну рану під час статевого акту та спричинити інфек- цію [42]. Набряк статевого члена характеризується на- бряком шкіри статевого члена. Набряк статевого члена після ін’єкції ГК супроводжувався пробле- мами надлишкової крайньої плоті, що може спри- чинити дистальне накопичення введеного гелю ГК і призвести до стиснення лімфи [46]. Набряк статевого члена можна зняти за допомогою елас- тичного бинта протягом 2 тижнів без серйозних побічних ефектів. Як показують літературні джерела найчасті- шими ускладненнями після ін’єкцій ГК є набряк статевого члена – 9,1 % пацієнтів та підшкірні ву- злики – 3,8 % пацієнтів (від 2,2 % до 5,4 % за різ- ними джерелами літератури) (рис.1). Фактори ін'єкції, такі як розмір канюлі та місце ін'єкції, відіграють вирішальну роль в інфікуванні. На відміну від результатів Jeong H.G. та ін. [44] та Kwak T.I. та ін. [45], такі ускладнення, як підшкі- рні вузлики та інфекції, частіше зустрічалися у до- слідженнях [43]. У дослідженні Kim J.J. et al. [38], початкову та додаткову ін’єкції ГК через 2 тижні робили через канюляцію з розмірами 27G та 30G Найменшою частотою ускладнень виявляється розвиток інфекції – у 1,9% пацієнтів (від 1,5 % до 2,4 % за різними джерелами літератури) та підш- кірні кровотечі – у 2,8 % пацієнтів (від 1,3 % до 4,3 % за різними джерелами літератури) (рис.1). Підшкірна кровотеча Підшкірні вузлики Інфекція Набряк статевого члена Рисунок 1 – Узагальнення літературних даних щодо частоти виникнення ускладнень після процедури збільшення статевого члена ін’єкціями гіалуронової кислоти 2,8 3,8 1,9 9,1 Кількість пацієнтів, % Кількість пацієнтів, % Рисунок 1 – Узагальнення літературних даних щодо частоти виникнення ускладнень після процедури збільшення статевого члена ін’єкціями гіалуронової кислоти Рисунок 1 – Узагальнення літературних даних щодо частоти виникнення ускладнень після процедури збільшення статевого члена ін’єкціями гіалуронової кислоти 237 Lytvak O, Shaprynskyi V, Markova M, Zasieda Yu East. Ukr. Med. J. 2023;11(3):232-240 https://doi.org/10.1371/JOURNAL.PONE.0110239. 7. Furr J, Hebert K, Gelman J. Complications of Genital Enlargement Surgery. Journal of Sexual Medicine 2018;15:1811–7. 15. Sito G, Marlino S, Santorelli A. Use of Macrolane VRF 30 in emicircumferential penis enlargement. Aesthetic Surgery Journal. 2013;33:258–64. 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https://openalex.org/W4321210947	https://www.nature.com/articles/s41598-023-29641-6.pdf	English	null	An observational and Mendelian randomisation study on iron status and sepsis	Scientific reports	2,023	cc-by	10,343	An observational and Mendelian randomisation study on iron status and sepsis OPEN Fergus Hamilton 1,2, Ruth Mitchell 1, Haroon Ahmed 3, Peter Ghazal 4 & Nicholas J. Timpso Iron deficiency is associated with a substantial burden of morbidity. However, supplementation of iron has been linked to increased rates of serious infection in randomised trials of children in sub-Saharan Africa. Randomised trials in other settings have been inconclusive and it is unknown if changes in levels of iron biomarkers are linked to sepsis in these other settings. We used genetic variants associated with levels of iron biomarkers as instrumental variables in a Mendelian randomisation (MR) analysis to test the hypothesis that increasing levels of iron biomarkers increase the risk of sepsis. In observational and MR analyses we found that increases in iron biomarkers increase the odds of sepsis. In stratified analyses, we show that this risk may be larger in those with iron deficiency and/ or anaemia. Taken together, results here suggest a required caution in supplementation of iron and underline the role of iron homeostasis in severe infection. Iron is a critical element for many biological processes and potentially harmful effects occur with iron deficiency or overload. Globally, iron deficiency remains a major problem in both low to middle and high-income countries1. Although iron supplementation for those with iron deficiency anaemia (IDA) is uncontroversial, uncertainty remains about the role of supplementation in other clinical scenarios, particularly in iron deficiency without anaemia (IDWA). Despite the lack of clear evidence of benefit in randomised trials, there is increasing use of intravenous and/or oral iron both on and off label; often for symptoms attributed to iron deficiency2–4. However, it is also clear that iron excess can be harmful, with people who are genetically predisposed to high iron (e.g. patients with mutations in HFE) developing a complex and broad range of syndromes, including an increased risk of infection5.h There has been longstanding interest in iron levels and the risk and outcome of infection due to the critical role of iron as a nutrient for both fungal and bacterial pathogens and the balance between the host and pathogen in management of iron. Hepcidin is positively regulated by the inflammatory cytokine interleukin-6, resulting in rapid reduction in serum iron and increases in serum ferritin resulting in iron replete macrophages6. The inflammatory regulated hepcidin-ferroportin axis is a common response to bacterial infection in humans7. www.nature.com/scientificreports www.nature.com/scientificreports Observational data Demographics and cohort definition i In our observational analysis, we used UK Biobank, a large adult volunteer cohort with linked primary and secondary care data that includes blood tests taken in primary care for routine reasons16. We aimed to investigate whether higher levels of ferritin (the only iron biomarker routinely clinically tested, and so available for our analysis) were associated with increased rates of sepsis, using regression models adjusted for relevant covariates.i We identified 222,081 participants in UK Biobank with linked primary care and hospital admission data. Amongst this cohort, 195,444 ferritin tests were recorded in the primary care records of 82,678 participants. Supplementary Fig. S1describes the flow through the study. The median ferritin level was 105 ug/L for men, and 68 ug/L for women, while the distribution was approximately log-normal (Supplementary Fig. S2). For our analysis population, we included those with ferritin levels taken after each participant was recruited to UK Biobank [2006–2014], leading to 72,865 participants. The tested cohort (those with a recorded ferritin test) differed from the cohort who were not tested, with more females (64% vs 53%) and with a different pattern of comorbidities (Table 1), reflecting the differences in testing conditional on illness. l gf g We then used linked hospital admission data provided by UK Biobank (Hospital Episode Statistics data) to identify cases of sepsis. We used ICD-10 coding to identify admissions (codes A02, A39, A40, A41) in this cohort. There were 2399 subsequent diagnoses of sepsis recorded in the tested cohort with a median time between ferritin result and sepsis diagnosis of 1943 days (IQR 1060–3105). Associations with sepsis Associations with sepsis We went on to perform logistic regression to test the association between ferritin (untransformed) and subsequent sepsis diagnoses, adjusted for age, UK Biobank recruitment centre, smoking status, alcohol usage, deprivation and clinical comorbidities. p In this model increasing ferritin level was associated with an increased odds of sepsis (OR 1.05; 95% CI 1.04–1.06, p < 1.1 × 10–11 for every 100 ug/L increase in ferritin) (Supplementary Table S1 for full regression output), with similar results in unadjusted models. We then went on to explore non-linearity, given that both low and high ferritin are associated with disease. In restricted cubic spline models (Fig. www.nature.com/scientificreports/ www.nature.com/scientificreports/ The alteration of the iron homeostasis setpoint in infection and inflammation raises valid concerns about iron supplementation. The strongest evidence for the relationship between iron supplementation and adverse infection outcomes was found from a large randomised controlled trial of iron and zinc supplementation in children from Zanzibar10. The trial was stopped early due to increased risks of severe illness and death in those assigned to the iron and zinc arm, with much of the increased risk attributed to malaria.if However, subsequent randomised trials have not identified this effect and the data remain controversial and inconsistent11. In adult settings, a recent meta-analysis of all randomised controlled trials of intravenous iron found increased risks of infection in those randomised to receive intravenous iron, but there was substantial uncertainty around the effect size12. Because of the limited number of studies, this meta-analysis was unable to compare oral iron vs no supplementation at all. Uncertainty therefore remains around whether increased iron is associated with increased rates of sepsis. Mendelian randomisation (MR)—a form of applied genetic epidemiology which deploys instrumental variable analysis using germline genetic variants—has been used as an approach to estimate causal relationships pertinent to iron status13–15. In this study, we set out to assess the relationship between iron status biomarkers and sepsis using a combination of observational and Mendelian randomisation analyses, in a large UK cohort study (UK Biobank). An observational and Mendelian randomisation study on iron status and sepsis OPEN This response is thought to be an attempt to reduce available iron to invading pathogens and aligns with evidence from multiple laboratory studies that many common pathogens grow more readily in serum that is high in iron8. p y y p g g y g Iron status is a complex trait and no single biomarker reliably represents total body iron state. Cellular location, redox state, and availability of iron are all relevant in understanding iron state. Markers used in clinical assessment of acute iron status, that rapidly (over hours) alter with iron supplementation or in response to physiological demands, include serum iron and the total iron binding capacity (TIBC), which, as a ratio (serum iron/TIBC), equates to the transferrin saturation (TSAT). TSAT levels clinically indicate the availability of iron for erythropoiesis and are low in iron deficiency and high in overload9. yi y g In contrast, ferritin—an iron storage protein—is generally considered a biomarker of iron body stores in a non-inflammatory state. TSAT and ferritin are used as the main diagnostic tests for iron deficiency anaemia worldwide and are measured more commonly than the other biomarkers. The master regulator for systemic iron homeostasis is the liver derived hepcidin that inhibits the only iron transporter, ferroportin, in iron-absorptive gut enterocytes and iron-recycling macrophages7. Although all four biomarkers are routine tests, ferritin is tested more commonly than the others as it is used as the initial diagnostic test for anaemia, and is used to stratify patients as iron deficient, with a traditional cut off of 50 ug/L as defining iron deficiency, although it is recognised this is a continuum9. 1MRC Integrative Epidemiology Unit, University of Bristol, Oakfield House, Oakfield Grove, Bristol BS8 2BN, UK. 2Infection Sciences, North Bristol NHS Trust, Bristol, UK. 3Division of Population Medicine, Cardiff University Medical School, Cardiff, UK. 4System Immunity Research Institute, Division of Infection and Immunity, Cardiff University, Cardiff, UK. email: Fergus.hamilton@bristol.ac.uk Scientific Reports \| (2023) 13:2867 \| https://doi.org/10.1038/s41598-023-29641-6 Observational data 1) we found evidence for a U-shaped relationship in the early part of the ferritin distribution, with those at both extremes of ferritin levels at higher risk of sepsis, although the large bulk of the distribution lies within the linear association between increasing ferritin and increasing odds of sepsis.ii g g p As the management of iron and incidence and causes of iron deficiency vary by sex, we re-ran models stratified by sex. There were some differences in the magnitude of risk, with women having comparatively more risk with the same ferritin level. The odds of sepsis start to increase in women at around 68 ug/L and in men at around 105 ug/L (levels that are generally considered physiologically normal) and are around the centre of distribution of ferritin levels for this cohort (median Ferritin for men 108 ug/L, for women 54 ug/L). These results are shown in Fig. 2, focussing on the range of 50–400 ug/L, with the full range visualised in Supplementary Fig. S3. As increased rates of sepsis in those with extremely low ferritin was likely to be confounded by disease state (i.e. the disease leading to iron deficiency could lead to sepsis) we re-ran the linear analyses excluding all with ferritin results below 50 ug/L. Effect estimates were similar, although larger, with an OR of 1.07 (95% CI 1.05–1.08, p = 5.8 × 10–13 per 100 ug/L). However, interpretation of these estimates causally is difficult, due to conditioning by both disease state, demographics, and testing. Therefore, we went on to perform two sample Mendelian randomisation, to assess whether the association between iron status and sepsis was causal, and to generate estimates for comparison with iron supplementation. Generation of instruments Mendelian randomisation uses single nucleotide polymorphisms (SNPs) as instrumental variables in an instrumental variable analysis to estimate the causal effect of a change in an exposure (in this case iron levels) on an outcome (in this case sepsis)15. In this case, the association between each SNP and the exposure and outcome are measured in separate independent datasets (Two Sample MR). https://doi.org/10.1038/s41598-023-29641-6 Scientific Reports \| (2023) 13:2867 \| www.nature.com/scientificreports/ Table 1. Associations between ferritin testing status and level on potential confounders. 1 Median (IQR); n (%); Kruskal–Wallis rank sum test; Pearson’s Chi-squared test. Generation of instruments Ferritin quartile Quartile 1, N = 24,858 Quartile 2, N = 24,857 Quartile 3, N = 24,857 Quartile 4, N = 24,857 Not tested, N = 146,600 p-value1 Ferritin (ug/L) 14 (8, 21) 46 (38, 56) 91 (78, 107) 199 (156, 280) n/a < 0.001 Age at recruitment (years) 54 (47, 62) 58 (51, 64) 60 (53, 65) 61 (55, 65) 58 (51, 63) < 0.001 Sex < 0.001 Female 19,903 (80%) 18,755 (75%) 15,922 (64%) 10,254 (41%) 74,156 (51%) Male 4955 (20%) 6102 (25%) 8935 (36%) 14,603 (59%) 72,444 (49%) Body Mass Index at recruitment (kg/ m2) 26.4 (23.5, 30.3) 26.4 (23.8, 29.9) 26.9 (24.3, 30.4) 27.9 (25.2, 31.1) 26.9 (24.3, 29.9) < 0.001 Unknown 6050 6403 6345 6150 36,096 Creatinine at recruitment (umol/L) 69 (60, 78) 69 (61, 81) 72 (61, 82) 77 (66, 90) 74 (64, 85) < 0.001 Unknown 24,367 24,323 24,235 24,308 143,255 CRP at recruitment (umol/L) 1.11 (0.57, 2.37) 1.25 (0.59, 2.12) 1.35 (0.64, 3.04) 1.67 (0.82, 3.33) 1.18 (0.62, 2.35) < 0.001 Unknown 24,371 24,327 24,237 24,311 143,259 History of diabetes 1548 (8.2%) 1342 (7.2%) 1216 (6.5%) 1308 (7.0%) 4762 (4.3%) < 0.001 History of cancer 1271 (5.1%) 1571 (6.3%) 1615 (6.5%) 1887 (7.6%) 15,779 (11%) < 0.001 History of liver disease: 154 (0.6%) 203 (0.8%) 264 (1.1%) 430 (1.7%) 146,600 (100%) < 0.001 Townsend deprivation index: − 1.80 (− 3.47, 0.95) − 1.84 (− 3.50, 0.94) − 1.99 (− 3.58, 0.83) − 2.03 (− 3.60, 0.76) − 2.23 (− 3.68, 0.28) < 0.001 Unknown 5,946 6,264 6,231 6,048 35,646 Alcohol intake frequency < 0.001 Prefer not to answer 24 (0.1%) 31 (0.2%) 22 (0.1%) 27 (0.1%) 109 (< 0.1%) Daily or almost daily 1931 (10%) 2512 (14%) 3266 (18%) 4847 (26%) 22,836 (21%) Three or four times a week 3144 (17%) 3401 (18%) 3851 (21%) 4543 (24%) 27,058 (24%) Once or twice a week 4850 (26%) 4710 (25%) 4695 (25%) 4586 (24%) 29,677 (27%) One to three times a month 2771 (15%) 2449 (13%) 2354 (13%) 1662 (8.8%) 12,296 (11%) Special occasions only 3403 (18%) 3085 (17%) 2565 (14%) 1821 (9.7%) 11,496 (10%) Never 2786 (15%) 2404 (13%) 1855 (10.0%) 1321 (7.0%) 7504 (6.8%) Unknown 5949 6265 6249 6050 35,624 Smoking status < 0.001 Prefer not to answer 81 (0.4%) 101 (0.5%) 84 (0.5%) 83 (0.4%) 403 (0.4%) Never 11,539 (61%) 10,521 (57%) 9815 (53%) 8883 (47%) 61,084 (55%) Previous 5693 (30%) 5960 (32%) 6616 (36%) 7582 (40%) 37,828 (34%) Current 1596 (8.4%) 2010 (11%) 2093 (11%) 2259 (12%) 11,661 (11%) Unknown 5949 6265 6249 6050 35,624 Sepsis diagnosis 889 (3.6%) 786 (3.2%) 818 (3.3%) 1322 (5.3%) 4651 (3.2%) < 0.001 Table 1. Generation of instruments Associations between ferritin testing status and level on potential confounders. 1 Median (IQR); n (%); Kruskal–Wallis rank sum test; Pearson’s Chi-squared test. To generate instruments for ferritin, we extracted linkage disequilibrium independent (r2 < 0.01), genome- wide significant (5 × 10–8) SNP’s from a recent meta-analysis of iron status, where each biomarker had been rank-based inverse normal transferred prior to analysis18. Details of the cohort, genetic quality control, and methodology are in the methods19. For serum ferritin we included 50 SNPs. Although we were unable to perform observational analyses for them, were also able to derive instruments for iron (23 SNPs), TSAT (17 SNPs), and (TIBC 26 SNPs). All included SNPS are available in Supplement S2. There was no sample overlap between this exposure dataset and the two outcome datasets. Development of outcome GWAS To generate outcome summary statistics for use in Mendelian randomisation analyses, we identified cases of sepsis using the same methodology as in the observational analysis, but in all participants in UK Biobank of European ancestry (case and ancestry definitions in the methods). In total, 12,664 cases were included, with 462,869 controls, giving a frequency of incident cases of 2.5%. We subsequently performed a case–control GWAS on quality controlled genetic data, with details of QC here20 and in the methods using regenie. regenie was chosen as it has better performance than traditional linear mixed models (e.g. BOLT-LMM) where there is a large case–control imbalance (as in this case)17. In total, we tested 12,224,3568 variants. For our main analysis, we only included participants with sepsis before the age of 75, as genetic influences on sepsis are likely to wane at the extremes of age. Manhattan and quantile–quantile plots are shown in Supplementary Figs. S4 and S5. Figure 3 describes the instruments and outcomes used for this analysis, with detailed description of the included cohorts in the methods. https://doi.org/10.1038/s41598-023-29641-6 Scientific Reports \| (2023) 13:2867 \| www.nature.com/scientificreports/ nature.com/scientificreports/ Figure 1. Restricted cubic spline plots of ferritin level and odds ratio for sepsis. The dashed line represents an odds ratio of 1, representing a null association. ww.nature.com/scientificreports/ Figure 1. Restricted cubic spline plots of ferritin level and odds ratio for sepsis. The dashed line represents an odds ratio of 1, representing a null association. Figure 1. Restricted cubic spline plots of ferritin level and odds ratio for sepsis. The dashed line represents a odds ratio of 1, representing a null association. Inverse variance weighted MR g We then went on to perform Two Sample Mendelian randomisation. We extracted SNPs from the outcome GWAS and harmonised effect alleles with the exposure SNPs, for each biomarker (Ferritin, Iron, TIBC, and TSAT) individually, and ran MR for each SNP on sepsis individually21. To generate summary estimates for each biomarker, we performed fixed effects inverse variance weighted (IVW) meta-analysis of each included SNP using the TwoSampleMR package in R22. Development of outcome GWAS g p p g In IVW meta-analysis, increasing ferritin was associated with increased odds of sepsis (OR 1.17; 95% CI 1.04–1.29, for each SD increase in Ferritin, Table 3), with similar results for serum iron (OR 1.13; 95% CI 1.03–1.23), TSAT (OR 1.17; 95% CI 1.09–1.27), and with the opposite result for TIBC (OR 0.91; 95% CI 0.83–0.99), which would be expected as this marker decreases with increasing availability of iron. Scatter plots for TSAT—the biomarker with the strongest association is shown in Fig. 4, while scatter plots for other biomarkers are available in Supplementary Fig. S6. Parallel analyses y We ran parallel analyses using outcomes derived in another cohort, FinnGen. FinnGen is a large, Finnish cohort study, with linked genetic and hospitalisation data. Details of the inclusion criteria are with the original study22. Cases of sepsis were identified with the same ICD-10 coding as in UK Biobank, while details of the, study design, and GWAS methodology are in the methods and available at the FinnGen website22. In FinnGen, there were 7643 cases of sepsis and 234,755 controls, leading to a frequency of incident cases of 3.1%. As some SNPs were not available in this GWAS, we utilised LD proxies (r2 > 0.8), if SNPS were not available.if p ( ) In IVW meta-analysis, we identified broadly similar effect sizes for iron (OR 1.24; 95% CI 1.03–1.45), although effect estimates for the other biomarkers were weaker, although all estimates were concordant. Scatter plots are available in Supplementary Fig. S7.if pp y g We then went on to perform fixed effects meta-analysis across both cohorts. In this analysis, the summary estimates for each SD increase in iron biomarker was OR 1.09 (95% CI 1.02–1.15) for TSAT, OR 1.12 (95% CI 1.01–1.19) for iron, OR 1.08 (95% CI 0.98–1.18) for ferritin, and OR 0.93 (95% CI 0.93–0.98) for TIBC. These results are shown in Fig. 5 and Table 2. Effect of measured iron status on estimates of riskf Effect of measured iron status on estimates of riskf ff f f It is possible that the effect of iron related genetic variants varies by iron status. That is, it may be that increases in each biomarker have more or less of an effect in those who are clinically iron deficient or iron overloaded or those with and without anaemia. This analysis is important as the majority of iron supplementation occurs in https://doi.org/10.1038/s41598-023-29641-6 Scientific Reports \| (2023) 13:2867 \| www.nature.com/scientificreports/ those who either have biochemical iron deficiency (e.g. measured low ferritin), anaemia (e.g. low haemoglobin), or both. Initially, we compared estimates in those with or without anaemia on recruitment to UK Biobank. d d h b f fi d l h b f b k ( h h h Figure 2. Odds ratios for sepsis with increasing ferritin between 50 and 450 ug/L, with the dashed line representing an odds ratio of 1, a null association. Figure 3. Flow chart of included studies, exposures, and outcomes. Figure 2. Odds ratios for sepsis with increasing ferritin between 50 and 450 ug/L, with the dashed line representing an odds ratio of 1, a null association. Figure 2. Odds ratios for sepsis with increasing ferritin between 50 and 450 ug/L, with the dashed line representing an odds ratio of 1, a null association. Figure 3. Flow chart of included studies, exposures, and outcomes. Figure 3. Flow chart of included studies, exposures, and outcomes. those who either have biochemical iron deficiency (e.g. measured low ferritin), anaemia (e.g. low haemoglobin), or both. Initially, we compared estimates in those with or without anaemia on recruitment to UK Biobank. We did this by performing stratified analyses within substrata of UK Biobank (e.g. those with or without anaemia) using a polygenic risk score (PRS) derived and weighted from the same variants used in the two sample MR24. As we cannot reliably estimate the PRS-exposure association in UK Biobank, we cannot estimate a causal https://doi.org/10.1038/s41598-023-29641-6 Scientific Reports \| (2023) 13:2867 \| www.nature.com/scientificreports/ Figure 4. Scatter plot for increasing TSAT and risk of sepsis. Figure 4. Scatter plot for increasing TSAT and risk of sepsis. Figure 4. Scatter plot for increasing TSAT and risk of sepsis. Figure 5. Summary IVW MR associations across both FinnGen and UK Biobank. Figure 5. Summary IVW MR associations across both FinnGen and UK Biobank. Effect of measured iron status on estimates of riskf (MR) estimate using this approach, but can compare the effect sizes of the PRS in logistic regression in different strata. As we would not expect the PRS-exposure association to vary by exposure strata, this allows us to compare the effect size across two strata, and our effect sizes are reported per each change in standard deviation (SD) of the PRS, which was scaled to have a mean of 0 and an SD of 1. (MR) estimate using this approach, but can compare the effect sizes of the PRS in logistic regression in different strata. As we would not expect the PRS-exposure association to vary by exposure strata, this allows us to compare the effect size across two strata, and our effect sizes are reported per each change in standard deviation (SD) of the PRS, which was scaled to have a mean of 0 and an SD of 1. In this approach, described in detail in the methods, strata are generated using the residuals of the regression of a PRS for each biomarker on haemoglobin (i.e. isolating a form of non-genetic variation in haemoglobin), in order to avoid potential bias possibly generated by stratification using a variable correlated with those in main https://doi.org/10.1038/s41598-023-29641-6 Scientific Reports \| (2023) 13:2867 \| www.nature.com/scientificreports/ Table 2. Meta-analysed IVW MR estimates for each biomarker across both FinnGen and UK Biobank. Odds ratio Lower CI Upper CI P value FinnGen TSAT 1.05 0.94 1.16 0.41 Iron 1.24 1.03 1.45 0.04 TIBC 0.94 0.84 1.04 0.23 Ferritin 1.04 0.85 1.23 0.70 UK Biobank TSAT 1.17 1.09 1.24 < 0.001 Iron 1.13 1.03 1.23 0.02 TIBC 0.91 0.83 0.99 0.02 Ferritin 1.17 1.04 1.29 0.01 Combined—IVW meta-analysis TSAT 1.11 1.05 1.20 < 0.001 Iron 1.15 1.05 1.26 0.003 TIBC 0.92 0.87 0.98 0.01 Ferritin 1.13 1.02 1.25 0.02 Table 2 Meta analysed IVW MR estimates for eac Odds ratio Lower CI Upper CI P value FinnGen TSAT 1.05 0.94 1.16 0.41 Iron 1.24 1.03 1.45 0.04 TIBC 0.94 0.84 1.04 0.23 Ferritin 1.04 0.85 1.23 0.70 UK Biobank TSAT 1.17 1.09 1.24 < 0.001 Iron 1.13 1.03 1.23 0.02 TIBC 0.91 0.83 0.99 0.02 Ferritin 1.17 1.04 1.29 0.01 Combined—IVW meta-analysis TSAT 1.11 1.05 1.20 < 0.001 Iron 1.15 1.05 1.26 0.003 TIBC 0.92 0.87 0.98 0.01 Ferritin 1.13 1.02 1.25 0.02 Table 2. Meta-analysed IVW MR estimates for each biomarker across both FinnGen and UK Biobank. analysis25. Effect of measured iron status on estimates of riskf Normal (n = 413,264) Anaemic (n = 34,122) Estimate p value Estimate p value TSAT 1.17 (1.09–1.26) < 0.001 1.3 (1.07–1.52) 0.022 Iron 1.13 (1.02–1.24) 0.035 1.46 (1.16–1.76) 0.014 Ferritin 1.13 (1–1.25) 0.072 1.36 (1.02–1.7) 0.073 TIBC 0.88 (0.79–0.97) 0.006 0.96 (0.72–1.2) 0.735 Normal (n = 413,264) Anaemic (n = 34,122) Estimate p value Estimate p value TSAT 1.17 (1.09–1.26) < 0.001 1.3 (1.07–1.52) 0.022 Iron 1.13 (1.02–1.24) 0.035 1.46 (1.16–1.76) 0.014 Ferritin 1.13 (1–1.25) 0.072 1.36 (1.02–1.7) 0.073 TIBC 0.88 (0.79–0.97) 0.006 0.96 (0.72–1.2) 0.735 Table 3. Odds ratios for each PRS in the anaemic and non-anaemic population in UK Biobank. To explore this result further we then ran analyses in those with available ferritin levels from the observational analysis above (n = 75,277), in order to assess the impact of chronic iron stores on odds ratios generated by our PRS. As iron prescription is routinely indicated in those only with low ferritin, but occasionally given in those with higher levels, we chose to split this population into three strata using clinical definitions that are likely to affect the chance of a patient receiving treatment: iron deficient (< 50 ug/L), normal ferritin (50–450 ug/L), and high ferritin (> 450 ug/L). g g In this analysis, we generated PRS as for the haemoglobin analysis above, and used the same analysis approach using residuals to generate strata to avoid collider bias.if g g For those with iron deficiency (low ferritin) and with normal ferritin, effect estimates were positive and similar (Fig. 7, Table 4), suggesting that increasing levels of iron biomarkers are associated with an increased rate of sepsis even in those with low/normal ferritin. In contrast, for the small number of participants with a Figure 7. Odds ratios per unit SD increase in each PRS within each stratum of ferritin for each iron biomarker. igure 7. Odds ratios per unit SD increase in each PRS within each stratum of ferritin for each iron biomarker. Table 4. Odds ratios for a one SD increase in each PRS in each stratum of ferritin level. Effect of measured iron status on estimates of riskf Haemoglobin levels were available on admission to UK Biobank for nearly all European ancestry participants with genetic data (n = 448,978) and we stratified patients into anaemic and non-anaemic as per NICE definitions (Hb < 125 g/L for women, Hb < 135 g/L for men). Although the potential for collider bias was present, we found that each PRS had very little effect on haemoglobin levels, with the correlation between the original haemoglobin and the residuals of the regression of > 0.99 in all cases. analysis25. Haemoglobin levels were available on admission to UK Biobank for nearly all European ancestry participants with genetic data (n = 448,978) and we stratified patients into anaemic and non-anaemic as per NICE definitions (Hb < 125 g/L for women, Hb < 135 g/L for men). Although the potential for collider bias was present, we found that each PRS had very little effect on haemoglobin levels, with the correlation between the original haemoglobin and the residuals of the regression of > 0.99 in all cases. g g g For both anaemic and non-anaemic participants, we then performed logistic regression using our PRS. We identified—contrary to our expectations—that estimates of the odds of sepsis were generally higher in those with anaemia. For ferritin, the OR per one SD increase in the PRS for the anaemic cohort was 1.36 (95% CI 1.02–1.7), while it was only OR 1.13 (1.00–1.25) for the non-anaemic cohort. We identified similar effect changes for both TSAT and Iron (Fig. 6, Table 3), although effect estimates were similar for TIBC in both cohorts. Figure 6. Odds ratios for each SD increase in each PRS for sepsis in anaemic and non-anaemic population in UK Biobank. Figure 6. Odds ratios for each SD increase in each PRS for sepsis in anaemic and non-anaemic population in UK Biobank. https://doi.org/10.1038/s41598-023-29641-6 Scientific Reports \| (2023) 13:2867 \| www.nature.com/scientificreports/ Table 3. Odds ratios for each PRS in the anaemic and non-anaemic population in UK Biobank. Discussion In both observational and MR estimates, we found evidence of increasing odds of sepsis with increasing levels of iron and associated biomarkers. There was a suggestion that effect sizes were larger in those who were iron deficient or anaemic for both TSAT and iron, which may indicate that increases in iron even in those who have reduced iron stores (e.g. low ferritin) are associated with an increased risk of sepsis. Effect sizes were broadly similar in FinnGen, despite the well-recognised heterogeneity in sepsis presentation, definition, and coding across cohorts26. One previous study has used two sample Mendelian randomisation to evaluate the relationship between iron biomarkers and sepsis, using a smaller cohort for identification of variants, and using a previously performed GWAS of sepsis in UK Biobank27. In common with our study, they identified associations with iron biomarkers and the odds of sepsis (OR for iron = 1.21, 95% CI = 1.13–1.29, p = 3.16 × 10−4). However, this study included much fewer SNPs, as the GWAS used was around five to ten times smaller (n = 23,986 vs n = 246,139 for ferritin), meaning that only two SNPs were included for iron status, and three SNPS included for transferrin saturation and ferritin. This study was therefore heavily reliant on the inclusion of the rs1800562 SNP in HFE, which is the causative SNP for 85% of cases of hereditary haemochromatosis28,29, making these results difficult to interpret outside this condition. In addition, a further study performed an MR-PheWAS of iron levels in UK Biobank, with the strongest association being skin and soft tissue infection (OR 1.25; 95% CI 1.1–1.42 for each SD increase in serum iron)27,28. Although this study did not identify an association with sepsis, there were fewer cases (7628 vs 15,614 in our analysis) at the time in UK Biobank. Secondly, a recent study of patients who carry mutations in the gene HFE leading to hereditary hemochromatosis, the most common cause of iron overload in the UK, identified an increased risk of pneumonia in male patients carrying HFE variants5. In non-genetic literature, there have been a number of randomised trials of iron supplementation, some of which have measured infection as an outcome. www.nature.com/scientificreports/ www.nature.com/scientificreports/ ferritin of > 450 ug/L, effect estimates were reversed, although there was substantial uncertainty about the effect estimates due to a small sample size. ferritin of > 450 ug/L, effect estimates were reversed, although there was substantial uncertainty about the effect estimates due to a small sample size. Testing assumptions of MR g p f We then performed sensitivity analyses. Using our polygenic risk score (derived above) in all UK Biobank participants, we looked for associations with potential confounders of sepsis using the PHESANT software25 (Supplementary Table S2), testing for horizontal pleiotropy. This software allows for the generation of the PRS- outcome association in UK Biobank for a large number of quantitative and binary outcomes in UK Biobank, and is equivalent to performing MR for these traits to identify potential associations between iron status and confounders. We found no strong evidence for association between age, sex and most clinical comorbidities, although there were associations identified for liver disease (OR 1.03; 95% CI 1.01–1.05, p = 0.002), and a history of cancer (OR 1.01; 95% CI 1.00–1.03, p = 0.01). Both are known associations of iron overload. p Subsequently, we performed a leave one out (LOO) analysis; whereby each SNP is removed from the MR one at a time, to ensure no individual SNP is driving the results. All results were robust to the LOO analysis, with little change in estimates (Supplementary Fig. S8). Importantly, results were robust with or without inclusion of variants in the HFE gene, the commonest genetic cause of iron overload in the U.K. g g Subsequently we meta-analysed the results using the MR-Egger approach, MR-PRESSO and weighted median approach (Fig. 3, Supplementary Table 3), which have differing underlying assumptions than the IVW method. Although the effect size of the estimates differed slightly, we found similar results regardless of statistical approach. We did not identify any outliers with the MR-PRESSO global test. Finally, we looked at whether the intercept of the MR-Egger analysis differed strongly from 0, which can indicated unbalanced pleiotropy15. However, all intercepts were close to 0, with formal tests for heterogeneity negative (Intercepts 0.002 for TSAT; − 0.006 for iron; − 0.001 for ferritin, − 0.002 for TIBC, all p > 0.3). Sensitivity analyses y y As a sensitivity analysis for case definition, we ran the same analysis including cases of sepsis diagnosed in those of all ages (including those aged over 75). Discussion These are summarised in a recent meta-analysis of 154 randomised trials of intravenous iron that found an increased risk of infection (RR 1.16; 95% CI 1.03–1.29), although there was marked heterogeneity across studies, and intravenous iron was compared against both “no iron”, and iron supplementation12. As intravenous iron is associated with around a one SD increase in TSAT, our estimates are remarkable concordant with the trial data.f In complementary in-vitro work, one recent experimental study explored the effect of bacterial growth in serum taken from oral iron supplementation in healthy adults and identified an increase in TSAT from 42.1% (SD 12.5%) to 75.7% (SD 18%) and total serum iron increased from 30.3 μmol/L (SD 10.2) to 53.0 μmol/L (SD 15.8) at 4 h after iron supplementation, and identified TSAT as the key driver of bacterial growth, with positive associations at all levels of transferrin saturation. www.nature.com/scientificreports/ This showed broadly the same effect estimates as our primary analyses, although confidence intervals were broader. Supplementary Table S4 includes these estimates, and the IVW weighted MA results with FinnGen. These are visualised in Supplementary Fig. S9. Effect of measured iron status on estimates of riskf Normal (50–450 ug/L) Low Ferritin (< 50 ug/L) High ferritin (> 450 ug/L Estimate p value Estimate p value Estimate p value TSAT 1.19 (0.97–1.42) 0.128 1.33 (1.03–1.64) 0.062 0.83 (0.29 to 1.37) 0.498 Iron 1.31 (1.01–1.62) 0.077 1.43 (1.02–1.85) 0.084 0.41 (− 0.3 to 1.13) 0.016 Ferritin 1.19 (0.85–1.53) 0.317 1 (0.52–1.47) 0.985 0.52 (− 0.34 to 1.37) 0.130 TIBC 0.99 (0.75–1.23) 0.956 0.92 (0.6–1.25) 0.636 1 (0.45 to 1.55) 0.989 Table 4. Odds ratios for a one SD increase in each PRS in each stratum of ferritin level. Normal (50–450 ug/L) Low Ferritin (< 50 ug/L) High ferritin (> 450 ug/L Estimate p value Estimate p value Estimate p value TSAT 1.19 (0.97–1.42) 0.128 1.33 (1.03–1.64) 0.062 0.83 (0.29 to 1.37) 0.498 Iron 1.31 (1.01–1.62) 0.077 1.43 (1.02–1.85) 0.084 0.41 (− 0.3 to 1.13) 0.016 Ferritin 1.19 (0.85–1.53) 0.317 1 (0.52–1.47) 0.985 0.52 (− 0.34 to 1.37) 0.130 TIBC 0.99 (0.75–1.23) 0.956 0.92 (0.6–1.25) 0.636 1 (0.45 to 1.55) 0.989 Table 4. Odds ratios for a one SD increase in each PRS in each stratum of ferritin level. Table 4. Odds ratios for a one SD increase in each PRS in each stratum of ferritin level. https://doi.org/10.1038/s41598-023-29641-6 Scientific Reports \| (2023) 13:2867 \| www.nature.com/scientificreports/ in observational analyses, it is clear that there are large differences in covariates across ferritin levels as a result of the sampling frame for available testing results, and as a result of the observational cohort. For instance, the relatively complex pattern of association, identified in other biomarkers (e.g. lymphocyte count, BMI28,29), with increased odds of incident sepsis cases in those with severe iron deficiency likely represents the increased risk associated with the disease driving severe iron deficiency (e.g. malignancy). i Secondly, common to all MR based studies, interpretation of an estimate for lifelong exposure to iron via germline genetic variation when assessed in other contexts, such as iron supplementation, requires justification. Iron exposure via supplementation is generally short lived, increases iron biomarkers over a short period (hours to days), and by a much larger amount than via most common SNPs. It is difficult to directly compare iron supplementation (individual events leading to short, time limited increases in iron levels) with lifelong increased iron exposure from genetic variants. However, the odds ratio for sepsis with each increasing standard deviation of TSAT from our meta-analysis of both cohorts (OR 1.11, 95% CI 1.05–1.20), should be put in the above context that oral supplementation increases TSAT by around 2–3 standard deviations over 4–8 h, and has longer term effects30,31; while intravenous iron increases TSAT by around a standard deviation for at least three months in non-anaemic populations with chronic kidney disease32.h p p y Thirdly, the heterogeneity of sepsis presents both a strength and a weakness. Sepsis is caused by a variety of bacteria, fungi, and viruses, only some of which scavenge free iron as a nutritional source (e.g. S. aureus, a common and important pathogen, uses largely heme-derived iron, and bacterial growth has been shown to be independent of serum iron levels8). As we were unable to access pathogen specific data, we could not identify pathogen-specific effects. This is compounded by the heterogeneity of clinical presentation and coding of sepsis, which has the effect of also reducing power significantly26,33. In that context, we were reassured to see similar effect sizes across both UK Biobank and FinnGen, supporting our main findings and supporting the biological plausibility of the effect. Data sources For both analyses we primarily used UK Biobank, a volunteer cohort of approximately 500,000 participants, that contains both genetic, physical, and biomarker information, and is linked to UK electronic health records. Recruitment started in 2006, ended in 2010, and participants continue to be followed up17. Data for this study was extracted in October 2021. UK Biobank received ethical approval from the Research Ethics Committee (REC reference for UK Biobank is 11/NW/0382). This study was performed under application number 52643. Informed consent was was obtained from all participants in UK Biobank. All analyses were performed in line with the relevant guidelines and regulations and in line with the ethical approval. g g pp For our MR analysis, exposures sources and our parallel analyses in FinnGen are detailed below. Implications p Although these estimates support the randomised trial literature, ongoing research should focus on estimating absolute risks of severe infection across differing situations and with differing baseline iron levels. Further mechanistic understanding of how iron homeostasis setpoint changes are linked to the risk of sepsis, and the implications for iron management within infection, are warranted. Given that iron homeostasis is critical for both pathogens and hosts, it is important to understand whether these changes in iron homeostasis alter sepsis incidence via simply increasing iron availability to pathogens, or by an altered immune response to pathogens. Our findings and those from recent trial and in-vitro data, provide further evidence that iron state is a predictor of risk of severe infection, and also suggest caution in iron supplementation in areas without robust evidence of benefit. www.nature.com/scientificreports/ yf Finally, our approach in using residuals from a PRS of iron to develop strata in which to generate strata specific estimates allows us to make inference on whether the effect of increasing levels of iron biomarkers are likely to be the same in important subgroups (e.g. anaemic patients). However, the reversal of the effect estimate for increasing iron in those with high (> 450 ug/L) ferritin was unexpected. Although it is possible this effect estimate is real, it may be that this represents an unaccounted-for collider bias (those who have high ferritin despite a low PRS for iron have a more sinister cause for their hyperferritinaemia). Alongside this, we make the assumption that the PRS-exposure association remains similar across all strata of iron and ferritin levels, although this assumption is also made in non-linear MR approaches18. Study design y g An observational cohort study and two sample Mendelian randomisation analysis. Conclusion Observational data shows ferritin levels have a U-shaped relationship with sepsis, but the odds of sepsis increase even within the normal range of iron levels. Mendelian randomisation analyses show that physiologically relevant increases in iron biomarkers causally associated with an increased risk of sepsis, regardless of baseline iron status. These findings are in keeping with the hypothesis that iron supplementation increases the risk of severe infection and suggest further research into the implications of perturbing iron homeostasis. Strengths and weaknessesh g The current work combines both traditional observational epidemiology with two sample MR using high quality genetic and phenotypic data, and the Hospital Episode Statistic (HES) based coding, but has limitations. Firstly, https://doi.org/10.1038/s41598-023-29641-6 Scientific Reports \| (2023) 13:2867 \| www.nature.com/scientificreports/ Statistical approachh The observational analysis used the raw ferritin level to estimate the risk of sepsis using logistic regression models adjusted for age, sex, UK Biobank recruitment centre, and the potential confounders listed above.i As very low ferritin (due to illness leading to iron deficiency) may alter the linearity of estimates, we also used restricted cubic spline modelling, using the rms package in R, and calculated specific estimates excluding those with likely iron deficiency (serum ferritin < 50 ug/L)34. Exposure statistics p For our primary analysis, we included independent (R < 0.1) SNPs that met genome wide significance (P < 5 × 10–8) in the recent iron meta-analysis34. Details of included SNPs, F-statistics, and data on the other biomarkers is included in Supplement S2. Outcome definition Cases of sepsis were identified in UK Biobank linked ICD-coded hospital admission data. ICD-10 codes A02, A39, A40 and A41 were used to identify sepsis. Cases were included if the code was in either the primary or secondary diagnostic position in the linked Hospital Episode Statistic (HES) data. All patients who had a code for sepsis before the age of 75 were included (13,260/16,173, or 82% of total cases), as the relative contribution of germline genetic variation on iron status on sepsis in extreme age was expected to be much smaller. As a sensitivity analysis, we included cases of sepsis at all ages. www.nature.com/scientificreports/ data linked to UK Biobank participants. We excluded extreme values (> 10,000). The index ferritin level was taken as the first recorded test result after the date the participant was recruited to UK Biobank (between 2006 and 2010). For participants with multiple ferritin test results, only the index ferritin level was used. All participants with available, linked data were included. data linked to UK Biobank participants. We excluded extreme values (> 10,000). The index ferritin level was taken as the first recorded test result after the date the participant was recruited to UK Biobank (between 2006 and 2010). For participants with multiple ferritin test results, only the index ferritin level was used. All participants with available, linked data were included. We extracted demographic information and potential confounders from UK Biobank data: smoking status, alcohol usage, Townsend Deprivation Index, BMI, renal function, liver disease, presence of malignancy, and baseline inflammation (CRP on recruitment to UK Biobank). Variables were derived from UK Biobank questionnaire or algorithmic data. Included variables were all measured on recruitment and thus preceded ferritin testing and outcome ascertainment. Study design—Mendelian randomisation Source of instruments f For our instruments, we extracted GWAS summary statistics from a recent meta-analysis of three separate GWA studies on four iron biomarkers (iron, transferrin saturation (TSAT), total iron binding capacity (TIBC), and ferritin)33. The original studies were performed in Iceland (deCODE genetics, n = 285,664), Denmark (Danish Blood Donor Study, n = 33,727), and the UK (Interval Study, n = 43,059). Details on each studies recruitment, follow up, GWAS and the meta-analytic methodology are included in their publication19. Each biomarker was inverse rank-normalised transformed before GWAS, so the effect sizes are reported as per standard deviation change. For each biomarker, this genetic heritability (the proportion of variance explained by the SNPs alone) was estimated to be 16% for Iron, 22% TSAT, 25% for TIBC, and 30% for Ferritin. Choice of primary instrument Study design—observational cohorti y g Data sources and definitions fi Approximately half of all participants in UK Biobank have associated primary care data (n = 222,081), includ laboratory results. For our observational cohort, we extracted all serum ferritin levels recorded in primary https://doi.org/10.1038/s41598-023-29641-6 Scientific Reports \| (2023) 13:2867 \| www.nature.com/scientificreports/ Choice of primary instrument f p y For a primary biomarker of acute iron status we chose TSAT as this represents the biomarker most rapidly altered by iron supplementation, is the best predictor of haemoglobin response due to iron supplementation, and has the strongest prior relationship with infection outcomes8,35,36. Definition of instrument Definition of instrument i For each biomarker, we extracted all SNPs that were available in the HRC-imputed UK Biobank genetic data and exposure data and met a threshold of p < 5 × 10–8, then performed LD-clumping to remove correlated variants (max R2 0.01), using the TwoSampleMR package20. p p (max R2 0.01), using the TwoSampleMR package20. g g 17 SNPs for TSAT with a mean F-statistic of 325 were included. (Conventionally, scores of > 10 are consistent with strong instruments)21. The minimum F-statistic included was 36.4. For serum iron, we included 23 SNPs (mean F statistic 186.7), for TIBC 26 SNPs (mean F statistic 214.3), and 50 SNPs for Ferritin (mean F statistic 86.7). As not all SNPs were available in FinnGen, we used harmonised, proxy SNPs where LD proxied SNPs were available, resulting in 16 SNPs available for TSAT, 22 for Iron, 25 for TIBC and 50 for Ferritin.f As the original GWAS were performed using transformed data, the effect sizes are in relation to standard deviation increases in rank-normalised transformed values. In the DECODE Icelandic cohort, the standard deviation on untransformed data for TSAT was 11.6%, for serum iron 6 µmol/L, for ferritin 224 ug/L, and for TIBC it was 12.6 µmol/L. Exposure statistics Ancestry y We restricted the sample to individuals of ‘European’ ancestry as defined by an in-house kmeans cluster analysis performed using the first 4 principal components provided by UK Biobank in the statistical software environment R. The current analysis includes the largest cluster from this analysis (n = 464,708)23. Statistical approach pp Univariable two sample, Mendelian randomisation (MR) analyses were conducted using the R package TwoSampleMR to investigate total effect estimates between genetically predicted iron status (for each biomarker individually) with sepsis38. This was based on the inverse variance weighted (IVW) method, which estimates the causal effect of an exposure on an outcome by combining ratio estimates using each variant in a fixed effect meta-analysis. Testing for interactions with respect to ferritin and haemoglobin levelsii Testing for interactions with respect to ferritin and haemoglobin levels As total iron state (e.g. iron deficiency defined by low ferritin) might affect MR estimates for the acute iron biomarkers, we ran specific analyses in those with varying levels of Hb and ferritin. g f p f g As total iron state (e.g. iron deficiency defined by low ferritin) might affect MR estimates for the acute iron biomarkers, we ran specific analyses in those with varying levels of Hb and ferritin. i To do this, we generated weighted allelic polygenic risk score (PRS) generated for each UK Biobank participant for each biomarker. All SNPs from the MR analysis were included, with weighting of each SNP by the effect size of that SNP. With certain underlying assumptions, this allelic risk score can be then used to perform MR38. We then extracted haemoglobin levels (from blood taken on recruitment to UK Biobank), and ferritin levels (from the primary care data, as detailed above). We also extracted sepsis cases occurring after blood sampling, using the same approach as in the observational data. pp We then performed ordinary least-squares (OLS) regression for each PRS on haemoglobin and ferritin, individually, and extracted the residuals from this regression. In effect, this removes the PRS (“genetic”) effect on each biomarker, leaving only the non-genetic components, an approach previously used to assess the dose–response relationship of other biomarkers13,24,39. We then stratified these residuals. For haemoglobin, we stratified into anaemic and non-anaemic (Hb < 125 g/L for women, Hb < 135 g/L for men). Ancestry For ferritin, we split into three cohorts: Iron deficient (< 50 ug/L), normal ferritin (50–450 ug/L), and high ferritin (> 450 ug/L). This use of residuals for strata is required as a naïve analysis stratifying on raw data would be potentially biased, as the PRS will associate with each strata (e.g. those with lower PRS for iron more are likely to be anaemic).f Subsequently, within the generated strata, we ran a logistic regression for each PRS on sepsis cases. In effect, this allows us to estimate whether MR estimates are altered in those who have differing levels of haemoglobin and/or ferritin. GWAS A case–control GWAS was performed of using regenie v2.2.4, on all UK Biobank participants of European ancestry13,23,35. In house algorithms were used to perform quality control and define ancestry with details available elsewhere19,37. GWAS was performed adjusting for age, sex, genetic chip, UK Biobank assessment centre and the first ten principal components13,24,36. https://doi.org/10.1038/s41598-023-29641-6 Scientific Reports \| (2023) 13:2867 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ Full details on the methodology are available elsewhere, but are described below in brief21. Regenie was chosen as it reduces type 1 error when there is significant case control imbalance, as compared to other methods such as BOLT-LMM37. Manhattan and QQ-plots for GWAS performed as part of this study are available in Supplementary Figs. 4 and 5. Summary statistics are available at the IEU Open GWAS repository (https://gwas. mrcieu.ac.uk/datasets/ieu-b-5066/). Genotyping and imputationh yp g p The data release for this analysis contains the cohort of successfully genotyped samples (n = 488,377). 49,979 individuals were genotyped using the UK BiLEVE array and 438,398 using the UK Biobank axiom array. Pre- imputation QC, phasing and imputation are described elsewhere23. In brief, prior to phasing, multiallelic SNPs or those with MAF ≤ 1% were removed. Phasing of genotype data was performed using a modified version of the SHAPEIT2 algorithm. Genotype imputation to a reference set combining the UK10K haplotype and HRC reference panels was performed using IMPUTE2 algorithms.hi p p g g The analyses presented here were restricted to autosomal variants using a graded filtering with varying imputation quality for different allele frequency ranges. Therefore, rarer genetic variants are required to have a higher imputation INFO score (Info > 0.3 for MAF > 3%; Info > 0.6 for MAF 1–3%; Info > 0.8 for MAF 0.5–1%; Info > 0.9 for MAF 0.1–0.5%) with MAF and Info scores having been recalculated on an in-house derived ‘European’ subset23. Data quality control q y Individuals with sex-mismatch (derived by comparing genetic sex and reported sex) or individuals with chromosome aneuploidy were excluded from the analysis (n = 814). Data availability Access to individual data for UK Biobank is via application to the independent data access committee. For this paper, summary outcome GWAS were released to the OpenGWAS repository (https://gwas.mrcieu.ac.uk/datas ets/ieu-b-5066/), and summary exposure data are available at deCODE.genetics website (https://www.decode. com/summarydata/), and therefore all MR results can be replicated using the TwoSampleMR package21. Received: 27 September 2022; Accepted: 8 February 2023 Received: 27 September 2022; Accepted: 8 February 2023 References References 1. Cappellini, M. D., Musallam, K. M. & Taher, A. T. Iron deficiency anaemia revisited. J. Intern. Med. 287(2), 153–170 (2020).hi 1. Cappellini, M. D., Musallam, K. M. & Taher, A. T. Iron deficiency anaemia revisited. J. Intern. 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Reporting Th d Reporting This study is reported in line with the STROBE-MR reporting guidelines, available in the supplement43. Sensitivity analyses Mendelian randomization analysis assumes that a genetic instrument used to proxy a risk factor (1) is associated with the risk factor (“relevance”), (2) does not share a common cause with the outcome (“exchangeability”), and (3) affects the outcome only through the risk factor (“exclusion restriction”). To test relevance, we measured the F-statistic for each exposure for each biomarker, while the previous meta-analysis has shown a reasonable proportion of variance explained (> 15% for each biomarker)40. p p p To test the assumption of exclusion restriction we tested a weighted allelic risk score (described below) against confounders (sex, BMI, diabetes, deprivation and, smoking status) by OLS regression using a specific MR-PheWAS software (PHESANT)25. Finally we also performed a set of statistical sensitivity analyses that test assumptions of MR, including different meta-analytic methods: MR-Egger, weighted median, MR-PRSSO, and iterative leave one out analyses, all of which test various assumptions of the MR approach15,40. Scientific Reports \| (2023) 13:2867 \| https://doi.org/10.1038/s41598-023-29641-6 www.nature.com/scientificreports/ Parallel analysesh y The linear analysis was run in parallel using the FinnGen R6 summary statistics for sepsis22. As some SNPs were not available in this cohort, SNP proxies (LD > 0.8) were selected and harmonised to ensure the effect alleles matched using the gwasVCF and TwoSampleMR package if a SNP was not available40.if We report both the FinnGen results, and a fixed effects meta-analysis of both cohorts using the meta package in R41. 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Int. 18(1), 38–46 (2014). 37. Mitchell, R., Elsworth, B. L. & Mitchell, R. et al. MRC IEU UK Biobank GWAS pipeline version 2 [Internet] (2019). https://data.bris. ac.uk/data/dataset/pnoat8cxo0u52p6ynfaekeigi/.h ac.uk/data/dataset/pnoat8cxo0u52p6ynfaekeigi/.h p p y g 8. Burgess, S. & Thompson, S. G. Funding g FH’s time was funded by the GW4-CAT Wellcome Doctoral Fellowship Scheme (202802/Z/16/Z). UK Biobank was funded by the Wellcome Trust, the Medical Research Council, the NIHR, and a variety of other charities (https://www.ukbiobank.ac.uk/learn-more-about-uk-biobank/about-us/our-funding). FinnGen is a public– private partnership (https://www.finngen.fi/en/access_results) funded by multiple instititions across Finland. We want to acknowledge the participants and investigators of the FinnGen study. PG’s time was funded by the Ser Cymru programme, the Welsh Government, and the EU-ERDF. The funder had no role in the design, analysis, or reporting of this study. Author contributions F.H. devised the study with N.T., P.G., and R.M. H.A. provided clinical expertise and review. F.H. wrote the manuscript, which all authors reviewed. N.T. and P.G. supervised F.H. www.nature.com/scientificreports/ Use of allele scores as instrumental variables for Mendelian randomization. Int. J. Epidemiol. 42(4) 1134–1144 (2013). 9. Aune, D. et al. BMI and all cause mortality: Systematic review and non-linear dose-response meta-analysis of 230 cohort studies with 3.74 million deaths among 30.3 million participants. BMJ 353, i2156 (2016). 40. Verbanck, M., Chen, C.-Y., Neale, B. & Do, R. Detection of widespread horizontal pleiotropy in causal relationships inferred from Mendelian randomization between complex traits and diseases. Nat. Genet. 50(5), 693–698 (2018). 41. Schwarzer, G., Carpenter, J. R. & Rücker, G. Meta-Analysis with R (Springer International Publishing, 2015). l d l l d l d h l l 2. Burgess, S. Sample size and power calculations in Mendelian randomization with a single instrumental variable and a binary outcome. Int. J. Epidemiol. 43(3), 922–929 (2014). 3. Skrivankova, V. W. et al. Strengthening the reporting of observational studies in epidemiology using mendelian randomisation (STROBE-MR): Explanation and elaboration. BMJ 375, n2233 (2021). References Causal effects of genetically predicted iron status on sepsis: A two-sample bidirectional mendelian rando study. Front. Nutr. 8, 747547 (2021). y y g y 7. Hu, Y. et al. Causal effects of genetically predicted iron status on sepsis: A two-sample bidirectional mendelian randomization study. Front. Nutr. 8, 747547 (2021). y 28. Hamilton, F., Arnold, D. & Payne, R. Association of prior lymphopenia with mortality in pneumonia: A cohort study in UK primary care. Br. J. Gen. 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W3107568192.txt	https://www.degruyter.com/document/doi/10.1515/soci-2020-0005/pdf	en		University language policy in Estonia and Catalonia.<b> Is there a north-south divide in European higher education?</b>	Sociolinguistica	2,020	cc-by	6,674	Josep Soler University language policy in Estonia and Catalonia. Is there a north-south divide in European higher education? Abstract: Within the context of the internationalisation of higher education, language matters have become increasingly prominent for universities across a wide variety of contexts. This has made the higher education domain an attractive site for applied linguists and sociolinguists to investigate in close detail. One relatively consolidated idea is that contexts in which English has made further incursions in universities are more internationalised than those settings where English has penetrated to a lesser extent. In line with that, a supposed north-south divide in European higher education has been conceived of, with universities in the north leading in terms of internationalisation efforts, while those in the south lag behind. In this article, I problematise this idea and suggest instead that a narrower and more focused contrastive analysis between language ecologies across different countries and contexts might be more useful. In doing that, we can thus avoid the dangers of binarism and lumping that can come associated to holding preconceived geographical borders. In particular, the comparative analysis proposed here allows us to highlight two key issues that seem to be highly pressing in present-day higher education domains: the language-ideological and the socio-economic. I build the argument in the paper by utilising empirical material from Estonia and Catalonia, two contexts that have been at the centre of my own work in recent years. Keywords: Language policy, higher education, internationalisation, Estonia, Catalonia. 1 Introduction One relatively wide-spread idea about the internationalisation of higher education and its connection to the English language is that, bluntly put, more English translates into more internationalisation. This, of course, has been problematised already, but somehow, the idea persists. More narrowly, a common-sense conclusion derived from this widespread idea is that the more a university system incorporates English, particularly (but not exclusively) for teaching purposes, the more it can be considered an internationalised environment. From this conclusion, it follows that universities in the north of Europe are more internationalised than those in the south, with the Josep Soler, Department of English, Stockholm University, SE-106 91 Stockholm, Sweden, josep.soler@english.su.se Open Access. © 2020 Soler, published by De Gruyter. the Creative Commons Attribution 4.0 International License. https://doi.org/10.1515/soci-2020-0005 This work is licensed under 58 Josep Soler former having more institutions offering more English-taught programmes than those in the latter (e. g. Wächter/Maiworm 2014). Such an oversimplification, however, may lead scholars to think that because institutions and their members operate in very distinct sociolinguistic realities, the issues that they may face are essentially different as well. Partly, that can certainly be true. However, it is also possible to argue that if we scratch a bit deeper than the surface level provided by numbers, we may see that regardless of geographical location, institutions and their members are dealing with very similar concerns, responding through language policy developments to issues connected with ideological and economic matters. This is the argument that I shall illustrate in this article, utilising empirical material from the higher education contexts of Estonia and Catalonia. Estonia and Catalonia are not a frequent pairing in comparative sociolinguistics, although some authors have in the past analysed in some detail their differences and similarities from that very same point of view (e. g. Branchadell 2011; Laitin 1992; Shafir 1995; Skerrett 2010; Soler 2013). The rationale behind looking at the two cases comparatively rests mainly on historical, socio-political, and sociolinguistic factors. During an extended period in the 20th century, the two nations found themselves dominated by authoritarian regimes (Francoism, 1939–1975, and the Soviet totalitarianism, 1940– 1991, respectively). In the course of those decades, the social, demographic and sociolinguistic landscapes of the two contexts were substantially modified by the arrival of migrant workers who settled in the growing industrial areas in the urban centres of the two places (Branchadell 2011). Moreover, those arrived during the period were speakers of the dominant language, Spanish and Russian respectively, who would in most cases be able to lead their lives without an important need to become proficient in the local language. Nevertheless, and this is an important difference, the influx of newcomers in Estonia stopped and even receded in the late 1980s and 1990s. In Catalonia, by contrast, the number of residents of migrant origin has continued to grow, almost exponentially, over the past decade in particular. It is therefore possible to state that Catalonia features a socially and linguistically more heterogeneous society than Estonia, despite the fact that the latest Population and Housing Census (Statistics Estonia 2011) has documented a total number of 157 different languages spoken presently in Estonia. In 1998, only 2.4 % of the population in Catalonia were of foreign origin (i. e. from outside Spain); by 2013, that percentage had increased up to 14.6 % (Idescat n.d.). In addition to historical and socio-demographic factors, another similarity between Estonia and Catalonia (particularly important for the purposes of this article) is the specific kind of language contact situation that exists in the two contexts: a medium-sized language (Catalan and Estonian, respectively) coexisting with a more widely spoken language (Spanish and Russian, respectively) in a position of contested power relations. Such contested power relations are not the exact same ones in Estonia as in Catalonia, but again, similarities can be observed, especially from the point of view of the institutional efforts in order to keep reinforcing the societal position of University language policy in Estonia and Catalonia. 59 the medium-sized language in both contexts. Even after the socio-political changes referred to above, dating back several decades, institutions in both Estonia and Catalonia are busy making sure speakers’ linguistic rights are preserved, and that the languages continue to be employed in public domains as resources for social cohesion. The label ‘medium-sized language’ might not be familiar to all readers. It is, however, a concept that has been theorised for some time now by researchers at the University of Barcelona (see e. g. Bastardas et al. 2018; Boix-Fuster 2015; Vila 2013). The main theoretical thrust behind this concept is to try to capture the sociolinguistic challenges facing language communities that fall in-between the categories of ‘majority’ and ‘minority’ languages. Medium-sized languages are primarily conceived of as languages in post-industrial, technologically advanced societies, many of which enjoy the quality of statehood (i. e. they are state-level, national languages). These languages have a strong degree of sociolinguistic vitality; in most domains, they are viable languages, likely to continue being transmitted from generation to generation. However, in the context of globalisation, increased transnational connections and global flows of goods, people, and information, societies with medium-sized languages find themselves facing similar concerns. Among others, such shared issues for speakers of medium-sized languages include the need to acquire languages of wider communicative reach in a more systematic way, making more active use of their plurilingual repertoire than it might have been the case in the past. In some domains in particular, the more intense contact with languages of wider communication might lead to questioning the long-term sustainability of the medium-sized language in that context. One specific domain that has been identified as particularly sensitive for the long-term presence of medium-sized languages is higher education (Vila/Bretxa 2015). Indeed, if we look at it in strict quantitative terms, only a handful of medium-sized languages around the world are used in the context of higher education, the majority of them in Europe, and mostly for teaching and learning purposes at lesser advanced levels (i. e. at undergraduate rather than graduate and postgraduate levels) (Laponce 2006). With this in mind, the question that follows almost inevitably is: will these medium-sized languages that have made it up to the level of higher education keep their presence in that domain, or will they eventually ‘quit the club’, as Vila (2015) puts it. In what follows, I take Estonia and Catalonia as illustrative cases of two medium-sized language communities and analyse some current issues in connection to their languages in the domain of higher education. In building my argument, I shall also tackle the perceived north-south divide in European higher education and the debates around the internationalisation of higher education. I shall show that perhaps such divide is less clear cut than is sometimes presented and that, whether in the north or in the south of Europe, higher education systems are shot through by issues of very similar nature, frequently with concerns that have to do with both identity and economic matters. 60 Josep Soler 2 Language, internationalisation, and higher education In the context of universities becoming increasingly international, as higher education institutions turn into internationally competitive and more attractive study and workplaces for a wider variety of students and staff, questions about language are increasingly more relevant. Indeed, language matters are laden by a multiple number of factors, including (but not limited to) practical, political, and socio-economic ones. At a practical level, questions about language in internationalising university environments typically are connected to issues of teaching and learning (if teaching takes place in an L2 or foreign language, debates emerge as to what best strategies should be followed to ensure that students’ learning process is not hampered). At a political level, in the Nordic countries in particular, concerns are normally aired in connection to the notion of domain loss, together with the idea that universities should continue to uphold their national responsibilities and make sure that the respective national language is used for meaningful purposes in the higher education domain. Finally, at a socio-economic level, universities, and particularly university officials, may worry about the sustainability of their budget bases, and in that respect, they may think of alternative ways of expanding their sources of income for example via the introduction of English-taught programmes (ETPs) for fee-paying students. With the above in mind, it is not surprising that, in recent times, universities have become attractive research sites for sociolinguists and applied linguists. Plenty of studies have been conducted in the past few years in connection to the three strands broadly outlined above: the practical, the political, and the socio-economic dimensions of higher education. The relevance of language, and language-connected issues, in all three areas has been more than attested by research with contributions from a wide variety of contexts. From all three perspectives, one particular sociolinguistic dynamic that has centred the focus of analyses is the increasing incorporation of the English language in more and more higher education systems outside Anglophone countries, and the effects that this has had at a national/local level. Discussions about the Englishization of higher education are nowadays abundant (e. g. Hultgren et al. 2014). However, the idea that the higher education domain deserved to be analysed from a sociolinguistic and language-political perspective is not entirely new. The same holds true for the focus on English. Ammon’s (2001) collection of essays is perhaps one of the earliest volumes on this topic, attesting that already at the turn of the century, in the context of a then growing literature on language and globalisation (e. g. Maurais/Morris 2003), universities were seen as a key site where issues of sociolinguistic nature, and specifically the impact of the English language, should be analysed in greater detail. Indeed, in the context of the post-national university (Bull 2004; Haberland/ Mortensen 2012) and in non-Anglophone settings, English seems to be behind many University language policy in Estonia and Catalonia. 61 of the debates of all kinds of nature (practical, political, socio-economic) in the higher education domain. On the one hand, there is the empirical reality that more universities in more countries are actively engaged in expanding the number of ETPs that they offer. Wächter and Maiworm’s 2014 report indicated a rise in the number of MA ETPs offered in European higher education from N=2389 in 2007 to N=8089 in 2014 (and only N=725 in 2003). Even with a methodological note of warning (more institutions were covered over the years in which the different reports were elaborated), the growth in absolute numbers of ETPs at the MA level is certainly without doubt. On the other hand, we see in parallel a development of language-ideological debates and controversies that emerge in higher education systems across a variety of countries, from Italy (Santulli 2015; Pulcini/Campagna 2015), to the Netherlands (de Rechtspraak 2018), to Latvia (The Baltic Times 2018). It might be far-fetched to suggest that English is the single or even the main cause of these debates, but it certainly is possible to trace a connection between a greater use of the language for different functions at universities and the emergence of language-related tensions and ambiguities in the context of higher education. Needless to say, thinking about higher education as one single, monolithic domain, would be misguided. Within universities, there is an ample range of contexts where different languages may come into play in complementary ways. In that regard, Hamel (2008) outlines three different spheres with a number of different activities and sub-activities within these spheres in the context of higher education (see also Darquennes/du Plessis/Soler 2020). Depending on the context, each of these activities can be carried out more frequently in one language or another. In Hamel’s model, the three main spheres in higher education consist of (a) scientific research/knowledge production, (b) diffusion and communication/knowledge consumption, and (c) scientific training. Haberland (2014) adds a fourth dimension to Hamel’s model: administration, which is by now increasingly seen as another key part of universities’ language ecologies in the context of higher education internationalisation (Llurda et al. 2014; Siiner 2016). All these different spheres are context-dependent and they may entail different linguistic practices and ideologies (Haberland 2014: 253–254). When conducting research, for example, everyday conversations in laboratories and departments, discussing project developments and so on can be certainly done in languages other than English, in non-Anglophone universities (Bretxa et al. 2016). Scientific communication (diffusion) and knowledge consumption is increasingly more dominated by English, a trend that is discipline specific (Kuteeva/Airey 2014: 539) and more recent in the humanities than in natural sciences. In terms of scientific training (i. e. education), again there is field-specific variation, as well as degree level variability, with more English being used in the hard sciences and in graduate level courses than in the humanities and in undergraduate degrees (Wächter/Maiworm 2014). So, there is a variety of situations and contexts and there is a variability of languages used in them that is contingent on a great number of factors. With this in mind, it is not surprising that university officials and administrators may find it hard 62 Josep Soler to address current sociolinguistic challenges at universities. In the Nordic countries, the approach taken from a language policy point of view has been characterised by the strategy known as ‘parallel language use’ (Linn 2010), a strategy that in principle entails that languages can be used in parallel in all the different contexts of the higher education domain. Inherently vague and ambiguous in nature, this approach has been already problematized (e. g. Fabricius et al. 2017; Kuteeva 2014). Indeed, in the context of Swedish higher education, Hult/Källkvist (2015) show that ‘parallel language use’ may be discursively shaped differently depending on the scale in which it is projected: local/national versus global. In the former, ‘parallel language use’ might frequently mean ‘more Swedish’, whereas in the latter, it might mean ‘more English’. Nevertheless, this is the approach that, formally, the vast majority of universities in Sweden have taken when developing their specific language policy documents (Björkman 2014). 3 E stonia and Catalonia: language policies and socio-economic factors in higher education With the above in mind, how do institutions of higher education in Estonia and Catalonia address language-related questions? How are language matters framed in university policy documents in the two contexts? How is language made relevant against the background of socio-economic concerns? In what follows, I tackle these questions by revisiting some of my own work on these topics, adding some original nuances to the arguments I have already presented elsewhere, in particular in connection to the socio-economic dimension. I shall, however, restrict myself to the most relevant elements of my analyses for the sake of the argument I construct in the article. Thus, I spare the reader of the methodological details of the work I draw on (mostly revolving around ethnographic and discursive approaches to language policy analysis); these are, nevertheless, readily available to anyone interested in these more intricate details in my recent work (see, in particular, Soler [2019], for the Estonian case, and Soler/ Gallego-Balsà [2019], for the Catalan case). Let us start with university language policy documents in the first place. With a combination of content and thematic analysis (Braun/Clarke 2006; Hsieh/Shannon 2005), the main themes that emerge from the language policy documents in Estonian and Catalan higher education are not drastically different (see also Soler et al. 2018; Soler 2019: chapter 2; Soler/Gallego-Balsà 2019: chapter 3). Table 1 below summarises the list of the four key themes from each of the two contexts. University language policy in Estonia and Catalonia. 63 Table 1: Main themes from university language policy documents in Estonia and Catalonia Estonia Catalonia Theme 1: Estonian needs to be protected, promoted, and developed. Theme 1: Catalan is the own and official language of Catalonia and of Catalan universities. It deserves to be given priority. Theme 2: Spanish as a right: Individually, speakers have the right to use and to be understood in Spanish, which is also an official language in Catalonia. Theme 3: English as a resource: key in achieving goals of internationalization and enhancing multilingualism at the university. Theme 4: Multilingualism: the ultimate goal is to attain a balance between the different languages, functionally distributed at the university. Theme 2: Competitiveness and internationalization demand an increase of ‘foreign languages’. Theme 3: English as a resource: speakers should have more access to it and enhance their competence in this language. Theme 4: ‘English is not enough’: English should coexist with other languages. In other words, if we take the most relevant themes that can be extracted from a set of policy documents as an indication of the underlying narrative that is presented in them (Prior 2015), we can see that the narrative that frames language-related matters at the higher education domain in the two contexts follows a rather similar logic. Indeed, in both Estonia and Catalonia, priority is given to the idea that the national/ local language deserves special attention within universities, a space in which it might require to be protected, promoted, and developed (Theme 1). In both settings we see an acknowledgement of the fact that a degree of engagement with the English language is nowadays unescapable, so English is seen as a resource of chief importance both institutionally and individually (Theme 3). In addition to English, however, the language policy documents in both settings emphasise the notion that multilingualism is the ideal scenario that should be aimed at (Theme 4). Of course, differences can be noted as well. For example, the way English is framed shows a degree of nuances: it appears as a more ‘foreign’ element in Estonia than in Catalonia, where it is conceived as an element of diversity in situ. Another noticeable difference between the two contexts is with regards to the attention paid to the other major societal language in each context, namely Spanish (in Catalonia) and Russian (in Estonia). As seen, Catalan university policy documents do acknowledge the importance of Spanish, mainly in connection to the idea that speakers have the right to use and to be understood in the language in all contexts within higher education (Theme 2). In Estonia, by contrast, Russian is virtually erased from university policy documents, a move by policymakers that would seem to mirror the national level language policy, with a marked Estonian monolingual overtone. Instead, the more euphemistic label ‘foreign language(s)’ is frequently used in the Estonian documents, a label that might in theory encompass any given foreign language, but much like Finnish higher education language policy, it tends to be used in order not to refer 64 Josep Soler explicitly to English all the time (Saarinen 2012; for actual examples illustrating each of the themes and further details in support of these claims, see Soler 2019). The key message from the similarity between the language policy narratives in the two contexts is that, despite some nuances, the concerns policymakers in both settings seem to share are very close: the idea that the national/local language deserves a degree of protection, the inescapability of the English language, and the desired goal of multilingualism. This is despite the fact that Estonia and Catalonia are geographically apart, with different degrees of incorporation of the English language in their higher education systems (especially for teaching purposes). So, despite these differences, we see rather similar language-ideological concerns at the policy level. This is the first argument to question the north-south divide in European higher education. Another factor that brings Estonia and Catalonia together is the socio-economic dimension and the impact it has on language management strategies at the university level. In both cases, we see socio-economic factors that trigger language-related debates that move clearly into language management strategies within the higher education domain. Let us start with Estonia first. Since 2011–2012, there has been a clear and steady trend of a decrease in the total number of students enrolled at universities, a reduction of as many as 20,000 students in total, from 67,000 in 2012 to 47,000 in 2018 (Klaas-Lang 2018). Coupled with a national higher education reform of 2013 (one of whose prerogatives was to abolish university fees for students registered in state-funded study places), university officials and stakeholders began to worry about the long-term sustainability of Estonian-medium higher education. The 2013 reform enabled the possibility for universities to charge registration fees for those programmes taught in a language other than Estonian, notably English. In that context, it was perhaps not a coincidence that the late rector of the University of Tartu, Professor Volli Kalm, publicly wondered about how sustainable Estonian-medium higher education would be in the future. At a leadership conference in Pärnu, he was quoted with the following statement: “The difficult questions regarding the future of Estonian higher education are tied to the competitiveness of Estonian higher education compared to English higher education. Master’s and doctoral studies are inevitably becoming more English-based. Students will increasingly go abroad for niche disciplines... We are often unaware of the price of preserving Estonian-language higher education” (Kalm 2012). This quickly sparked a number of responses in different media outlets, some in support of Kalm’s view, but many more critical of it. To tackle those criticisms, Kalm was compelled to issue another statement, explaining himself better and saying that while it is true that supporting higher education in Estonian is not inexpensive, it is also a responsibility that the country’s universities, and particularly the University of Tartu, cannot escape from (see Soler/Vihman [2018] for a more detailed analysis of this specific language-ideological debate). University language policy in Estonia and Catalonia. 65 In short, we see clearly how societal concerns, combined with economic matters, trickle down into language-related issues, a conflict of non-linguistic interests between different stakeholders in a given language management situation, something already noted in the language planning literature for some time now (e. g. Jernudd/ Neustupny 1987; Neustupny 1983). We can see how language-ideological positions become malleable and articulate (implicitly, but perhaps also more easily) societal, demographic, and economic concerns. In the case of Catalonia, we see how economic measures are both channelled via language management strategies and, at the same time, have an impact on language issues at universities. In this case, the trigger was not a decrease in the number of students enrolled at higher education institutions, but a reduction of the public funding for universities starting in 2010. That year, the growing trend of public spending on Catalan universities was interrupted at just above 700 million euros, and it went down to slightly over 600 million euros in 2014–2015, at which point the decreasing trend was stopped and slightly reverted (see http:// universitatsirecerca.gencat.cat). Just like in Estonia, higher education reforms also took place in Catalonia at the same time that the budgetary restrictions were being implemented, and much like in Estonia, these reforms came together with language-related concerns, in the case of Catalonia perhaps even more explicitly. In fact, even before the 2010 budgetary restrictions, universities had already started drafting their individual language policy documents. By that time, a public discussion began to consolidate itself around the role of universities in ensuring that future generations in Catalonia would be fluent in a ‘third’ language (meaning, almost invariably, English). Initially, a policy reform was passed so that universities were prompted to recommend their graduates in all disciplines that, by the time they would complete their studies, they would possess a B2 level of the CEFR in a ‘third language’. In 2014, this recommendation became legally binding and economically transcendental: the requirement became part of the law that regulates the fiscal, administrative, and financial measures of Catalonia’s public sector (Article 211 of Law 2/2014 of 27 January). Needless to say, the reform did not go down well with university administrators, who saw yet another obstacle imposed on them in order to access the already decreasing available funding. After some degree of confrontation between university and government officials, an agreement was reached in May 2018, and the Catalan parliament passed an amendment to the 2014 law so that the requirement for students to possess a B2 level in a ‘third’ language was postponed for another four years. In theory, those students who began their university degrees in the academic year 2018–2019 will have to possess such a language certificate by the time they graduate (by 2021–2022). In reality, it remains largely unclear how strictly universities will be able to enforce such a regulation, unless the syllabi of all the programmes in all the disciplines in all the public universities in Catalonia are modified to include some language proficiency requirements. Without the proper resources to put in place these changes, it does not seem that higher education officials in Catalonia are likely to go down that route, 66 Josep Soler and without those changes, it seems legally impossible to prevent a student who has completed all the credit course requirements not to be awarded their university degree just because they do not have a B2 certificate in a ‘third’ language. In sum, it seems very likely that the requirement will simply be postponed ad infinitum, or at least until another policy reform is put in place in order to bring it back to a recommendation (for more details on language policy in Catalan higher education see also Pons-Parera 2015; Vila 2015; Soler/Gallego-Balsà 2019). All in all, in addition to the narratives underlying university policy documents in Estonia and Catalonia, another important similarity can be brought up for discussion: the socio-economic dimension and its impact on language management strategies. Although prompted by apparently different concerns (a demographic decrease in number of students, in the case of Estonia, and a decrease in the public funding available for universities, in the case of Catalonia), we see how language policy initiatives become a tool for public officials to gear decisions in a given direction and to manage a given context, in this case the domain of higher education, with specific agendas in mind. In the next and final section of the article, I elaborate on this further, linking it to the implications it has in connection to how we conceptualise higher education language policy, specifically with regards to the so-called north-south divide in European higher education. 4 Conclusions At the start of the article, I set out to problematise the often taken-for-granted existence of a north-south divide in the internationalisation of higher education in Europe. I hope the above section has made it clear that such a divide is more perceived than real. Indeed, while at first sight Estonia and Catalonia would appear to show very distinct higher education settings (particularly in connection to the percentage of English-taught programmes), when we look beyond the numeric and statistical overviews, we start to notice important similarities. Readers interested in more detailed, in-depth analyses of the two cases should find enough relevant material in Soler (2019) and Soler/Gallego-Balsà (2019). In this article, I have shown that some of the similarities between the two cases have to do with two dimensions that are crucial in any sociolinguistic context, but particularly more so in settings with medium-sized languages. These dimensions are the language-ideological, on the one hand, and the socio-economic, on the other. For all the numeric differences, Estonian and Catalan higher education stakeholders are concerned with the sustainability of their own language at the university domain, even if there is an acknowledgement of the inescapability of English, and a shared desire of multilingualism as a final goal. In both settings, socio-economic concerns are pressing (decreasing number of students, on one hand, and decrease in public spending on universities, on the other), and these concerns University language policy in Estonia and Catalonia. 67 materialise themselves and are channelled through language policy developments. With this in mind, statements such as the following seem to go a bit too far in their assessment of the reality of higher education internationalisation across different geographical areas in Europe: “There remain huge differences between individual countries, especially in the form of a NorthSouth divide. As in our earlier reports, the Nordic countries and the Netherlands are the leading providers [of English-taught programmes]. The runners-up are the Baltic States. Southern European countries are still largely ‘abstentious’” (Wächter/Maiworm 2014: n.p.). There certainly is an element of truth in this depiction of the current situation in European higher education, but it is also equally important to remind ourselves that “while estimates on EMI [English-medium instruction] are useful in their own right, they often conceal a highly complex and linguistically diverse reality at internationalised universities” (Hultgren et al. 2015: 5). Not only that. I would add that such representations of clear-cut differences between geographical regions carries two important dangers: a danger of binarism and a danger of lumping. Translated into the domain of higher education and the supposed geographical divide, binarism implies the notion that just because two institutions are located in distinct geopolitical areas, they will by default show different realities. Lumping, on the other hand, is the reverse process: the notion that whenever two institutions share the same geopolitical location, they will, also by default, share very similar realities. These two conceptual processes may add to the difficulty of finding similarities across institutions in preconceived borders and of looking for differences between institutions within the same areas. At least since the foundational work of Einar Haugen (1972), we know of the importance of examining and understanding linguistic ecologies in their locally sit uated terms. This seems particularly important in order to better grasp the kind of language contact situations in present-day globalised times, and to understand the competition, or indeed the conflict, between different types of languages. In that regard, Vila (2015) has argued that environments with medium-sized languages are highly sensitive to societal changes and developments, and this seems particularly true for the domain of higher education. Certainly, in a relatively very short period of time, the domain of higher education in medium-sized language contexts, especially in Europe, has become increasingly complex and sociolinguistically challenging. Some of the challenges of such contexts have been highlighted in this article, and these pertain to: (a) the centrality of language-ideological concerns, and (b) the prevalence of socio-economic matters and the impact they have on language and language policy developments. On both levels, the language-ideological and the socio-economic, it seems clear that language may easily lend itself as an element that can be manipulated by specific stakeholders in a given context in order to advance specific agendas. In sum, in addition to looking at general trends and the overall picture offered by large-scale statistical reports, certainly valuable and something that should continue to be conducted, we should also not lose sight of the importance of narrowing down 68 Josep Soler our analyses into specific language configurations, and try to point out which agents are behind what kind of language policy initiatives and for what purposes. The above analysis has hopefully hinted at the usefulness of taking such an angle, particularly from the point of view of the socio-economic dimension of higher education institutions. 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https://openalex.org/W3179305256	https://www.researchsquare.com/article/rs-283967/v1.pdf?c=1631893193000	English	null	Serjania marginata Casar. hydroalcoholic extract reduced cytokine and inflammatory parameters in experimental models of inflammation and infection in mice	Research Square (Research Square)	2,021	cc-by	8,932	Serjania marginata Casar. hydroalcoholic extract reduced cytokine and inflammatory parameters in experimental models of inflammation and infection in mice reduced cytokine and inflammatory parameters in experimental models of inflammation and infection in mice Maicon Matos Leitão (  mleitao02@hotmail.com ) UFGD FCS: Universidade Federal da Grande Dourados Faculdade de Ciencias da Saude https://orcid.org/0000-0003-2699-1511 Joyce Alencar Santos Radai UFGD FCS: Universidade Federal da Grande Dourados Faculdade de Ciencias da Saude Luis Fernando Benitez Macorini UFGD FCS: Universidade Federal da Grande Dourados Faculdade de Ciencias da Saude Thiago Leite Fraga Centro Universitario da Grande Dourados Silvia Cristina Heredia Vieira Anhanguera University Centre of Campo Grande: Centro Universitario Anhanguera de Campo Grande Claudia Andrea Lima Cardoso UEMS: Universidade Estadual do Mato Grosso do Sul Candida Aparecida Leite Kassuya UFGD FCS: Universidade Federal da Grande Dourados Faculdade de Ciencias da Saude Research Article Keywords: Mycobacterium tuberculosis, Bacillus Calmette-Guerin (BCG), cipó-timbó. inflammation, tuberculosis Posted Date: March 13th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-283967/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Maicon Matos Leitão (  mleitao02@hotmail.com ) Maicon Matos Leitão (  mleitao02@hotmail.com ) UFGD FCS: Universidade Federal da Grande Dourados Faculdade de Ciencias da Saude https://orcid.org/0000-0003-2699-1511 UFGD FCS: Universidade Federal da Grande Dourados Faculdade de Cien https://orcid.org/0000-0003-2699-1511 Research Article License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/21 Page 1/21 Abstract This study investigated the antimycobacterial, anti-inflammatory and antihyperalgesic effects of EESM in in vitro and in vivo models. EESM (0.98–1000 µg/ml) was evaluated in in vitro against Mycobacterium tuberculosis, M. bovis, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus epidermidis. The EESM oral administration (p.o.) (30, 100 and 300 mg/kg) and dexamethasone subcutaneous injection (s.c.) (1 mg/kg) were tested against the carrageenan-induced inflammatory paw edema and pleurisy in Swiss mice. The EESM (30 and 100 mg/kg, p.o.) and dexamethasone (1 mg/kg, s.c.) were tested against the CFA-induced paw inflammation and M. bovis (bacillus Calmette-Guerin - BCG)-induced pleurisy in C57bL6 mice. The minimum inhibitory concentration (MIC) of EESM in the presence of M. tuberculosis was 62.4 µg/ml. The values of MIC of EESM in the presence S. epidermidis, K. pneumoniae were 1000 µg/mL while EESM did not interfere with against P. aeruginosa growth. EESM significantly inhibited paw edema/mechanical hyperalgesia in carrageenan induced paw inflammation and leukocytes migration/proteins exudation in carrageenan-induced pleurisy model. In the BCG-induced pleurisy model, the daily treatment for 7 days, with EESM inhibited the levels of IL-1β in blood and in pleural exudate. The EESM did not alter the mycobacterial growth in the cell culture from pleural lavage, spleen and liver samples collected from BCG-treated animals. The EESM significantly inhibited the persistent edema and mechanical hyperalgesia induced by CFA. This study confirms the EESM anti-inflammatory property and showed that EESM has high potency in inducing inhibition of mycobaterial growth and low potency or no effects in relation to other microorganisms. 1. Introduction The immune and inflammatory process respond promptly to the presence of infectious pathogens. If the infectious pathogens persist, the body develops an adaptative immune and inflammatory response (Frizinsky et al., 2019, Doddam et al., 2019). For the discovery of potential anti-inflammatory/anti- infective agents, the infectious pathogens or PAMPs (Pathogen Associated Molecular Patterns) could be used in experimental models. The inflammatory response to live infectious agents and PAMPs depends on several components, such as leukocytes, receptor pattern recognition receptors (PRR), the complement system, pro-inflammatory mediators, cytokines, among others (Frizinsky et al., 2019; Sonnenberg and Hepworth, 2019; Kumar et al., 2020; Mookherjee et al., 2020). Pharmacological control of an inflammatory response to an aggressive pathogen is carried out using antibiotics. Depending on the development of multi-resistant strategies of pathogen and host response, endogenous and pharmacological control is often not fully effective. In relation to the prevalence of tuberculosis, a disease caused by Mycobacterium tuberculosis has increased due to the development of resistant strains, the use of anti-tumor necrosis fator (TNF)s and the co-infection by the human immunodeficiency virus (Doddam et al., 2019; Pérez et al., 2020; Ryndak and Laal, 2019). In addition, the currently recommended vaccine, M. bovis - bacillus Calmette-Guerin (BCG), attenuates the development of tuberculosis in severe form in children, however it does not prevent Page 2/21 Page 2/21 primary infection or reactivation of latent tuberculosis infection in adults and new vaccines have been developed (Ryndak and Laal, 2019; Pérez et al., 2020). primary infection or reactivation of latent tuberculosis infection in adults and new vaccines have been developed (Ryndak and Laal, 2019; Pérez et al., 2020). The alternative strategies are needed to eliminate the pathogens and also harmful inflammation (Mookherjee et al., 2020). The plant candidate as alternative source is Serjania marginata Casar. (Sapindaceae) found in Brazil, Paraguay, Bolívia and Argentina (Acevedo-Rodríguez, 1990; Ferrucci, 1991; Flora do Brasil 2020, 2020). The traditional knowlegde calls this plant as "cipó-uva" or "cipó-timbó" (Corrêa, 1984; Heredia-Vieira et al., 2015) or blancuzco (Ferrucci, 1991) and the leaves, prepared in the form of juice, are used to treat stomachache by the Guarani Indians (Bourdy et al., 2004). Gastroprotective activity of S. marginata leaves aqueous (Ota et al., 2019) and hydroalcoholic extracts (Périco et al., 2015) were scientifically shown. Périco and colleagues (2015) also showed that hydroalcoholic extract from leaves of S. marginata was antimicrobial, antidiarrheal, and (anti)mutagenic. Moreira et al. (2019) demonstrated that S. 1. Introduction marginata leaves aqueous extract was not toxic after a single exposure, however if it was used after prolonged periods it affected some parameters. Périco et al. (2015) showed that after 14 days of administration hydroalcoholic extract also affected some parameters such as induce gastric lesions. The present work was motivated since the description of in vitro antimicrobial property of S. marginata was related by Périco et al., 2015, and by Serjania sp are used against other infections and inflammatory conditions in folk medicine (Bourdy et al., 2004; De Lima et al., 2006; Arruda et al., 2009). This study investigated the anti-inflammatory and anti-mycobacterial activity of hydroalcoholic extract from leaves of S. marginata (EESM) in several models contributing to the pharmacological knowledge of this plant. 2.1 Vegetal material and extract preparation 2.1 Vegetal material and extract preparation Leaves from S. marginata were collected in February 2011 in an area of Cerrado in Dourados, Mato Grosso do Sul, Brazil (longitude of 55°19′24.9″ W, and an altitude of 429 m). The dried (500 g) and pulverized leaves were extracted by percolation at room temperature with EtOH/H2O (7:3, v/v). The hydroalcoholic extract was filtered, concentrated under vacuum (± 40 °C), and lyophilized (yield: 33% w/w). The phytochemical analysis of EESM was performed by Heredia-Vieira et al. (2015). 2.2 Chemical reagents Complete Freund's Adjuvant (CFA), dexamethasone (DEXA), isoniazid, and carrageenan were brought from Sigma-Aldrich (St Louis, MO, USA). Ketamine and xylasine were obtained from Syntec (Santana de Paraíba, SP, Brazil). BCG was obtained from Fundação Ataulpho de Paiva (Rio de Janeiro, Brazil). Dimethylsulfoxide (DMSO) and other reagents were acquired from other suppliers. 2.3 In vitro antimycobacterial and antibacterial activity Page 3/21 The in vitro antimycobacterial activity was performed according to Palomino et al. (2002). The EESM was dissolved in 5% of DMSO in a solution of pure water and the minimum inhibitory concentration (MIC) values for EESM was assayed at range of 0.98 – 250 μg/ml and for isoniazid between 0.004 – 1 μg/ml in the presence of the M. tuberculosis strain H37Rv (ATCC27294). The in vitro antibacterial activity methodology was performed according to National Committee for Clinical Laboratory Standards 2002 and Campos e cols, 2014. The EESM was dissolved in 5% of tween 80 in a solution of pure water and the minimum inhibitory concentration (MIC) values for EESM was assayed at 8.125, 16.25, 32.5, 75, 125, 250, 500, 1000 μg/ml. Bacterial cultures (Klebsiella pneumoniae ATCC 700603, Pseudomonas aeruginosa ATCC 27853, and S. epidermidis ATCC 12228) will be activated through subcultures on Mueller-Hinton agar for 24 h at 37 °C. After activation and/or subculture, the bacterial inoculum will be standardized, which consisted of the preparation of a bacterial suspension in sterile saline, standardized on the McFarland 0.5 da scale (1 x 108 CFU/ml) and diluted 1:10 also in saline (1 x 107 CFU/ml). After dilution, 10 µL volumes will be transferred to the sterile microplate wells, containing a final volume of 100 µL of Mueller-Hinton broth plus the different final concentrations of EESM, resulting in a final inoculum of approximately 10 x 105 CFU/ml and later taken to greenhouse at 37 ºC for 24 h. The MICs (minimum inhibitory concentrations) in microdilution will be determined by reading in a spectrophotometer at 610 nm in a microplate observing the lowest concentration in which the EESM will completely inhibit microbial growth (National Committee for Clinical Laboratory Standards 2002; Campos et al., 2014). 2.4 Animals, control drugs’ administration, EESM dilution and doses for in vivo experiments Female and male Swiss (28 – 32 g) and C57BL/6 mice (25 – 30 g) were provided by the central biotherium at the Federal University of Grande Dourados (UFGD). Animals were kept in polypropylene boxes in a sectorial Biotherium at Faculty of Health Sciences at UFGD with the following conditions: 22 °C, in the presence of a 12 h light/dark cycle, with food and water ad libitum. The EESM was dissolved in saline solution (0.9 %) and the dose was calculated according to mice weight. Vehicle solution is the saline solution since it was used to dissolve the EESM. The EESM oral administration (p.o.) doses used in the carrageenan-induced paw inflammation and pleurisy were 30, 100 and 300 mg/kg to verify if the EESM presents a dose-response profile. As the doses of 30 and 100 mg/kg of EESM were effective against inflammatory parameters, these doses were tested also against BCG-induced pleurisy and CFA- induced inflammatory reaction. To verify the inhibitory efficacy of the commercial drugs, the dose of 1 mg/kg (by subcutaneous route – s.c.) of dexamethasone was used as control group in in vivo models while the dose of 25 mg/kg (p.o.) of isoniazid was used in BCG model. 2.6 Leukocyte migration and protein analysis in pleural exudate collected from carrageenan induced pleurisy 2.6 Leukocyte migration and protein analysis in pleural exudate collected from carrageenan induced pleurisy Female Swiss mice were distributed in five experimental groups (n = 6) and one hour before the pleurisy, they were treated (p.o.) with 30, 100 and 300 mg/kg of EESM, with the vehicle and dexamethasone (1 mg/kg, s.c.). Subsequently, with the aid of an adapted needle, 100 μL of carrageenan (containing 100 μg) was injected into the right side of the animals' chest cavity to induce inflammation (Vinegar et al., 1973). Four hours after the injection of carrageenan, the animals were euthanized (100 mg/kg of ketamine and 10 mg/kg of xylasine by intraperitoneal route (i.p.)) and, afterwards, the exudate was collected by washing the chest cavity with 1 mL of phosphate buffered saline (PBS). Having made the collection, the total number of leukocytes in the exudate was determined by the KX-21N Sysmex apparatus and the protein exudation was evaluated by the Bradford reaction, using the commercially available Bradford kit (Bioagency, São Paulo, Brazil). 2.8 Paw edema, mechanical and cold hyperalgesia evaluation in CFA induced paw inflammation for 22 2.5 Paw edema, mechanical and cold hyperalgesia evaluation in carrageenan induced paw inflammation The male Swiss mice were distributed in five experimental groups (n = 6) and one hour before the carrageenan injection, they were treated (p.o.) with 30, 100 and 300 mg/kg of EESM, with the vehicle and dexamethasone (1 mg/kg, s.c.). Subsequently, the animals received a subcutaneous injection (100 μL) of Page 4/21 Page 4/21 Page 4/21 a solution containing 300 μg of carrageenan (Winter et al., 1962) dissolved in sterile 0.9 % saline in the right hind paw. The contralateral paw, which was used as a control, received 100 μL of saline. Paw edema was analyzed using a plethysmometer (Panlab, Spain) at 1, 2 and 4 h after injection. In addition, mechanical hyperalgesia was assessed using an electronic version of the von Frey test (Analgesimeter von frey, InSight) and cold sensitivity was analyzed with the acetone drop test (Decosterd and Woolf, 2000) at 3 and 4 h after carrageenan injection. 2.8 Paw edema, mechanical and cold hyperalgesia evaluation in CFA induced paw inflammation for 22 days 2.7 Leukocyte migration, Il-1β dosage and CFU growth in BCG-induced pleurisy model Groups of six male C57BL/6 mice were treated orally with EESM (30 or 100 mg/kg), isoniazid (25 mg/kg), saline solution (0.9 %; control group) and saline solution (0.9 %; naive group). Pleurisy was induced 1 h after the previous treatment with an intrapleural injection with 0.1 mL of a suspension of BCG (4 X 105 colony forming units (CFU) into the right pleural cavity. In the naïve group, instead of a BCG injection, the mice received a saline injection in the pleural space. The animals were treated orally with EESM, isoniazid, saline solution (0.9 %) once daily for 7 days (Andrade-Silva et al., 2016). After 7 days, the animals were euthanized (100 mg/kg of ketamine and 10 mg/kg of xylasine, i.p.) and the pleural cavity was washed with 1 ml of sterile phosphate buffered saline. The washed sample of 50 µL was diluted with Evans blue to determine the total number of leukocytes. The rest of the sample was centrifuged, and the supernatant was stored for the Il-1β measurements using ELISA. The blood was also collected from each animal for Il-1β measurements. The precipitate was suspended in 0.5 mL of sterile ultrapure water and 0.1 mL of Ogawa Kudu, and 0.1 mL of the suspension was plated onto 7H11 agar. The spleen and liver were macerated with 1 mL of sterile saline solution, and 0.1 mL of the suspension was plated on 7H11 agar. The cells were cultured for 60 days at 37°C in 5% CO2 (Kassuya et al., 2020). Page 5/21 Page 5/21 Groups of six male C57BL/6 mice were treated with vehicle (control group), EESM (30 and 100 mg/kg, p.o.), and dexamethasone 1 mg/kg (s.c., the positive control group) every day, once a day for 22 days. After the first treatment, the animals received 30 μL of Freund's complete adjuvant (CFA) (oil suspension containing inactive M.tuberculosis) via intraplantar injection in the right paw. Mechanical and cold sensitivity and paw edema were measured 6, 11, 16 and 22 days after the injection of CFA. 3.1 Effects of EESM on M. tuberculosis and other microorganisms in vitro The MIC value of EESM in the presence of the M. tuberculosis strain was 62.4 µg/ml, while the MIC value of isoniazid was 0.030 µg/ml. EESM was effective against S. epidermidis, K. pneumoniae in 1000 µg/ml and was not effective against P. aeruginosa. 2.9 Statistical analysis The data are presented as the mean ± standard error (SEM). The determination of significant differences among groups was made via one-way analysis of variance (ANOVA) and the Newman-Keuls test was chosen as a post hoc (GraphPad Prism Software). The percentage of inhibition was calculated from the control group. Differences were considered to be significant when p < 0.05. 3.4 Effects of EESM on Leukocyte migration, Il-1β dosage and CFU growth in BCG-induced pleurisy model After 7 days of oral treatment, EESM (30 and 100 mg/kg) did not decrease the leukocyte migration into the pleural spaces after intrathoracic injection of BCG, however isoniazid reduced significantly the leukocyte invasion to the pleura (Figure 4A). In pleural exudates, the reduction of IL-1β levels were significant with inhibition of 72 % and 76 % with the treatment of 30 and 100 mg/kg EESM (p.o.) and 76 % isoniazid (25 mg/kg, p.o.) (Figure 4B). In serum, the reduction of IL-1β levels were significant with inhibition 50 % and 59 % with the treatment of 30 and 100 mg/kg EESM (p.o.) and 67 % isoniazid (25 mg/kg, p.o.) (Figure 4C). The statistical comparison among groups showed that only the control group differed from the other groups in Figures 4B and 4C. During the cell culture of the pleural lavage, spleen and liver samples collected from BCG treated animals, the EESM (30 or 100 mg/kg) did not alter the mycobacteria growth (results not shown) and the UFC count did not differ from control group (results not shown) until 60 days. The negative control group samples presented mycobacterial growth in all cultivated plaques, with a mean of 54 CFU after 60 days of culture, while the isoniazid group samples did not show any growth after 60 days (results not shown). 3.2 Effects of EESM on paw edema induced by carrageenan The paw edema was significantly inhibited at doses of 100 and 300 mg/kg of EESM after 2 h of carrageenan injection with inhibition of 38 % and 44 %, respectively (p < 0.05) (Figures 1B). After 4 h, all doses showed significant differences from the control group with inhibition of 36 %, 33 % and 58 % in doses 30, 100 (p < 0.05) and 300 mg/kg (p < 0.01). The dexamethasone group showed significant inhibition at 1, 2 and 4 h. Dexamethasone was different from all treated groups when the comparison among groups was perfomed (Figures 1A-C). The groups treated with doses of 100 and 300 mg/kg differed from the control and from the group treated with 30 mg/kg of EESM (Figure 1B) and in figure 1C the groups treated with doses of 30 and 100 mg/kg differed from the control and from group treated with 300 mg/kg of EESM. In the carrageenan induced mechanical hyperalgesia, 100 % inhibition was observed in two evaluations at the dose of 300 mg/kg and the dexamethasone group (p < 0.001) (Figures 2A and 2B). The dose of 100 mg/kg also demonstrated prevention of hyperalgesia development, with 100 % (p < 0.001) and 96 % (p < 0.01) efficacy, in 3 and 4 h after carrageenan injection, respectively (Figures 2A and 2B). The dose of 30 mg/kg was not effective for the prevention of carrageenan paw hyperalgesia. The control and the group treated with 30 mg/kg of EESM differed significantly from the other treated groups (Figures 2A and 2B). The groups treated with doses of 100, 300 mg/kg and dexamethasone did not differ among themselves (Figure 2A) and in figure 2B the groups treated with doses of 300 mg/kg and dexamethasone differed from the other treated groups. Page 6/21 Page 6/21 In the cold nociceptive response, no doses showed a significant difference from control group, however the dexamethasone group obtained an inhibition of 52 % and 49 % (p < 0.05) in 3 and 4 h, respectively (Figures 2C and 2D). In the cold nociceptive response, no doses showed a significant difference from control group, however the dexamethasone group obtained an inhibition of 52 % and 49 % (p < 0.05) in 3 and 4 h, respectively (Figures 2C and 2D). 3.3 Effects of EESM on pleurisy induced by carrageenan In pleurisy induced by carrageenan, EESM 100 and 300 mg/kg significantly reduced leukocyte migration (p < 0.001) by 59 and 67 %, respectively, compared to the control group (Figure 3A). In protein exudation evaluation, EESM demonstrated significant difference (p < 0.001), with 53, 42 and 60 % of inhibition, at doses 30, 100 and 300 mg/kg, respectively. The dexamethasone group also demonstrated a significant decrease in both evaluations (p < 0.001) (Figure 3). The statistical comparison among groups showed that control and the group treated with 30 mg/kg of EESM differed from the other groups in Figure 3A while only control group differed from others groups in Figure 3B. 3.4 Effects of EESM on Leukocyte migration, Il-1β dosage and CFU growth in BCG-induced pleurisy model 3.4 Effects of EESM on Leukocyte migration, Il-1β dosage and CFU growth in BCG-induced pleurisy model 3.5 Effects of EESM on CFA induced paw inflammation The treatments with both doses of EESM did not differ from the control group on days 11, 16 and 22. 3.5 Effects of EESM on CFA induced paw inflammation A single daily oral treatment with EESM for 6 days at doses of 30 and 100 mg/kg and dexamethasone (1 mg/kg, s.c.) reduced mechanical hyperalgesia in 100 % respectively (Figure 5A). After 11 and 16 days from CFA injection, no significant inhibitions in mechanical response were observed with 30 mg/kg of EESM, however a significant inhibition was detected with dexamethasone (Figure 5B and 5C). At 22 days after CFA, the inhibition observed with 30 mg/kg of EESM was 100 % (Figure 5D). The groups treated with EESM (30 and 100 mg/kg) did not differ statistically among themselves at days 6, 11 and 16, however Page 7/21 Page 7/21 Page 7/21 they were different from the dexamethasone group on 11 and 16 days (Figures 5A-C). The group treated with 100 mg/kg differed from the group treated with 30 mg/kg at 22 days after CFA injection (Figure 5D). they were different from the dexamethasone group on 11 and 16 days (Figures 5A-C). The group treated with 100 mg/kg differed from the group treated with 30 mg/kg at 22 days after CFA injection (Figure 5D). The dose of 100 mg/kg, but not the dose of 30 mg/kg of EESM and dexamethasone groups showed a significant difference in paw edema on days 6 and 11 after the administration of CFA with the inhibition of 21 % and 47 %, respectively (Figures 6A and 6B). On days 16 and 22, the doses of 30, 100 mg/kg of EESM and dexamethasone groups were different from the control groups with inhibitions of 33 %, 50 % and 65 % on day 16 and 57 %, 67 % and 87 % on day 22, and 57 %, 67 % and 87 % on day 22, respectively (Figures 6C and 6D). The treatment with 100 mg/kg did not statistically differ from dexamethasone group on days 6, 11 and 16, however on day 22 these groups were statistically different (Figure 6). Both treatments with EESM were not statistically different to dexamethasone group on day 16, nevertheless on day 22 both treatments with EESM differed from dexamethasone group. In cold hyperalgesia induced by CFA, the treatment with EESM at dose of 100 mg/kg was statistically different from the control group, with the inhibition 52 % showing similarity inhibition to dexamethasone group on day 6. 4. Discussion Species of the Serjania genus have been used in folk medicine for their anti-inflammatory potential in Brazil and in Mexico (Salinas-Sánchez et al., 2017) while S. marginata leaves are squeezed for juice by the Bolivian people as an analgesic agent for stomach (Bourdy et al., 2004). The present study was the first one to show the anti-inflammatory, analgesic and potential antibiotic properties of EESM in inflammatory/infection/hyperalgesic models. The data of the present work corroborate with the traditional folk use of S. marginata as an analgesic and expand the information regarding its use. Some inflammatory models used in this present research involve carrageenan and CFA. The carrageenan was used in two acute inflammatory models to analyze the efficacy and potency of anti-inflammatory agents until 48 h (Posadas et al., 2004). When the carrageenan is administered into the mice paw the edema, mechanical and cold hyperalgesia can be measured (Santos el al., 2020) and when it is injected by intrapleural route the leukocyte and protein can be measured in pleural exudate (Leitão et al., 2020). In the present study the EESM showed to be effective against acute edema (2, and 4 h, Figure 1B and 1C), mechanical hyperalgesia (3 and 4 h, Figures 2A and 2B), however the cold hyperalgesia (Figures 2C and 2D) was not inhibited in carrageenan paw inflammatory model. In the carrageenan induced-pleurisy model, both leukocyte migration and protein exudation were inhibited by EESM (Figures 3A and 3B). In this model, the EESM induced a dose dependent inhibition (Figures 1B, 1C, 2A, 2B and 3A) in which the efficacy response to EESM differed significantly when its dose was increased. In Figure 2B the classical dose-response effect could be detected since each dose of EESM significantly differed in efficacy among themselves. As far as we know, only the research of Périco et al. (2015) showed the myeloperoxidase Page 8/21 activity inhibition by EESM in ulcer models. The present study showed the anti-inflammatory and mechanical anti-hyperalgesic (analgesic) response of EESM occurs a dose-dependent manner. The major compounds such as flavonoids, proanthocyanidins and saponins have already been described in EESM (Heredia-Vieira et al., 2015). Flavonoids and proanthocyanidins (tannins) are the chemical classes related to the antioxidant and anti-inflammatory activities of some plant species. 4. Discussion Flavonoids, due to their chemical structure and their redox capacity, can interfere in important mechanisms that are related to inflammatory processes such as enzyme inhibition (Hernández-Rodríguez et al., 2019; Malekia et al., 2019). Proanthocyanidins have anti-inflammatory potential that could modulate the arachidonic acid and the nuclear factor NF-kB (Cádiz-Gurrea et al., 2017) Périco et al. (2015) and Heredia-Vieira et al. (2015) showed that EESM gastroprotective property is associated with the presence of proanthocyanidins and flavonoids. Other substances present in EESM are pulsatilla D, hederacolchiside A1, salzmannianosid B and serjanioside D (saponins), epicatechin, cassiaoccidentalin A, tetrastigma B and serjanone A (flavonoids), proanthocyanidins A- 1 and A-2 and cinnamtannin B-1 (Heredia-Vieira et al., 2015). The flavonoids, proanthocyanidins and saponins could be responsible for the EESM anti- inflammatory activity. The major compounds such as flavonoids, proanthocyanidins and saponins have already been described in EESM (Heredia-Vieira et al., 2015). Flavonoids and proanthocyanidins (tannins) are the chemical classes related to the antioxidant and anti-inflammatory activities of some plant species. Flavonoids, due to their chemical structure and their redox capacity, can interfere in important mechanisms that are related to inflammatory processes such as enzyme inhibition (Hernández-Rodríguez et al., 2019; Malekia et al., 2019). Proanthocyanidins have anti-inflammatory potential that could modulate the arachidonic acid and the nuclear factor NF-kB (Cádiz-Gurrea et al., 2017) Périco et al. (2015) and Heredia-Vieira et al. (2015) showed that EESM gastroprotective property is associated with the presence of h d d fl d h b l ll h d l h d proanthocyanidins and flavonoids. Other substances present in EESM are pulsatilla D, hederacolchiside A1, salzmannianosid B and serjanioside D (saponins), epicatechin, cassiaoccidentalin A, tetrastigma B and serjanone A (flavonoids), proanthocyanidins A- 1 and A-2 and cinnamtannin B-1 (Heredia-Vieira et al., 2015). The flavonoids, proanthocyanidins and saponins could be responsible for the EESM anti- inflammatory activity. The EESM was also tested in vitro against gram-negative and gram-positive bacteria. The MIC of EESM in the presence of the M. tuberculosis was 62.4 µg/ml showing to be a promising antibacterial natural agent. Cardoso et al. (2013) indicated that an ethanolic extract of S. erecta, another species of Serjania genus, demonstrated a MIC of 128.0 µg/ml and EESM showed to be twice more potent than this extract. The values of MIC of EESM in the presence S. epidermidis and K. 4. Discussion Tumor necrosis factor alpha (TNF-α) and IL-1β are produced by immune cells that are involved in pulmonary tuberculosis (Jayaraman et al., 2014; Weiss et al., 2019). These results showed that doses of EESM controlled the BCG-inflammation. increase in IL-1β levels after 7 days in BCG model may be correlated with the mycobacterial growth, as showed previously by our group (Kassuya et al., 2020). Bourigault et al. (2013) indicated that mice deficient in interleukin (IL) showed reduction in bacterial load 35 days’ post infection. Tumor necrosis factor alpha (TNF-α) and IL-1β are produced by immune cells that are involved in pulmonary tuberculosis (Jayaraman et al., 2014; Weiss et al., 2019). These results showed that doses of EESM controlled the BCG-inflammation. increase in IL-1β levels after 7 days in BCG model may be correlated with the mycobacterial growth, as showed previously by our group (Kassuya et al., 2020). Bourigault et al. (2013) indicated that mice deficient in interleukin (IL) showed reduction in bacterial load 35 days’ post infection. Tumor necrosis factor alpha (TNF-α) and IL-1β are produced by immune cells that are involved in pulmonary tuberculosis (Jayaraman et al., 2014; Weiss et al., 2019). These results showed that doses of EESM controlled the BCG-inflammation. An aliquot of precipitate sample from pleural exudate and also spleen and liver macerate from all BCG- injected animals were platted in Middlebrook 7H9/Ogawa Kudu culture medium for analysis of BCG growth. The BCG culture was found positive for Ziehl-Neelsen stain confirming that this microorganism is a Mycobacterium sp. The isoniazid inhibited significantly all the aspects of in vivo inflammatory response (Figure 4) including mycobacterial growth in vitro and in vivo. The EESM was not able to inhibit the formation of CFUs in pleural exudate, liver, and spleen tissues from in vivo experiments. The absence of EESM antibacterial effects may be related to the route of administration, dose and pharmacokinetics/pharmacodynamics of the EESM. To verify if EESM inhibited the BCG in vitro, the attenuated BCG was reactived in Lowenstein Jensen medium (an inclined tube under standard growth conditions) (Njeru and Jackon, 2020) and subsequently EESM was added to evaluation of antimicrobial activity in vitro. Several concentrations until 4 mg/ml of EESM were tested against BCG in vitro therefore no inhibitions were detected demonstrating no EESM antibacterial and/or bacteriostatic effects. The EESM was also tested against CFA (an oil suspension containing killed M. 4. Discussion tuberculosis), an immunogenic/inflammatory stimulus since it is not possible to work with viable M. tuberculosis (Dubé et al., 2020). The CFA induced persistent edema, mechanical hyperalgesia and cold sensitivity in control animals for 22 days. We observed the inhibition of the formation of paw edema with similar results to the dexamethasone group for 22 days, with a significant difference in the dose of 100 mg/kg of EESM in all evaluations and on days 16 and 22 in the dose of 30 mg/kg, comparing to the group control (Figures 6A- D). In the assessment of mechanical hyperalgesia, the EESM showed a significant difference compared to the control group on day 22 (Figure 5D). These results demonstrated that chronic anti-inflammatory ability of EESM even also in the persistent CFA model of inflammation. 5. Conclusion The present study was the first one to show the anti-inflammatory, anti-hyperalgesic and potential antibiotic properties of EESM in inflammatory/infection/hyperalgesic in acute and persistent models. EESM has high potency in inducing inhibition of mycobaterial growth and low potency or no effects in relation to other microorganisms in in vitro. EESM chemical composition showed the presence of several classes of compounds such as flavonoids, proanthocyanidins and saponins could be responsible for the EESM anti-inflammatory activity. 4. Discussion pneumoniae was 1000 µg/ml indicating that EESM had low potency in inducing inhibition of other bacteria growth. Périco et al., 2015 showed that the hydroethanolic extract from S. marginata had in vitro antibacterial and antifungal activities of the HESM against S. aureus (MIC of 125 mg/mL), E. coli (MIC of 250 mg/mL), S. setubal (MIC of 250 mg/mL), and C. albicans (MIC of 250 mg/mL). EESM was not effective against P. aeruginosa growth. These set of results showed that EESM has high potency in inducing inhibition of mycobaterial growth and low potency or no effects in relation to other microorganisms. aeruginosa growth. These set of results showed that EESM has high potency in inducing inhibition of mycobaterial growth and low potency or no effects in relation to other microorganisms. Since the EESM activity seems to be more selective against M. tuberculosis than to other microorganisms the in vivo EESM potential against mycobacteria was performed. The EESM was tested in BCG-induced pleurisy since it is not possible to work with M. tuberculosis in vivo in our University. Another reason to work with BCG as a mycobacteria model, is that its immunization schedule is applied against tuberculosis at birth in various countries. The licensed vaccine with attenuated BCG is performed in health care and several new vaccines are in clinical phases (Pérez et al., 2020). BCG was inoculated in the interpleural space, inducing an infectious and inflammatory mycobacterial response confirmed by the results of the control groups compared to the naive group (Figure 4). The results of leukocyte migration into the pleural space, IL-1β levels in serum and in pleural exudate in the negative control group significantly differed from those of the naïve group after 7 days. In BCG pleurisy model, oral doses of 30 and 100 mg/kg of EESM did not interfere with leukocyte migration to the pleural exudate however it induced a significant inhibition on the interleukin-1 beta (IL-1β) levels in serum and pleural exudate. The Page 9/21 Page 9/21 increase in IL-1β levels after 7 days in BCG model may be correlated with the mycobacterial growth, as showed previously by our group (Kassuya et al., 2020). Bourigault et al. (2013) indicated that mice deficient in interleukin (IL) showed reduction in bacterial load 35 days’ post infection. Authors’ contributions All authors participated in the design, interpretation of the studies, analysis of the data and review of the manuscript; Maicon Matos Leitão and Silvia Cristina Heredia Vieira conducted the anti-inflammatory and analgesic evaluation; Luis Fernando Benitez Macorini, Thiago Leite Fraga and Joyce Alencar Santos Radai conducted the antimicrobial and antimycobacterial evaluation; Claudia Andrea Lima Cardoso and Silvia Cristina Heredia Vieira were involved in the preparation of extract; Candida Aparecida Leite Kassuya and Maicon Matos Leitão performed data analyses and wrote the manuscript. All authors read and approved the final manuscript. Code availability Not applicable Abbreviations Page 10/21 Complete Freund's Adjuvant (CFA), Dimethylsulfoxide (DMSO), Interleukin-1β (IL-1β), Mycobacterium bovis - Bacillus Calmette-Guerin (BCG), Receptor pattern recognition receptors (PRR), Tumor necrosis fator (TNF). Complete Freund's Adjuvant (CFA), Dimethylsulfoxide (DMSO), Interleukin-1β (IL-1β), Mycobacterium bovis - Bacillus Calmette-Guerin (BCG), Receptor pattern recognition receptors (PRR), Tumor necrosis fator (TNF). Funding Body information This work did not received a specific funding. Ethics approval The register code for research A9CDAAE was obtained from National System for the Management of Genetic Heritage and Associated Traditional Knowledge (SISGEN). A specimen was deposited at the Herbarium of the Federal University of the Mato Grosso do Sul, Brazil under the number 41054. Approval of the animal protocols were granted by the UFGD ethics committee (33/2016) and of University center of Grande Dourados (UNIGRAN) ethics committee (No. 050/2020). Conflict of interest The author(s) confirm that this article content has no conflicts of interest. Availability of data and material All data are described in the article. Consent to participate Not applicable Consent for publication Not applicable Consent for publication Page 11/21 Page 11/21 Not applicable Acknowledgments The authors would like to thank National Council for Scientific and Technological Development (CNPq), Support Foundation for the Development of Education, Science and Technology of the State of Mato Grosso do Sul (FUNDECT), and Coordination for the Improvement of Higher Education Personnel (CAPES), Fund for Support to Teaching, Research and Outreach Activities of University of Campinas (FAEPEX-UNICAMP). References 1. Acevedo-Rodriguez P (1990) Distributional patterns in Brazilian Serjania (Sapindaceae). ‎Acta Bot Bras 4(1):69–82. https://doi.org/10.1590/S0102-33061990000100005 2. 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Comp, pp 265–288 19. Jayaraman P, Sada-Ovalle I, Nishimura T, Anderson AC, Kuchroo VK, Remold HG, Behar SM (2013) IL- 1β promotes antimicrobial immunity in macrophages by regulating TNFR signaling and caspase-3 activation. J. Immun. J. (Baltimore, Md.: 1950), 190(8), 4196–4204. https://doi.org/10.4049/jimmunol.1202688 20. Kassuya R, Radai J, Macorini L, Nunes V, Salvador M, Leite P, Oliveira, Rodrigo, Croda J, Arena, Arielle, Kassuya C (2020) Blutaparon portulacoides ethanolic extract reduced IL-1β and inflammatory parameters induced by the Mycobacterium complex and carrageenan in mice. Inflammopharmacol. 10.1007/s10787-020-00752-0 21. Kumar D, Romero Y, Schuck KN, Smalley H, Subedi B, Fleming SD. Drivers and regulators of humoral innate immune responses to infection and cancer. 2020. Mol Immunol. May;121:99–110. doi: 10.1016/j.molimm.2020.03.005. Epub 2020 Mar 18 21. Kumar D, Romero Y, Schuck KN, Smalley H, Subedi B, Fleming SD. Drivers and regulators of humoral innate immune responses to infection and cancer. 2020. Mol Immunol. May;121:99–110. doi: 10.1016/j.molimm.2020.03.005. Epub 2020 Mar 18 22. Leitão MM, Joyce AS, Radai IC, Ferrari, Fábio J, Negrão SE, Silva-Filho RJ, Oliveira, Jonas daS, Mota, Candida AL, Kassuya (2020) Effects of an ethanolic extract and fractions from Piper glabratum (Piperaceae) leaves on pain and inflammation, Regul. Toxicol. Pharmacol., Volume 117, 104762, ISSN 0273–2300, https://doi.org/10.1016/j.yrtph.2020.104762 Page 13/21 Page 13/21 23. Malekia SJ, Crespob JF, Cabanillas B (2019) Anti-inflammatory effects of flavonoids. Food Chem 299:125124 24. Mookherjee N, Anderson M, Haagsman H, Davidson D (2020) Antimicrobial host defence peptides: functions and clinical potential. Nat Rev Drug Discov, 19. 10.1038/s41573-019-0058-8 25. Moreira SS, Tamashiro LK, Jorge BC, da Silva Balin P, Heredia-Vieira SC, de Almeida GL, Cardoso CAL, Kassuya CAL, Arena AC (2019) Toxicological safety evaluation in acute and 28-day studies of aqueous extract from Serjania marginata Casar. (Sapindaceae) leaves in rats. J Ethnopharmacol v 231:197–204. doi:10.1016/j.jep.2018.11.024 26. National Committee for Clinical Laboratory Standards (NCCLS) (2002) Performance standards for antimicrobial susceptibility testing. Inf Supplements. 100–112 26. National Committee for Clinical Laboratory Standards (NCCLS) (2002) Performance standards for antimicrobial susceptibility testing. Inf Supplements. 100–112 27. Njeru SN, Muema JM (2020) Antimicrobial activity, phytochemical characterization and gas chromatography-mass spectrometry analysis of Aspilia pluriseta Schweinf. Extracts. Heliyon. Oct 8;6(10). doi: 10.1016/j.heliyon.2020.e05195 28. References Ota EC, Honorato CA, Heredia-Vieira SC, Flores-Quintana CI, de Castro Silva TS, Inoue LAKA, Cardoso CAL (2019) Hepatic and gastroprotective activity of Serjania marginata leaf aqueous extract in Nile tilapia (Oreochromis niloticus). Fish Physiol Biochem Jun 45(3):1051–1065. doi:10.1007/s10695- 019-00622-9 29. Pérez I, Uranga S, Sayes F, Frigui W, Samper S, Arbués A, Aguiló N, Brosch R, Martín C, Gonzalo- Asensio J (2020) Live attenuated TB vaccines representing the three modern Mycobacterium tuberculosis lineages reveal that the Euro-American genetic background confers optimal vaccine potential. EBioMedicine May 55:102761. doi:10.1016/j.ebiom.2020.102761. Epub 2020 Apr 28. PMID: 32361249 30. Périco LL, Heredia-Vieira SC, Beserra FP, Santos RC, Weiss MB, Resende FA, Ramos MAS, Bonifácio BV, Bauab TM, Varanda Ea, Gobbi JIF, Rocha LRM, Vilegas W, Hiruma-Lima CA (2015) Does the gastroprotective action of a medicinal plant ensure healing effects? An integrative study of the biological effects of Serjania marginata Casar. (Sapindaceae) in rats. J Ethnopharmacol 172:312– 324 31. Posadas I, Bucci M, Roviezzo F, Rossi A, Parente L, Sautebin L, Cirino G (2004) Carrageenan-induced mouse paw oedema is biphasic, age-weight dependent and displays differential nitric oxide cyclooxygenase-2 expression. Br J Pharmacol 142:331–338. https://doi.org/10.1038/sj.bjp.0705650 32. Ryndak MB, Laal S (2019) Mycobacterium tuberculosis Primary Infection and Dissemination: A Critical Role for Alveolar Epithelial Cells. Front. Cell. Infect. Microbiol., v. 9 32. Ryndak MB, Laal S (2019) Mycobacterium tuberculosis Primary Infection and Dissemination: A Critical Role for Alveolar Epithelial Cells. Front. Cell. Infect. Microbiol., v. 9 33. Salinas-Sánchez DO, Jiménez-Ferrer E, Sánchez- Sánchez V, Zamilpa A, Ginzález-Cortazar M, Tortorielo J, Herrera-Ruiz M (2017) Anti-Inflammatory Activity of a Polymeric Proanthocyanidin from Serjania schiedeana. Mol 22:863 33. Salinas-Sánchez DO, Jiménez-Ferrer E, Sánchez- Sánchez V, Zamilpa A, Ginzález-Cortazar M, Tortorielo J, Herrera-Ruiz M (2017) Anti-Inflammatory Activity of a Polymeric Proanthocyanidin from Serjania schiedeana. Mol 22:863 4. Santos ED, Leitão MM, Aguero Ito CN, Silva-Filho SE, Arena AC, Maria de Souza Silva-Comar F, 34. Santos ED, Leitão MM, Aguero Ito CN, Silva-Filho SE, Arena AC, Maria de Souza Silva-Comar F, Nakamura Cuman RK, Oliveira RJ, Nazari Formagio AS, Leite Kassuya CA (2020) Analgesic and anti- inflammatory articular effects of essential oil and camphor isolated from Ocimum 34. References Santos ED, Leitão MM, Aguero Ito CN, Silva-Filho SE, Arena AC, Maria de Souza Silva-Comar F, Nakamura Cuman RK, Oliveira RJ, Nazari Formagio AS, Leite Kassuya CA (2020) Analgesic and anti- inflammatory articular effects of essential oil and camphor isolated from Ocimum Page 14/21 Page 14/21 kilimandscharicum Gürke leaves. J Ethnopharmacol. Dec 11:113697. doi: 10.1016/j.jep.2020.113697. Epub ahead of print. PMID: 33316364 kilimandscharicum Gürke leaves. J Ethnopharmacol. Dec 11:113697. doi: 10.1016/j.jep.2020.113697. Epub ahead of print. PMID: 33316364 35. Sonnenberg GF, Hepworth MR (2019) Functional interactions between innate lymphoid cells and adaptive immunity. Nat Rev Immunol v 19(10):599–613 35. Sonnenberg GF, Hepworth MR (2019) Functional interactions between innate lymphoid cells and adaptive immunity. Nat Rev Immunol v 19(10):599–613 36. Vinegar R, Truax JF, Selph JL (1973) Some quantitative temporal characteristics of carrageenin- induced pleurisy in the rat. Proc. Soc. Exp. Biol. Med., v. 143, n. 3, p. 711–714 36. Vinegar R, Truax JF, Selph JL (1973) Some quantitative temporal characteristics of carrageenin- induced pleurisy in the rat. Proc. Soc. Exp. Biol. Med., v. 143, n. 3, p. 711–714 37. Weiss DI, Ma F, Merleev AA, Maverakis E, Gilliet M, Balin SJ, Bryson BD, Ochoa MT, Pellegrini M, Bloom BR and Robert L. Modlin. 2019. IL-1β Induces the Rapid Secretion of the Antimicrobial Protein IL-26 from Th17 Cells. J. Immunol., June 24, ji1900318; DOI: https://doi.org/10.4049/jimmunol.1900318 37. Weiss DI, Ma F, Merleev AA, Maverakis E, Gilliet M, Balin SJ, Bryson BD, Ochoa MT, Pellegrini M, Bloom BR and Robert L. Modlin. 2019. IL-1β Induces the Rapid Secretion of the Antimicrobial Protein IL-26 from Th17 Cells. J. Immunol., June 24, ji1900318; DOI: https://doi.org/10.4049/jimmunol.1900318 38. Winter CA, Risley EA, Nuss GW (1962) Carrageenin-induced edema in hind paw of the rat as an assay for antiiflammatory drugs. Proc. Soc. Exp. Biol. Med., v. 111, p. 544–547 38. Winter CA, Risley EA, Nuss GW (1962) Carrageenin-induced edema in hind paw of the rat as an assay for antiiflammatory drugs. Proc. Soc. Exp. Biol. Med., v. 111, p. 544–547 Figures Figures Figure 1 Effect of EESM oral treatments at 1 (A), 2 (B) and 4 (C) hour after carrageenan-induced edema. The control (saline 0.9 %, p.o.), EESM (30, 100, or 300 mg/kg, p.o.), and DEXA (dexamethasone 1 mg/kg, i.p.) groups were treated 1 h before the carrageenan injection. Each bar represents the mean ± SEM. The letters a, b, c, and d indicate significant differences among groups according to One-way ANOVA followed by the Newman-Keuls test Figure 1 Effect of EESM oral treatments at 1 (A), 2 (B) and 4 (C) hour after carrageenan-induced edema. The control (saline 0.9 %, p.o.), EESM (30, 100, or 300 mg/kg, p.o.), and DEXA (dexamethasone 1 mg/kg, i.p.) groups were treated 1 h before the carrageenan injection. Each bar represents the mean ± SEM. The letters a, b, c, and d indicate significant differences among groups according to One-way ANOVA followed by the Newman-Keuls test Page 15/21 Figure 2 Effect of EESM oral treatment at 3 and 4 h after carrageenan-induced mechanical sensitivity (Figures 2A and 2B) and cold hypersensitivity (Figures 2C and 2D). The control (saline 0.9 %, p.o.), EESM (30, 100, or 300 mg/kg, p.o.), and DEXA (dexamethasone 1 mg/kg, s.c.) groups were treated before 1 h of carrageenan injection in the paw. Each bar represents the mean ± SEM. The letters a, b and c indicate significant differences among groups according to One-way ANOVA followed by the Newman-Keuls test Figure 2 Effect of EESM oral treatment at 3 and 4 h after carrageenan-induced mechanical sensitivity (Figures 2A and 2B) and cold hypersensitivity (Figures 2C and 2D). The control (saline 0.9 %, p.o.), EESM (30, 100, or 300 mg/kg, p.o.), and DEXA (dexamethasone 1 mg/kg, s.c.) groups were treated before 1 h of carrageenan injection in the paw. Each bar represents the mean ± SEM. The letters a, b and c indicate significant differences among groups according to One-way ANOVA followed by the Newman-Keuls test Page 16/21 Figure 3 Effect of EESM oral administration on (A) leukocyte migration x 106 cells/cavity, (B) proteins (mg/ml) induced by intrapleural injection of carrageenan in mice. The control group received saline solution (0.9 %), and the EESM groups received 30, 100, or 300 mg/kg of EESM. Each bar represents the mean ± SEM. The letters a and b indicate significant differences among groups according to One-way ANOVA followed by the Newman-Keuls test Figure 3 Effect of EESM oral administration on (A) leukocyte migration x 106 cells/cavity, (B) proteins (mg/ml) induced by intrapleural injection of carrageenan in mice. The control group received saline solution (0.9 %), and the EESM groups received 30, 100, or 300 mg/kg of EESM. Each bar represents the mean ± SEM. The letters a and b indicate significant differences among groups according to One-way ANOVA followed by the Newman-Keuls test Page 17/21 Figure 4 Page 18/21 Figure 4 Effects of EESM oral treatment on leukocyte (A) in the pleurisy, Il-1□ levels pleural exudate (B), and in blood (C) induced by BCG. The animals received EESM (30, or 100, p.o.), vehicle (control) or isoniazid (ISO, 25 mg/kg, p.o.) for 7 days and an intrathoracic injection of BCG was administered since the first day. The naive group received an intrapleural injection of sterile saline instead of BCG and was also treated with saline solution. Each bar represents the mean ± SEM of 6 animals. The letters a, b, and c Figure 4 Effects of EESM oral treatment on leukocyte (A) in the pleurisy, Il-1□ levels pleural exudate (B), and in blood (C) induced by BCG. The animals received EESM (30, or 100, p.o.), vehicle (control) or isoniazid (ISO, 25 mg/kg, p.o.) for 7 days and an intrathoracic injection of BCG was administered since the first day. The naive group received an intrapleural injection of sterile saline instead of BCG and was also treated with saline solution. Each bar represents the mean ± SEM of 6 animals. The letters a, b, and c Page 18/21 Page 18/21 indicate significant differences among groups according to One-way ANOVA followed by the Newman- Keuls test indicate significant differences among groups according to One-way ANOVA followed by the Newman- Keuls test Figure 5 Effects of oral administration of EESM on mechanical hyperalgesia induced by CFA. The animals received EESM (30, or 100, p.o., daily, once a day), vehicle (control) or dexamethasone (DEXA, 1 mg/kg s.c., daily, once a day) for 6 (A), 11(B), 16 (C), and 22 (D) days. The CFA injection was performed on the first day. Each bar represents the mean ± SEM of 6 animals. The letters a, b, and c indicate significant differences among groups according to One-way ANOVA followed by the Newman-Keuls test Figure 5 Figure 5 Effects of oral administration of EESM on mechanical hyperalgesia induced by CFA. The animals received EESM (30, or 100, p.o., daily, once a day), vehicle (control) or dexamethasone (DEXA, 1 mg/kg, s.c., daily, once a day) for 6 (A), 11(B), 16 (C), and 22 (D) days. The CFA injection was performed on the first day. Each bar represents the mean ± SEM of 6 animals. The letters a, b, and c indicate significant differences among groups according to One-way ANOVA followed by the Newman-Keuls test Page 19/21 Figure 6 Effects of oral administration of EESM on edema induced by CFA. The animals received EESM (30, or 100, p.o., daily, once a day), vehicle (control) or dexamethasone (DEXA, 1 mg/kg, s.c., daily, once a day for 6 (A), 11(B), 16 (C), and 22 (D) days. The CFA injection was performed on the first day. Each bar represents the mean ± SEM of 6 animals. The letters a, b, and c indicate significant differences among groups according to One-way ANOVA followed by the Newman-Keuls test Figure 6 Effects of oral administration of EESM on edema induced by CFA. The animals received EESM (30, or 100, p.o., daily, once a day), vehicle (control) or dexamethasone (DEXA, 1 mg/kg, s.c., daily, once a day for 6 (A), 11(B), 16 (C), and 22 (D) days. The CFA injection was performed on the first day. Each bar represents the mean ± SEM of 6 animals. The letters a, b, and c indicate significant differences among groups according to One-way ANOVA followed by the Newman-Keuls test Figure 6 Effects of oral administration of EESM on edema induced by CFA. The animals received EESM (30, or 100, p.o., daily, once a day), vehicle (control) or dexamethasone (DEXA, 1 mg/kg, s.c., daily, once a day) for 6 (A), 11(B), 16 (C), and 22 (D) days. The CFA injection was performed on the first day. Each bar represents the mean ± SEM of 6 animals. The letters a, b, and c indicate significant differences among groups according to One-way ANOVA followed by the Newman-Keuls test Page 20/21 Figure 7 Effects of oral administration of EESM on cold hyperalgesia induced by CFA. The animals received EES (30, or 100, p.o., daily, once a day), vehicle (control) or dexamethasone (DEXA, 1 mg/kg, s.c., daily, once day) for 6 (A), 11(B), 16 (C), and 22 (D) days. The CFA injection was performed on the first day. Each ba represents the mean ± SEM of 6 animals. The letters a and b indicate significant differences among groups according to One-way ANOVA followed by the Newman-Keuls test Figure 7 Effects of oral administration of EESM on cold hyperalgesia induced by CFA. The animals received EESM (30, or 100, p.o., daily, once a day), vehicle (control) or dexamethasone (DEXA, 1 mg/kg, s.c., daily, once a day) for 6 (A), 11(B), 16 (C), and 22 (D) days. The CFA injection was performed on the first day. Each bar represents the mean ± SEM of 6 animals. The letters a and b indicate significant differences among groups according to One-way ANOVA followed by the Newman-Keuls test Page 21/21
https://openalex.org/W4321495061	https://www.nature.com/articles/s41598-023-30137-6.pdf	English	null	Publisher Correction: Identification of key immune-related genes in dilated cardiomyopathy using bioinformatics analysis	Scientific reports	2,023	cc-by	287	www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Publisher Correction: Identification of key immune‑related genes in dilated cardiomyopathy using bioinformatics analysis OPEN Feng Li , Tong‑Yue Du , Li‑Da Wu , Lei Zhang , Huan‑Huan Liu , Zhen‑Ye Zhang , Jie Zhang , Zhi‑Yuan Zhang , Ling‑Ling Qian , Ru‑Xing Wang & Jian‑Feng Hao Feng Li , Tong‑Yue Du , Li‑Da Wu , Lei Zhang , Huan‑Huan Liu , Zhen‑Ye Zhang , Jie Zhang , Zhi‑Yuan Zhang , Ling‑Ling Qian , Ru‑Xing Wang & Jian‑Feng Hao Correction to: Scientific Reports https://doi.org/10.1038/s41598-022-26277-w, published online 01 February 2023 The original version of this Article contained an error in the order of the author names, which was incorrectly given as Feng Li, Tong-Yue Du, Li-Da Wu, Lei Zhang, Huan-Huan Liu, Zhen-Ye Zhang, Jie Zhang, Zhi-Yuan Zhang, Ling-Ling Qian, Jian-Feng Hao & Ru-Xing Wang. © The Author(s) 2023 The original Article has been corrected. The original Article has been corrected. 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/. \| https://doi.org/10.1038/s41598-023-30137-6 Scientific Reports \| (2023) 13:3096
https://openalex.org/W2242736313	http://pdf.blucher.com.br/engineeringproceedings/tic2015/036.pdf	Portuguese	null	O USO DO CIM (CITY INFORMATION MODELING) PARA GERAÇÃO DE IMPLANTAÇÃO EM CONJUNTOS DE HABITAÇÃO DE INTERESSE SOCIAL: UMA EXPERIÊNCIA DE ENSINO	null	2,015	cc-by	837	Resumo A questão da habitação e os paradigmas que envolvem o habitar humano na contemporaneidade têm sido uma temática recorrente no panorama arquitetônico do século XXI; assim, torna-se necessário entendê-los como elementos fundamentais na estruturação das cidades. Essa problemática se intensifica ao analisarmos o tema da habitação de interesse social (HIS) e seu impacto na qualidade de vida dos habitantes, e como consequência, das cidades brasileiras. O presente trabalho é parte dos resultados de uma pesquisa de doutorado que utilizou o formalismo da gramática da forma e a abordagem da modelagem paramétrica como métodos para a geração de espaços públicos em conjuntos de HIS no Brasil, assumindo que essa metodologia pode contribuir para uma exploração mais ampla e aprofundada de diferentes soluções projetuais. A fim de testar o método proposto foram organizados três workshops com alunos do curso de Arquitetura e Urbanismo da Faculdade de Engenharia Civil, Arquitetura e Urbanismo, da Universidade Estadual de Campinas. Cada workshop foi direcionado para diferentes escalas do projeto de um conjunto habitacional: (1) desenho urbano, (2) implantação urbana do conjunto habitacional e (3) relação entre os edifícios e os espaços externos. Este artigo apresentará os resultados obtidos no workshop (1), denominado Parametric Urban Design. Palavras-chave: Modelagem paramétrica. City Information Modeling. Habitação de Interesse Social. José Nuno Beirão Universidade de Lisboa (UL) jnb@fa.ulisboa.pt Leticia Teixeira Mendes Universidade Federal de Pernambuco (UFPE) leticia.mendes@ufpe.br Gabriela Celani Universidade Estadual de Campinas (UNICAMP) Leticia Teixeira Mendes Universidade Federal de Pernambuco (UFPE) leticia.mendes@ufpe.br Gabriela Celani Universidade Estadual de Campinas (UNICAMP) celani@fec.unicamp.br 1 BEIRÃO, J. N.; MENDES, L. T.; CELANI, G. O uso do CIM (City Information Modeling) para geração de implantação em conjuntos de habitação de interesse social: uma experiência de ensino. In: ENCONTRO BRASILEIRO DE TECNOLOGIA DE INFORMAÇÃO E COMUNICAÇÃO NA CONSTRUÇÃO, 7, 2015, Recife. Anais... Porto Alegre: ANTAC, 2015. CIM (CITY INFORMATION MODELING) FOR THE GENERATION OF SITE LAYOUTS FOR SOCIAL HOUSING COMPLEXES: A TEACHING EXPERIENCE José Nuno Beirão Universidade de Lisboa (UL) jnb@fa.ulisboa.pt Leticia Teixeira Mendes Universidade Federal de Pernambuco (UFPE) leticia.mendes@ufpe.br José Nuno Beirão Universidade de Lisboa (UL) jnb@fa.ulisboa.pt Leticia Teixeira Mendes Universidade Federal de Pernambuco (UFPE) leticia.mendes@ufpe.br Gabriela Celani Universidade Estadual de Campinas (UNICAMP) Abstract The issue of housing and the paradigms related to human dwelling in the contemporary world have been a recurrent topic in the 21st architectural scenario; therefore, it is necessary to understand them as fundamental elements in the structuring of cities. This issue is even more relevant when we consider low-income housing and its impact on the life quality of people, and therefore of Brazilian cities. This paper presents part of the results of a PhD research that used the Shape Grammar formalism and the parametric modeling approach as methods for the generation of public spaces in low-income housing developments in Brazil. The research assumed that these 1 BEIRÃO, J. N.; MENDES, L. T.; CELANI, G. O uso do CIM (City Information Modeling) para geração de implantação em conjuntos de habitação de interesse social: uma experiência de ensino. In: ENCONTRO BRASILEIRO DE TECNOLOGIA DE INFORMAÇÃO E COMUNICAÇÃO NA CONSTRUÇÃO, 7, 2015, Recife. Anais... Porto Alegre: ANTAC, 2015. methods could contribute to the generation of a broader number of design alternative solutions. Aiming to test the proposed method, three workshops were offered to the University of Campinas' Architecture and Urban Design students. Each workshop focused on a different design scale: (1) urban design, (2) site planning of the complex and (3) spatial relations between buildings and open spaces. In this paper the results of the first workshop, titled Parametric Urban Design, are described. Keywords: Parametric design. City Information Modeling. Low-income housing. DOI: http://dx.doi.org/10.11606/gtp.v10i2.102564 methods could contribute to the generation of a broader number of design alternative solutions. Aiming to test the proposed method, three workshops were offered to the University of Campinas' Architecture and Urban Design students. Each workshop focused on a different design scale: (1) urban design, (2) site planning of the complex and (3) spatial relations between buildings and open spaces. In this paper the results of the first workshop, titled Parametric Urban Design, are described. methods could contribute to the generation of a broader number of design alternative solutions. Aiming to test the proposed method, three workshops were offered to the University of Campinas' Architecture and Urban Design students. Each workshop focused on a different design scale: (1) urban design, (2) site planning of the complex and (3) spatial relations between buildings and open spaces. In this paper the results of the first workshop, titled Parametric Urban Design, are described. methods could contribute to the generation of a broader number of design alternative solutions. Aiming to test the proposed method, three workshops were offered to the University of Campinas' Architecture and Urban Design students. Each workshop focused on a different design scale: (1) urban design, (2) site planning of the complex and (3) spatial relations between buildings and open spaces. In this paper the results of the first workshop, titled Parametric Urban Design, are described. Keywords: Parametric design. City Information Modeling. Low-income housing. http://dx.doi.org/10.11606/gtp.v10i2.102564 DOI: http://dx.doi.org/10.11606/gtp.v10i2.102564
https://openalex.org/W3214286852	https://www.mdpi.com/2079-9292/10/21/2717/pdf?version=1636368287	English	null	Stock Market Prediction Using Machine Learning Techniques: A Decade Survey on Methodologies, Recent Developments, and Future Directions	Electronics	2,021	cc-by	18,449	Abstract: With the advent of technological marvels like global digitization, the prediction of the stock market has entered a technologically advanced era, revamping the old model of trading. With the ceaseless increase in market capitalization, stock trading has become a center of investment for many ﬁnancial investors. Many analysts and researchers have developed tools and techniques that predict stock price movements and help investors in proper decision-making. Advanced trading models enable researchers to predict the market using non-traditional textual data from social platforms. The application of advanced machine learning approaches such as text data analytics and ensemble methods have greatly increased the prediction accuracies. Meanwhile, the analysis and prediction of stock markets continue to be one of the most challenging research areas due to dynamic, erratic, and chaotic data. This study explains the systematics of machine learning-based approaches for stock market prediction based on the deployment of a generic framework. Findings from the last decade (2011–2021) were critically analyzed, having been retrieved from online digital libraries and databases like ACM digital library and Scopus. Furthermore, an extensive comparative analysis was carried out to identify the direction of signiﬁcance. The study would be helpful for emerging researchers to understand the basics and advancements of this emerging area, and thus carry-on further research in promising directions. Received: 17 October 2021 Accepted: 5 November 2021 Published: 8 November 2021 Keywords: generic review; machine learning; stock market prediction; support vector machine Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. eview Stock Market Prediction Using Machine Learning Techniques: A Decade Survey on Methodologies, Recent Developments, and Future Directions https://doi.org/10.3390/ electronics10212717 Academic Editor: George Angelos Papadopoulos Received: 17 October 2021 Accepted: 5 November 2021 Published: 8 November 2021 Citation: Rouf, N.; Malik, M.B.; Arif, T.; Sharma, S.; Singh, S.; Aich, S.; Kim, H.-C. Stock Market Prediction Using Machine Learning Techniques: A Decade Survey on Methodologies, Recent Developments, and Future Directions. Electronics 2021, 10, 2717. https://doi.org/10.3390/ electronics10212717 Academic Editor: George Angelos Papadopoulos Received: 17 October 2021 Accepted: 5 November 2021 Published: 8 November 2021 eview Stock Market Prediction Using Machine Learning Techniques: A Decade Survey on Methodologies, Recent Developments, and Future Directions Nusrat Rouf 1, Majid Bashir Malik 2, Tasleem Arif 3, Sparsh Sharma 4, Saurabh Singh 5 , Satyabrata Aich 6,* and Hee-Cheol Kim 7,* id Bashir Malik 2, Tasleem Arif 3, Sparsh Sharma 4, Saurabh Singh 5 , Satyabrata Aich 6,* m 7,* 1 Research Lab., Department of Computer Sciences, BGSB University, Rajouri 185234, India; nusratrouf@bgsbu.ac.in 1 Research Lab., Department of Computer Sciences, BGSB University, Rajouri 185234, India; nusratrouf@bgsbu.ac.in 2 Department of Computer Sciences, BGSB University, Rajouri 185234, India; majidbashirmalik@bgsbu.ac.in 3 Department of Information Technology, BGSB University, Rajouri 185234, India; t.arif@bgsbu.ac.in 4 Department of Computer Science and Engineering, NIT Srinagar 190001, India; sparsh.sharma@nitsri.net 5 Department of Industrial and System Engineering, Dongguk University, Seoul 04620, Korea; saurabh89@dongguk.edu 6 Department of Computer Engineering, Institute of Digital Anti-Aging Healthcare, Inje University, Gimhae 50834, Korea 7 College of AI Convergence, Institute of Digital Anti-Aging Healthcare, u-AHRC, Inje University, Gimhae 50834, Korea * Correspondence: satyabrataaich@gmail.com (S.A.); heeki@inje.ac.kr (H.-C.K.); Tel.: +82-55-320-3720 (H.-C.K.) g 2 Department of Computer Sciences, BGSB University, Rajouri 185234, India; majidbashirmalik@bgsbu.ac.in 3 Department of Information Technology, BGSB University, Rajouri 185234, India; t.arif@bgsbu.ac.in 4 Department of Computer Science and Engineering, NIT Srinagar 190001, India; sparsh.sharma@nitsri.net 5 Department of Industrial and System Engineering, Dongguk University, Seoul 04620, Korea; saurabh89@dongguk.edu 2 Department of Computer Sciences, BGSB University, Rajouri 185234, India; majidbashirmalik@bgsbu.ac.in 3 Department of Information Technology, BGSB University, Rajouri 185234, India; t.arif@bgsbu.ac.in 4 Department of Computer Science and Engineering NIT Srinagar 190001 India; sparsh sharma@nitsri net 2 Department of Computer Sciences, BGSB University, Rajouri 185234, India; majidbashirmalik@bgsbu.ac.in 3 Department of Information Technology, BGSB University, Rajouri 185234, India; t.arif@bgsbu.ac.in 4 Department of Computer Science and Engineering, NIT Srinagar 190001, India; sparsh.sharma@nitsri.net 5 Department of Industrial and System Engineering, Dongguk University, Seoul 04620, Korea; saurabh89@dongguk.edu 6 Department of Computer Engineering, Institute of Digital Anti-Aging Healthcare, Inje University, Gimhae 50834, Korea 7 College of AI Convergence, Institute of Digital Anti-Aging Healthcare, u-AHRC, Inje University, Gimhae 50834 Korea * Correspondence: satyabrataaich@gmail.com (S.A.); heeki@inje.ac.kr (H.-C.K.); Tel.: +82-55-320-3720 (H.-C.K. Citation: Rouf, N.; Malik, M.B.; Arif, T.; Sharma, S.; Singh, S.; Aich, S.; Kim, H.-C. Stock Market Prediction Using Machine Learning Techniques: A Decade Survey on Methodologies, Recent Developments, and Future Directions. Electronics 2021, 10, 2717. electronics 1. Introduction An advancement in the fundamental aspects of information technology over the last few decades has altered the route of businesses. As one of the most captivating inventions, ﬁnancial markets have a pointed effect on the nation’s economy [1]. The World Bank reported in 2018 that the stock market capitalization worldwide has surpassed 68.654 trillion US$ [2]. Over the last few years, stock trading has become a center of attention, which can largely be attributed to technological advances. Investors search for tools and techniques that would increase proﬁt and reduce the risk [3]. However, Stock Market Prediction (SMP) is not a simple task due to its non-linear, dynamic, stochastic, and unreliable nature [4]. SMP is an example of time-series forecasting that promptly Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/electronics Electronics 2021, 10, 2717. https://doi.org/10.3390/electronics10212717 Electronics 2021, 10, 2717 2 of 25 niques diction examines previous data and estimates future data values. Financial market prediction has been a matter of worry for analysts in different disciplines, including economics, mathematics, material science, and computer science. Driving proﬁts from the trading of stocks is an important factor for the prediction of the stock market [5]. The stock market is dependent on various parameters, such as the market value of a share, the company’s performance, government policies, the country’s Gross Domestic Product (GDP), the inﬂation rate, natural calamities, and so on [6]. The Efﬁcient Market Hypothesis explains that stock market costs are signiﬁcantly determined by new information, and follow a random walk pattern, such that they cannot be predicted solely based on past information [7]. This was a widely accepted theory in the past. With the advent of technology, researchers demonstrated that stock market prices could be predicted to a certain extent. Historical market data, combined with the data extracted from social media platforms, can be analyzed to predict the changes in the economic and business sectors [8]. The performance of stock market prediction systems relies intensely on the quality of the features it is using [9]. While researchers have used some strategies for enhancing the stock-explicit features, more attention needs to be paid to feature extraction and selection mechanisms. Figure 1 presents the outline of this article. 1.1.2. Technical Analysis Technical analysis is the study of stock prices to make a proﬁt, or to make better investment decisions [19]. Technical analysis predicts the direction of the future price movements of stocks based on their historical data, and helps to analyze ﬁnancial time series data using technical indicators to forecast stock prices. Meanwhile, it is assumed that the price moves in a trend and has momentum [20]. Technical analysis uses price charts and certain formulae, and studies patterns to predict future stock prices; it is mainly used by short-term investors. The price would be considered high, low or open, or the closing price of the stock, where the time points would be daily, weekly, monthly, or yearly. Dow theory puts forward the main principles for technical analysis, which are that the market price discounts everything, prices move in trends, and historic trends usually repeat the same patterns [21]. There are several technical indicators, such as the Moving Average (MA), Moving Average Convergence/Divergence (MACD), the Aroon indicator, and the money ﬂow index, etc. The evident ﬂaws of technical analysis as per [18] are that expert’s opinions deﬁne rules in technical analysis, which are ﬁxed and are reluctant to change. Various parameters that affect stock prices are ignored. p p g The prerequisite is to overcome the deﬁciencies of fundamental and technical anal- ysis, and the evident advancement in the modelling techniques has motivated various researchers to study new methods for stock price prediction. A new form of collective intelligence has emerged, and new innovative methods are being employed for stock value forecasting. The methodologies incorporate the work of machine learning algorithms for stock market analysis and prediction. 1.1. Classical Approaches for SMP According to [10], there exist two main traditional approaches to the analysis of the stock markets: (1) fundamental analysis and (2) technical analysis. 1. Introduction [ ] p g p p y p and estimates future data values. Financial market prediction has been a matter of worry for analysts in different disciplines, including economics, mathematics, material science, and computer science. Driving profits from the trading of stocks is an important factor for the prediction of the stock market [5]. The stock market is dependent on various parame- ters, such as the market value of a share, the company’s performance, government poli- cies, the country’s Gross Domestic Product (GDP), the inflation rate, natural calamities, and so on [6]. The Efficient Market Hypothesis explains that stock market costs are signif- icantly determined by new information, and follow a random walk pattern, such that they cannot be predicted solely based on past information [7]. This was a widely accepted the- ory in the past. With the advent of technology, researchers demonstrated that stock mar- ket prices could be predicted to a certain extent. Historical market data, combined with the data extracted from social media platforms, can be analyzed to predict the changes in the economic and business sectors [8]. The performance of stock market prediction sys- tems relies intensely on the quality of the features it is using [9]. While researchers have used some strategies for enhancing the stock-explicit features, more attention needs to be paid to feature extraction and selection mechanisms. Figure 1 presents the outline of this article. Figure 1. Article outline. Figure 1. Article outline. Figure 1. Article outline. Figure 1. Article outline. Electronics 2021, 10, 2717 3 of 25 1.1.1. Fundamental Analysis Fundamental analysis calculates a genuine value of a sector/company and determines the amount that one share of that company should cost. A supposition is made that, if given sufﬁcient time, the company will move to a cost agreeing with the prediction. If a sector/company is undervalued, then the market value of that company should rise, and conversely, if a company is overvalued, then the market price should fall [11]. The analysis is performed considering various factors, such as yearly ﬁscal summaries and reports, balance sheets, a future prospectus, and the company’s work environment [12]. If stocks are overvalued, then the market price will fall [13], e.g., the Dotcom bubble burst in the year 2000 [14]. The two most common metrics used to predict long-term price movements yearly for fundamental analysis are (a) the Price to Earnings ratio (P/E) and (b) the Price by Book ratio (P/B). The P/E ratio is used as a predictor. The companies with a lower P/E ratio yield higher returns than companies with a high P/E ratio [15]. Financial analysts also use this to prove their stock recommendations [16]. Fundamental analysis can be used for the consideration of ﬁnancial ratios to distinguish poor stocks from quality stocks [17]. The P/B ratio compares the company value speciﬁed by the market to the company value speciﬁed on paper. If the ratio is high, the company may be overvalued, and the company’s value might fall with time. Conversely, if the ratio is low, the company may be underestimated, and the price may rise with time. Of course, fundamental analysis is a powerful method. Still, it has some drawbacks. Fundamental analysis, ﬁrstly, lacks adequate knowledge of the rules governing the workings of the system, and secondly, there is non-linearity in the system [18]. 1.2.2. Sentiment Analysis Approach One of the phenomena of current times that is changing the world is the global availability of the internet. The most-used platforms on the internet are social media. It is estimated that social media users all over the world will number around 3.07 billion [27]. There is a high association between stock prices and events related to stocks on the web. The event information is extracted from the internet to predict stock prices; such an approach is known as event-driven stock prediction [28]. Through social networks, people generate tremendous amounts of data that is ﬁlled with emotions. Much of this data is related to user perceptions and concerns [29]. Sentiment analysis is a ﬁeld of study that deals with the people’s concerns, beliefs, emotions, perceptions, and sentiments towards some entity [30,31]. It is the process of analyzing text corpora, e.g., news feeds or stock market- speciﬁc tweets, for stock trend prediction. The Stock Twits, Twitter, Yahoo Finance, and so on are well-known platforms used for the extraction of sentiments. There is a signiﬁcant importance of using sentimental data for enhancing the prediction of volatility in the stock market. The ‘Wisdom of Crowds’ and sentiment analysis generate more insights that can be used to increase the performance in various ﬁelds, such as box ofﬁce sales, election outcomes, SMP, and so on [32]. This suggests that a good decision can be made by taking the opinions and insights of large groups of people with varied types of information [33]. The information generated through social media allows us to explore vast and diverse opinions. Exploring sentiments from social media in addition to numeric time-series stock data would enhance the accuracy of the prediction. Using time-series data as well as social media data would intensify the prediction accuracy. Different approaches and techniques have been proposed over time to anticipate stock prices through numerous methodologies, thanks to the dynamic and challenging panorama of stock markets [34]. 1.2. Modern Approaches for SMP There are some modern approaches that can be functional and fruitful for SMP that would enhance prediction accuracies. In this review, we will highlight some modern functional approaches. Electronics 2021, 10, 2717 4 of 25 1.2.1. Machine Learning Approach 1.2.1. Machine Learning Approach Because of global digitization, SMP has entered a technological era. Machine learning in stock price prediction is used to discover patterns in data [22]. Usually, a tremendous amount of structured and unstructured heterogeneous data is generated from stock mar- kets. Using machine learning algorithms, it is possible to quickly analyze more complex heterogeneous data and generate more accurate results. Various machine learning methods have been used for SMP [23]. The machine learning approaches are mainly categorized into supervised and unsupervised approaches. In the supervised learning approach, named input data and the desired output are given to the learning algorithms. Meanwhile, in the unsupervised learning approach, unlabeled input data is provided to the learning algorithm, and the algorithm identiﬁes the patterns and generates the output accordingly. Furthermore, different algorithmic approaches have been used in SMP, such as the Support Vector Machine (SVM), k Nearest Neighbors (kNN), Artiﬁcial Neural Networks (ANN), Decision Trees, Fuzzy Time-Series, and Evolutionary Algorithms. The SVM is a supervised machine learning technique that limits error and augments geometric margins, and is a pattern classiﬁcation algorithm [24]. In terms of accuracy, the SVM is an important machine learning algorithm compared to the other classiﬁers [25]. In the kNN, stock prediction is mapped into a classiﬁcation based on closeness. Using Euclidean distance, the kNN classiﬁes the “k” nearest neighbors in the training set. The ANN is a nonlinear computa- tional structure for various machine learning algorithms to analyze and process complex input data together. The FIS (Fuzzy Inference Systems) apply rules to fuzzy sets and then apply de-fuzziﬁcation to give crisp outputs for decision making [26]. The evolutionary algorithms include gene-inspired neuro-fuzzy and neuro-genetic algorithms, mimic the natural selection theory of species, and can give an optimal output. 2. Research Methodology This section explains the overall process of the literature collection on SMP using machine learning. Initially, the phrase “stock market prediction using machine learning” was keyed to various search engines, digital libraries and databases, including ‘google Electronics 2021, 10, 2717 5 of 25 using scholar’, ‘research gate’, ‘ACM digital library’, ‘IEEE Explore’, ‘Scopus’, and so on. During the process of literature collection, various phrases like “stock market prediction methods”, “impact of sentiments on stock market prediction”, and “machine learning-based approach for stock market prediction” were keyed. The OR and AND operators were used for the keyword searches in single and multiple classes, respectively. As a result, some of the fundamental papers in the ﬁeld of stock market prediction were retrieved. By the careful analysis of a few basic papers, a primary insight into the domain was obtained. The search criteria were further modiﬁed to collect the literature of the last decade, in order to enhance and improve the domain. In addition, the literature selected was screened by applying quality criteria, where metrics such as indexing, quartiles, impact factors and publishers were observed. Figure 2 presents the steps followed in the literature collection. y g g g g g scholar’, ‘research gate’, ‘ACM digital library’, ‘IEEE Explore’, ‘Scopus’, and so on. During the process of literature collection, various phrases like “stock market prediction meth- ods”, “impact of sentiments on stock market prediction”, and “machine learning-based approach for stock market prediction” were keyed. The OR and AND operators were used for the keyword searches in single and multiple classes, respectively. As a result, some of the fundamental papers in the field of stock market prediction were retrieved. By the care- ful analysis of a few basic papers, a primary insight into the domain was obtained. The search criteria were further modified to collect the literature of the last decade, in order to enhance and improve the domain. In addition, the literature selected was screened by applying quality criteria, where metrics such as indexing, quartiles, impact factors and publishers were observed. Figure 2 presents the steps followed in the literature collection. Figure 2. Literature collection process. Figure 2. Literature collection process. Figure 2. Literature collection process. Figure 2. Literature collection process. .1. Market Data 4.1. Market Data Market data are the temporal historical price-related numerical data of financial mar- kets. Analysts and traders use the data to analyze the historical trend and the latest stock prices in the market. They reflect the information needed for the understanding of market behavior. The market data are usually free, and can be directly downloaded from the mar- ket websites. Various researchers have used this data for the prediction of price move- ments using machine learning algorithms. The previous studies have focused on two ypes of predictions. Some studies have used stock index predictions like the Dow Jones ndustrial Average (DJIA) [35], Nifty [36], Standard and Poor’s (S&P) 500 [37], National Association of Securities Dealers Automated Quotations (NASDAQ) [38], the Deutscher Aktien Index (DAX) index [39], and multiple indices [40,41]. Other studies have used in- dividual stock prediction based on some specific companies like Apple [42], Google [43], or groups of companies [12 44] Market data are the temporal historical price-related numerical data of ﬁnancial markets. Analysts and traders use the data to analyze the historical trend and the latest stock prices in the market. They reﬂect the information needed for the understanding of market behavior. The market data are usually free, and can be directly downloaded from the market websites. Various researchers have used this data for the prediction of price movements using machine learning algorithms. The previous studies have focused on two types of predictions. Some studies have used stock index predictions like the Dow Jones Industrial Average (DJIA) [35], Nifty [36], Standard and Poor’s (S&P) 500 [37], National Association of Securities Dealers Automated Quotations (NASDAQ) [38], the Deutscher Aktien Index (DAX) index [39], and multiple indices [40,41]. Other studies have used individual stock prediction based on some speciﬁc companies like Apple [42], Google [43], or groups of companies [12,44]. r groups of companies [12,44]. Furthermore, the studies focused on time-specific predictions like intraday [45], daily 20], weekly [46], and monthly predictions [47], and so on. Moreover, most of the previous esearch is based on categorical prediction, where predictions are categorized into discrete lasses like up, down, positive, or negative [32,48]. Technical indicators have been widely used for SMP due to their summative representation of trends in time series data. Some tudies considered different types of technical indicators, e.g., trend indicators, momen- um indicators, volatility indicators and volume indicators [32,49,50]. .1. Market Data 4.1. Market Data Furthermore, nu- merous studies have used an amalgam of different types of technical indicators for SMP 42 51] Furthermore, the studies focused on time-speciﬁc predictions like intraday [45], daily [20], weekly [46], and monthly predictions [47], and so on. Moreover, most of the previous research is based on categorical prediction, where predictions are categorized into discrete classes like up, down, positive, or negative [32,48]. Technical indicators have been widely used for SMP due to their summative representation of trends in time series data. Some studies considered different types of technical indicators, e.g., trend indicators, momentum indicators, volatility indicators and volume indicators [32,49,50]. Furthermore, numerous studies have used an amalgam of different types of technical indicators for SMP [42,51]. . Types of Data 4. Types of Data SMP systems can be classified according to the type of data they use as the input. Most of the studies used market data for their analysis. Recent studies have considered extual data from online sources as well. In this section, the studies are classified based on he type of data they use for prediction purposes. At the end of this section, Table 1 points ut the comparison of the data sources, type of input and prediction duration used in the tudies so far. SMP systems can be classiﬁed according to the type of data they use as the input. Most of the studies used market data for their analysis. Recent studies have considered textual data from online sources as well. In this section, the studies are classiﬁed based on the type of data they use for prediction purposes. At the end of this section, Table 1 points out the comparison of the data sources, type of input and prediction duration used in the studies so far. 3. Generic Scheme for SMP 3. Generic Scheme for SMP Figure 3 describes the generic process involved in SMP. The process starts with the collection of the data, and then pre-processing that data so that it can be fed to a machine learning model. The prediction models generally use two types of data: market and tex- tual data. The literature of both types is discussed in the following section. The next sec- tion classifies the previous studies based on the type of data used. Furthermore, the next section surveys the previous studies based on the various data-preprocessing approaches applied. Moreover, the literature is further surveyed based on the machine learning algo- rithms used by different systems. Figure 3 describes the generic process involved in SMP. The process starts with the collection of the data, and then pre-processing that data so that it can be fed to a machine learning model. The prediction models generally use two types of data: market and textual data. The literature of both types is discussed in the following section. The next section classiﬁes the previous studies based on the type of data used. Furthermore, the next section surveys the previous studies based on the various data-preprocessing approaches applied. Moreover, the literature is further surveyed based on the machine learning algorithms used by different systems. 6 of 25 of 25 Electronics 2021, 10, 2717 lectronics 2021, 10, x FOR P Types of Data Machine Learning Data Pre-Processing Methods Figure 3. Generic Scheme for SMP (Stock Market Prediction). Figure 3. Generic Scheme for SMP (Stock Market Prediction). Types of Data Machine Learning Data Pre-Processing Methods igure 3. Generic Scheme for SMP (Stock Market Prediction). Figure 3. Generic Scheme for SMP (Stock Market Prediction). 2 T l D 4.2. Textual Data .2. Textual Data Textual data is used to analyze the effect of sentiments on the stock market. Public sentiments have been proven to affect the market considerably. The most challenging part is Electronics 2021, 10, 2717 7 of 25 to convert the textual information into numerical values so that it can be fed to a prediction model. Furthermore, the extraction of textual data is a challenging task. The textual data has many sources, such as ﬁnancial news websites, general news, and social platforms [52]. Most of the studies were carried out on textual data try to predict whether the sentiment towards a particular stock is positive or negative. The previous studies considered several textual sources for SMP, such as the Wall Street Journal [53], Bloomberg [22], CNBC and Reuters [54], Google Finance [55], and Yahoo Finance [56]. The extracted news may be either generalized news or some speciﬁc ﬁnancial news, but the majority of the researchers use ﬁnancial news, as it is deemed to be less susceptible to noise [57]. Some researchers have used less formal textual data, such as message boards [58,59]. Meanwhile, the textual data from microblogging websites and social networking websites are comparably less explored than other textual data forms for SMP. Besides this, one challenge faced for the processing of the textual data are that the information generated on these platforms is enormous, increasing the computational complexities [60,61]. For example, the researchers in [62] processed 1,00,000 tweets, and the researchers in [63] processed around 2,500,000 tweets, which was a complex task. Moreover, for the textual data, no proper standard format is followed while posting on social media, which increases the processing complexities. In addition, the detection of shorthand spellings, emoticons and sarcastic statements is yet another challenge. Machine learning algorithms come to the forefront to deal with all kinds of challenges faced while processing textual data. Previous studies have mostly considered the sentiment of textual data as positive or negative [35,48,64]. A few studies have considered mood words to determine the mood of a tweet, such as [8,58,65]. 5. Data Pre-Processing Once the data is available, it needs some pre-processing so that it can be fed to a machine learning model. The signiﬁcance of the output depends on the pre-processing of the data [66,67]. The textual data must be transformed into a structured format that can be used in a machine learning model. The previous studies revealed that there are three signiﬁcant pre-processing steps, i.e., feature selection, order reduction and the represen- tation of features. Table 1 presents the comparison of the data sources, type of input and prediction duration. Table 2 presents the comparison of the data pre-processing techniques used in the studies so far. Table 1. Comparison of the data sources, type of input and prediction duration. References Data Type of Input Prediction Duration [37] S&P 500 Market data Few days ahead [38] NASDAQ index Market data Few days ahead [39] DAX 30 broker house newsletters, RSS market feeds, and stock exchange data Intraday [56] Yahoo Finance Financial News Intraday [44] DGAP, Euro-Adhoc Corporate announcements ﬁnancial new Daily [58] Yahoo ﬁnance (18 Stock Companies data) Market data, yahoo ﬁnance message board data Daily [35] DJIA Market data and Twitter Daily [32] BSE and NSE stocks Market data, technical indicators, Twitter data Intraday [36] Nifty and Sensex Market data and news Intraday Table 1. Comparison of the data sources, type of input and prediction duration. Electronics 2021, 10, 2717 8 of 25 Table 1. Cont. References Data Type of Input Prediction Duration [47] Yahoo Finance Market data, Twitter data, and news data Daily and monthly [49] S&P, NYSE, DJIA Market data, Technical Indicators, Social media data Daily weekly [51] Apple, yahoo Market data, technical indicators 60 day and 90-day prediction [63] Microsoft company Twitter Daily [42] NASDAQ, DJIA, Apple Stock (AAPL) Market data, technical indicators, news. One-day ahead [43] Google stock Market data Five days horizon [45] Taiwan Stock Exchange CWI Market data High-frequency trading [68] S&P 500 Market data Daily [50] Columbia Stock Market Market data, Technical indicators Next day [69] S&P 500 Financial news from Noodle, Reuters Intraday [70] Enron Corpus Sentiment data Daily, weekly [46] BSE, Tech Mahindra Market data Daily and weekly [71] Apple stock data Market data Daily [72] United States stock exchange Market data, technical indicator Daily [73] KSE, LSE, Nasdaq, NYSE Twitter, yahoo ﬁnance, Wikipedia Weekly [74] Google stock Market data Daily Table 1. Cont. 5.1. Feature Selection Feature selection is a crucial step in textual data processing. Most of the studies on SMP have used basic feature extraction techniques such as Bag of Words, where the text is broken into words and each word is converted into a numeric feature. The feature selection depends on the number of occurrences of a word. Table 2 points out the various feature selection methods used in the literature so far. As in [67,70], most of the previous literature used feature selection techniques where the order of words is discarded, causing the loss of context. Another feature selection method is Word2Vec, as proposed by [32,81]. It is a word embedding technique based on a multi-layer perceptron. This technique takes into consideration the order and co-occurrence of words, and hence retains the context. Word2Vec has been used in some of the works, such as [63,77]. Moreover, a few studies have used Latent Dirichlet Allocation (LDA) [82–84]., where words are viewed as a probabilistic collection of concepts, and the concepts are used as features [82–84]. Some works [44,76,79] used the N-grams technique. N-grams is the contagious collection of N words from a given sequence of text. Other methods like genetic algorithms and particle swarm optimization have also been used for feature selection, like in [80,85]. 5. Data Pre-Processing References Data Type of Input Prediction Duration [47] Yahoo Finance Market data, Twitter data, and news data Daily and monthly [49] S&P, NYSE, DJIA Market data, Technical Indicators, Social media data Daily weekly [51] Apple, yahoo Market data, technical indicators 60 day and 90-day prediction [63] Microsoft company Twitter Daily [42] NASDAQ, DJIA, Apple Stock (AAPL) Market data, technical indicators, news. One-day ahead [43] Google stock Market data Five days horizon [45] Taiwan Stock Exchange CWI Market data High-frequency trading [68] S&P 500 Market data Daily [50] Columbia Stock Market Market data, Technical indicators Next day [69] S&P 500 Financial news from Noodle, Reuters Intraday [70] Enron Corpus Sentiment data Daily, weekly [46] BSE, Tech Mahindra Market data Daily and weekly [71] Apple stock data Market data Daily [72] United States stock exchange Market data, technical indicator Daily [73] KSE, LSE, Nasdaq, NYSE Twitter, yahoo ﬁnance, Wikipedia Weekly [74] Google stock Market data Daily Table 2. Comparison of various pre-processing techniques. References Feature Selection Order Reduction Feature Representation [39] Bag of Words Stemming Sentiment value [56] Opinion Finder overall tone and polarity Minimum Occurrence per document Boolean [44] Bag-of-words, noun phrases, word combinations, n-gram Frequency for news, Chi2-approach and bi-normal separation (BNS) for exogenous-feedback-based feature selection, dictionary TF-IDF [75] Bag-of-words WordNet to replace words TF-IDF [76] N-grams Document frequency Boolean [32] Context based approach SentiWordNet Sentiment value Table 2. Comparison of various pre-processing techniques. References Feature Selection Order Reduction Feature Representation [39] Bag of Words Stemming Sentiment value [56] Opinion Finder overall tone and polarity Minimum Occurrence per document Boolean [44] Bag-of-words, noun phrases, word combinations, n-gram Frequency for news, Chi2-approach and bi-normal separation (BNS) for exogenous-feedback-based feature selection, dictionary TF-IDF [75] Bag-of-words WordNet to replace words TF-IDF [76] N-grams Document frequency Boolean [32] Context based approach SentiWordNet Sentiment value Table 2. Comparison of various pre-processing techniques. Electronics 2021, 10, 2717 9 of 25 Table 2. Cont. References Feature Selection Order Reduction Feature Representation [58] Bag of words, LDA, JST, Aspect Based - TF-IDF [47] Correlation Lemmatization Boolean [69] Bag of Words Chi2, Information Gain, Document Frequency, Occurrence TF-IDF [77] Bag-of-word, Word2vec TF-IDF [78] GA PCA, FA, FO - [79] N-grams SVM based Recursive Feature Elimination, PCA, KPCA, and XGB - [73] Bag-of-words Occurrence TF-IDF [80] GA, Feature Ranking PCA-SVM, DA-RNN - 6.1. Artiﬁcial Neural Networks (ANN) The ANN is a biological brain-inspired technique in which a large number of artiﬁcial neurons are strongly interconnected in order to solve complex problems [90]. These models understand the context of a problem by creating multiple transformations on the feature space, followed by non-linearity, to create its simpliﬁed representations [91]. Numerous studies have employed ANN models for SMP [38,40,92–95]. For example, the authors in [68] employed ANN for daily trend prediction of the S&P 500 index. Three- dimensional reduction techniques—e.g., PCA, Fuzzy Robust Principal Component Analysis (FRPCA), and Kernel-based Principal Component Analysis—were applied to streamline the dataset. The results suggested that combining the ANNs with the PCAs is more efﬁcient. Furthermore, the selection of an appropriate kernel function directly affects the performance of KPCA [68]. Multilayer perceptron (MLP) is a frequently used technique for SMP [42,43,96,97]. MLP is an ANN with one input and output layer, and one or more intermediate layers. Generally, the MLP uses the backpropagation method for training, in which predicted errors are back propagated from the output layer to the input layer to minimize the errors [98,99]. Multilayer perceptron (MLP) is a frequently used technique for SMP [42,43,96,97]. MLP is an ANN with one input and output layer, and one or more intermediate layers. Generally, the MLP uses the backpropagation method for training, in which predicted errors are back propagated from the output layer to the input layer to minimize the errors [98,99]. A study compared three ANN models—MLP, dynamic artiﬁcial neural network (DAN2) and autoregressive conditional heteroscedasticity (GARCH)—for NASDAQ price prediction [38]. All three models were evaluated using the Mean Absolute Deviate (MAD) and Mean Square Error (MSE). The results demonstrated that the MLP outperformed DAN2 and GARCH-MLP. Furthermore, it provides a future direction for researchers by suggesting that they focus on ﬁnding out whether GARCH has a remedying impact on forecasts or other correlated variables that have a remedying impact on forecasts. A study compared three ANN models—MLP, dynamic artiﬁcial neural network (DAN2) and autoregressive conditional heteroscedasticity (GARCH)—for NASDAQ price prediction [38]. All three models were evaluated using the Mean Absolute Deviate (MAD) and Mean Square Error (MSE). The results demonstrated that the MLP outperformed DAN2 and GARCH-MLP. Furthermore, it provides a future direction for researchers by suggesting that they focus on ﬁnding out whether GARCH has a remedying impact on forecasts or other correlated variables that have a remedying impact on forecasts. 6. Machine Learning Methods This section attempts to summarize the machine learning models used in previous studies for stock prediction and forecasting. After the data is pre-processed and trans- formed to a standard representation, it is fed to machine learning models for further processing. Table 3 presents the distribution of various machine learning techniques used in literature so far. The following section brieﬂy summarizes the different machine learning approaches presented: • Artiﬁcial Neural Networks (ANN) • Artiﬁcial Neural Networks (ANN) • Support Vector Machine (SVM) • Naïve Bayes (NB) • Genetic Algorithms (GA) • Fuzzy Algorithms (FA) • Deep Neural Networks (DNN) • Regression Algorithms (RA) • Hybrid Approaches (HA) • Regression Algorithms (RA) • Hybrid Approaches (HA) 5.2. Order Reduction The feature selection process for the textual data leads to an increase in the number of features. High dimensional features are extremely difﬁcult to process, and leads to the poor efﬁciency of most of the learning algorithms [86]. This phenomenon is known as the curse of dimensionality [87]. Lower numbers of features will decrease the training complexity of the algorithms. Table 2 points out the order reduction techniques used in previous studies. A well-known form of multi-variant analysis, Principal Component Analysis (PCA), is used to select the most relevant features, reducing the dimensionality of the features. In [68], the daily direction of the S&P 500 index is predicted using 60 features. The authors used three variants of PCA, and concluded that the inclusion of PCA not only reduced the overall training complexity but also increased the accuracy of the predictions. In [78], numerous feature reduction techniques, e.g., PCA, Factor Analysis (FA), Genetic Algorithms (GA), and Fireﬂy Optimization (FO), we used to solve the data complexity. 10 of 25 10 of 25 Electronics 2021, 10, 2717 5.3. Feature Representation 5.3. Feature Representation Feature representation is one of the important factors for the efﬁcient training of machine learning algorithms. Once the number of required features is determined, the input data is converted to a numeric representation so that machine learning algorithms can readily process it. Table 2 presents the type of representation or the weighing used in the literature so far. Boolean representation is one of the most basic techniques of feature representation, in which the presence and absence of the feature (word) are represented by 1 and 0, respectively, for Bag of words [56]. Another technique, Term Frequency-Inverse Document Frequency (TF-IDF), has been used in numerous studies [44,69,73]. Generally, the text pre-processing phase is considered to be a crucial phase, and may signiﬁcantly impact the model’s accuracy [88,89]. 6.4. Genetic Algorithms (GA) GA are a heuristic approach to problem-solving that mimic the natural evolution process. The algorithms apply the concept of natural selection to select the optimal possible solution. In SMP, GA is used to fine-tune the parameters for the generation of the best trading rule. Numerous studies have used GA to enhance SMP accuracies [4,26,78,80,97,109–111]. For example, the authors in [112] developed an intelligent decision support system for stock trading. This study employed rough sets and GA for non-linear and complex stock data to ﬁnd the features that can be used to generate the optimal trading rules. These rules are applied to generate optimal buy or sell strategies. 6.3. Naïve Bayes (NB) NB is a classification method that classifies the data points based on the Bayesian Theorem of probability. This classification method is extremely fast, and can scale over large datasets. This classification approach has been used widely for SMP [49,69,103,105–107]. For example, the authors in [108] employed the Naïve Bayes algorithm for the sentiment analysis of textual data from multiple sources. The authors compared the effect of conventional and social media data sources on different companies and their interrelatedness. 6.2. Support Vector Machine (SVM) The SVM is a supervised machine learning technique that limits error and augments geometric margins. It is a pattern classiﬁcation and regression algorithm that was given by [24]. In terms of accuracy, the SVM is an important linear separation algorithm compared to other classiﬁers [25]. As presented in Table 4, it is the most popular method used for SMP [39–41,44,50,51,72,103,104]. The authors in [47] developed a daily and monthly SMP model using historical and sentimental data for the bank, mining, and oil sectors. The historical prices were obtained from yahoo ﬁnance, and a sentiment dataset was created by using news and tweets for one year. PCA with multiple factors was applied to the sparse dataset considered for the sentiment analysis. In this study, three algorithms—i.e., Decision-Boosted Tree, SVM, and Logistic Regression—were compared, and the accuracy was used as a performance metric. The Decision-Boosted Tree outﬂanked the Logistic Regression and SVM. The Decision- Boosted Tree achieved accuracies of 54.8%, 76%, and 76.9% for the bank, mining, and oil sectors, respectively. The Logistic regression attained accuracies of 65.4%, 61%, and 44.2%, respectively, and the SVM achieved accuracies of 51%, 59%, and 44.2% for the respective sectors. The study ﬁnally suggested the consideration of the impact of intra-day price movement for the next-day stock price to improve the accuracy. 6.1. Artiﬁcial Neural Networks (ANN) y g p Another study used Generalized Feed Forward (GFF) and MLP models for the predic- tion of the Istanbul Stock Exchange (ISE) market index [100], where the data were taken from the Central Bank of Turkey. A total of eight sets of predictions (six ANN and two Electronics 2021, 10, 2717 11 of 25 11 of 25 MAs) were performed by changing the number of hidden layers. Two sets of predictions were based on MA. The accuracy of the prediction was calculated using the coefﬁcient of determination, and the highest accuracy for both MLP and GFF was achieved using one hidden layer. MAs) were performed by changing the number of hidden layers. Two sets of predictions were based on MA. The accuracy of the prediction was calculated using the coefﬁcient of determination, and the highest accuracy for both MLP and GFF was achieved using one hidden layer. In addition, often-used ANN technique for SMP is the Radial Basis Function network (RBF). It is a layered network where hidden layers use a radial activation function [101,102]. For example, the authors in [91] used the RBF neural network to predict the Shanghai and NASDAQ index by using an extension of LPP (Locality Preserving Projection) known as two-dimensional LPP for the selection of most relevant features for the prediction. The proposed method performed well on both of the market indices. 6.5. Fuzzy Algorithms (FA) Fuzzy logic is a human reasoning-based method where all of the intermediate possi- bilities between 0 and 1 are used for decision making. It is a powerful approach in which the degree of belongingness to a certain category is considered. The adaptive neuro-fuzzy inference system (ANFIS) is the most popular fuzzy algorithm that is used for SMP. Some example studies in which ANFIS was employed for SMP include [72,113,114]. The authors Electronics 2021, 10, 2717 12 of 25 12 of 25 in [29] developed a fuzzy logic approach to analyze the sentiments on social media for SMP. Furthermore, several studies have used hybrid fuzzy approaches for SMP [115–117]. As an example of a hybrid fuzzy approach, Sedighi et al. (2019) proposed a novel model for prediction using an Artiﬁcial Bee Colony (ABC), SVM, and ANFIS. In this study, data from 50 companies were taken from the US Stock Exchange from 2008–2018. The model used 20 technical indicators as the input. The criteria for the performance measures were accuracy and quality. Furthermore, the model had a more exact forecasting accuracy than the others. 6.6. Deep Neural Networks (DNN) DNN are an improvement over conventional neural networks where more hidden layers and neurons are employed for automatic feature extraction and transformation. The increase in the number of hidden layers with non-linear processing units improves the efﬁciency of learning from raw data [118]. DNN have been used frequently for ﬁnancial predictions using textual and numeric data [74,119]. Different studies have used DNN algorithms such as Convolutional Neural Networks (CNNs) [120–122], Long-Short Term Memory (LSTM) [123,124], and Deep Belief Networks (DBNs) [106,125–127]. For example, a recent study by [128] made a comparison of four prediction models for stock market price prediction, including an Auto-Regressive Integrated Moving Average (ARIMA), Vector Auto Regression (VAR), LSTM, and Nonlinear Auto-Regressive with exogenous inputs (NARX). The model performance was evaluated using an accuracy metric. The data used for the analysis were the closing price of the NASDAQ. The results revealed that NARX made accurate predictions for the short term but failed in long-term predictions. It also concluded that models that integrate machine learning and technical indicators could predict more accurately. LSTM networks are able to learn long-term dependencies, such that they have a vigilant effect on time series prediction. Moreover, the authors in [43] compared three Recurrent Neural Network (RNN) models on Google stock price data, namely basic RNN, Gated Recurrent Unit (GRU) and the LSTM. The results revealed that LSTM outperformed other techniques and achieved an accuracy of 72% on a 5-day horizon. Furthermore, the authors in [129] applied the dynamic LSTM network to predict Nifty prices using Open, High, Low, and Close as features, and achieved a Root Mean Square Error (RMSE) of 0.00859 in terms of daily percentage changes. 6.7. Regression Algorithms (RA) Regression is a predictive approach that models the relationship between a dependent variable and independent variables [130]. Different regression approaches have been used in previous studies: simple linear regression [131–133], multiple regression [134,135], decision tree regression [17,136], logistic regression [137], support vector regression (SVR) [56,138], and ensemble regression [41,69,139]. For example, the authors in [140] developed a model that predicts the stock price of a user-speciﬁed company a few days ahead. Regression analysis and candlestick pattern detection were applied to the data, which were collected from multiple sources. The model predicted the market movement to a satisfactory level of efﬁciency. Furthermore, different machine learning algorithms were used, and an improved accuracy of 85% was achieved. 6.8. Hybrid Approaches (HA) The hybrid approach is the amalgam of various techniques used for the enhance- ment of performance in prediction models. Hybrid algorithms increase the efﬁciency of prediction models, as suggested by [141]. A few studies have used various hybrid approaches [9,59,71,72,114,142–144]. For example, the authors in [145] proposed a novel, intelligent, hybrid model for stock prediction by combining the predictions of the linear and non-linear models. The authors used an exponential smoothing model as a linear model and an autoregressive moving reference neural network as a non-linear model. The initial predictions were performed by a linear model, and the prediction errors were calculated Electronics 2021, 10, 2717 13 of 25 13 of 25 and then fed to an autoregressive moving reference neural network. This model minimized the errors due to non-linear processing. Summation and multiplication methods were used for the generation of predictions from the prediction errors. The NSE data were used for the testing of the model, and the results indicated that the model could be a promising and a new approach for the prediction of stock returns. rison of the different techniques applied in the studies. Artiﬁcial Neural Networks (ANN), Support Vector Naïve Bayes (NB), Fuzzy Logic (FL), Hybrid Algorithms (HA), Genetic Algorithms (GA), Regression , Ensemble Algorithms (EA). Table 3. Comparison of the different techniques applied in the studies. Artiﬁcial Neural Networks (ANN), Support Vector Machine (SVM, Naïve Bayes (NB), Fuzzy Logic (FL), Hybrid Algorithms (HA), Genetic Algorithms (GA), Regression Algorithms (RA), Ensemble Algorithms (EA). References ANN SVM NB DNN FL HA GA RA EA [134] [38] [44] [39] [56] [92] [40] [143] [58] [63] [103] [51] [68] [43] [102] [114] [42] [45] [69] [77] [133] [106] [71] [72] [124] [41] [74] [73] [80] In terms of price predictions, the model outperformed the RNN and achieved a lower MSE and Mean Absolute Error (MAE) compared to the constituent models. In terms of price predictions, the model outperformed the RNN and achieved a lower MSE and Mean Absolute Error (MAE) compared to the constituent models. 7. Evaluation Metrics The accuracy of almost all of the studies lies in a range of 50–90%, as presented in Table 4. The accuracy metric is handy to use and has less computation complexity than the other metrics. However, it doesn’t consider the importance of Type 1 and Type 2 errors in the case of skewed data distributions [147]. The MSE measures the mean difference between the predicted and actual output. It is an important metric in regression analysis because it measures how close the predicted value is to the actual value. An area under curve measures the degree of separability between classes. It is an important metric for classiﬁcation problems. The higher the AUC, the better the model’s predictability [148]. Precision is the ratio of correctly predicted positives to the total positives predicted [42]. Recall is the ratio of correctly predicted positives to the total number of positives [73]. The F-measure is the harmonic mean of the precision and recall, and indicates the importance of false positives and false negatives in the confusion matrix [42,49,69]. The R2, or the coefﬁcient of determination, is the measure of the closeness of the data to the predicted regression line [41,133]. The MAE measures the average difference between the predicted and actual data [133]. more flexible, hence avoiding the risk of overfitting. The majority of the reviewed studies applied regularization approaches to prevent overfitting [23,25,83]. A few recent studies applied the procedure of data augmentation to prevent overfitting [154,155]. 9. Comparative Analysis The distribution of the number of papers published in recent years is presented in Figure 5. The number of publications increased from 2009, and was at its peak in 2019, but over the previous two years, the publication number was low. The distribution of machine learning algorithms used for SMP is shown in Figure 6, where the SVM was the most popular technique used. However, the ANN and DNN have attracted the research com- munity’s attention for the last few years. Traditional neural network approaches may not make accurate SMPs as initially; the weight of the randomly selected problems may suffer Table 4. Comparison of the evaluation metrics applied to different SMP methods. 7. Evaluation Metrics Generally, two approaches are used for SMP: classiﬁcation and regression. The former approach classiﬁes the market trend into categories like Up and Down. For the latter, output is a numerical value predicting the ups and downs of the price. Figure 4 presents the taxonomy of the evaluation metrics used in the studies so far. Table 4 points out the different evaluation parameters used in the reviewed studies, as well as the time frame of the prediction. For the most part, the studies used accuracy as an evaluation metric, which is the percentage ratio of correct predictions over the total number of test instances [146]. 14 of 25 dictions d hit Electronics 2021, 10, 2717 Figure 4. Taxonomy of the performance metrics. Figure 4. Taxonomy of the performance metrics. Figure 4. Taxonomy of the performance metrics. Figure 4. Taxonomy of the performance metrics. 8. Overfitting One of the most well-known and challenging issues in machine learning models is overfitting. In this phenomenon, the model tries too hard to learn from training data. This means that the model picks up on noise or random fluctuations in the training data and learns them as ideas. These ideas don’t apply to the new data that is to be predicted, thereby resulting in poor model generalization. Because stock market data is highly sto- chastic, it is imperative to explain the methods used to resolve this issue. The most com- mon approach to mitigate the issue of overfitting is cross validation. A few studies have applied this approach, like in [38,42,150–152]. In a typical k-fold cross-validation, the data is partitioned into k subsets, or folds. The model is trained iteratively on k-1 folds, and the remaining fold—also known as the hold-out fold—is treated as a test set. Numerous stud- ies have used the early stopping method to overcome overfitting [153]. Another method is to remove irrelevant features and noise from the data, which greatly increases the model’s generalizability. A few studies have implemented these procedures to avoid over- fitting, such as [42,44,68,121]. The most important preventive measure against overfitting is regularization. This technique removes the extra weights from the selected features and redistributes them uniformly It discourages the learning of models that are complex or Moreover, other metrics like MSE, the Area Under Curve (AUC), Akaike Information Criterion (AIC), R-Squared (R2), Precision, Recall, F-measure, MAE and Mean Absolute Percentage Error (MAPE) are used as well. 8. Overﬁtting One of the most well-known and challenging issues in machine learning models is overﬁtting. In this phenomenon, the model tries too hard to learn from training data. This means that the model picks up on noise or random ﬂuctuations in the training data and learns them as ideas. These ideas don’t apply to the new data that is to be predicted, thereby resulting in poor model generalization. Because stock market data is highly stochastic, it is imperative to explain the methods used to resolve this issue. The most common approach to mitigate the issue of overﬁtting is cross validation. A few studies have applied this approach, like in [38,42,150–152]. In a typical k-fold cross-validation, the data is partitioned into k subsets, or folds. The model is trained iteratively on k-1 folds, and the remaining fold—also known as the hold-out fold—is treated as a test set. Numerous studies have used the early stopping method to overcome overﬁtting [153]. Another method is to remove irrelevant features and noise from the data, which greatly increases the model’s generalizability. A few studies have implemented these procedures to avoid overﬁtting, such as [42,44,68,121]. The most important preventive measure against overﬁtting is regularization. This technique removes the extra weights from the selected features and redistributes them uniformly. It discourages the learning of models that are complex or more ﬂexible, hence avoiding the risk of overﬁtting. The majority of the reviewed studies applied regularization approaches to prevent overﬁtting [23,25,83]. A few recent studies applied the procedure of data augmentation to prevent overﬁtting [154,155]. 7. Evaluation Metrics Reference Performance Measure Prediction Type Output [38] MSE, MAD% Few days ahead MAD% (2.32) MLP [56] Accuracy, trading return Intraday 59.0%, 3.30% [44] Accuracy Daily 65.1% Kalyanaraman, V., 2014 Accuracy Daily 81.82% [58] Accuracy Daily Average accuracy of 54.4% [40] Accuracy, RMSE Daily 59.6% [47] Accuracy Daily and monthly DBT achieved better accuracy (76.9%) than SVM and LR [63] Accuracy and correlation Daily Accuracy of around 70% [51] Accuracy, RMSE Long-term 99% accuracy for yahoo data (XGBoost) [49] Error Rate, F-measure Next Month, Next Week 0.85 [42] Accuracy, f-measure, precision, AUC One day ahead 85% [43] Log loss and accuracy Daily, weekly 72% accuracy (LSTM) [68] Accuracy, Return Daily 58.1% more flexible, hence avoiding the risk of overfitting. The majority of applied regularization approaches to prevent overfitting [23 25 83] Table 4. Comparison of the evaluation metrics applied to different SMP methods. n recent years is presented in nd was at its peak in 2019, but w. The distribution of machine where the SVM was the most e attracted the research com- network approaches may not selected problems may suffer MAD% (2.32) MLP 59.0%, 3.30% 65.1% 81.82% Average accuracy of 54.4% 59.6% DBT achieved better accuracy (76.9%) than SVM and LR Accuracy of around 70% 99% accuracy for yahoo data (XGBoost) 0.85 85% 72% accuracy (LSTM) 58.1% 15 of 25 Electronics 2021, 10, 2717 Table 4. Cont. Table 4. Cont. Reference Performance Measure Prediction Type Output [123] Accuracy, MSE Long short-term 56.7% (LSTM),57.2% (ELSTM) [70] Accuracy Daily, weekly 80% [69] Accuracy, f-measure 0.84 [50] Accuracy Daily 72% [133] MSE, MAE, MAPE and R2 Daily LR 0.73SVM 0.93 [106] MAPE Daily 2.03–2.17 [71] training error, testing error Daily 0.03, 0.072 [41] RMSE, Accuracy, AUC, R2, MAE Monthly >90%(Ensemble) [74] Accuracy Monthly 87.32 Seethalakshmi, R., 2020 R2, AIC . . . . . . . . . 0.992 (R2) [140] Accuracy Next few days 90–96% (KNN Regression) [73] Precision, recall, f-measure, accuracy Weekly 76.5% [80] MSE Daily 0.0039(GA-LSTM) Moreover, the MAPE is used as a performance indicator in a few studies that measure the mean of absolute error percentages in predictions [38,106]. Furthermore, a few of the reviewed studies used trading return or return on investment (ROI) as an evaluation metric, where the trading technique was tested to measure the profitability of predictions [56,68]. Other studies have used Prediction of Change in Direction (POCID) [149] and hit ratios [144]. 9. Comparative Analysis The distribution of the number of papers published in recent years is presented in Figure 5. The number of publications increased from 2009, and was at its peak in 2019, but over the previous two years, the publication number was low. The distribution of machine learning algorithms used for SMP is shown in Figure 6, where the SVM was the most popular technique used. However, the ANN and DNN have attracted the research community’s attention for the last few years. Traditional neural network approaches may not make accurate SMPs as initially; the weight of the randomly selected problems may suffer from the local optimal, and results in incorrect predictions [123]. The deep learning approaches are used to analyze complicated patterns in the stock data, and provide much Electronics 2021, 10, 2717 16 of 25 l faster results. Furthermore, there is no such single technique that can promise to give the optimum results. The comparative analysis between the type of data used and the performance of the models is represented in Figure 7. Data alone from social media do not perform better than using market data and technical indicators. However, if data from textual sources is combined with them, then the model performance increases. faster results. Furthermore, there is no such single technique that can promise to g optimum results. The comparative analysis between the type of data used and the mance of the models is represented in Figure 7. Data alone from social media do n form better than using market data and technical indicators. However, if data from sources is combined with them, then the model performance increases. p y p p p faster results. Furthermore, there is no such single technique that can promise to g optimum results. The comparative analysis between the type of data used and the mance of the models is represented in Figure 7. Data alone from social media do n form better than using market data and technical indicators. However, if data from sources is combined with them, then the model performance increases. p y p p , p faster results. Furthermore, there is no such single technique that can promise to g optimum results. The comparative analysis between the type of data used and the mance of the models is represented in Figure 7. Data alone from social media do n form better than using market data and technical indicators. 9. Comparative Analysis Number of publications per ye Figure 5. Number of publications per ye Figure 5. Number of publications per ye Figure 5. Number of publications per year. Figure 5. Number of publications per ye Figure 5. Number of publications per ye Figure 6 Distribution of the SMP techniques 14% 16% 8% 9% 9% 11% 9% 10% ANN SVM NB GA FL DNN RA HA Fi 6 Di t ib ti f th SMP t h i 14% 16% 8% 9% 9% 11% 9% 10% ANN SVM NB GA FL DNN RA HA Fi Di ib i f h SMP h i Fi D b f h SMP h 14% 16% 8% 9% 9% 11% 9% 10% ANN SVM NB GA FL DNN RA HA Figure 6. Distribution of the SMP techniqu Figure 6. Distribution of the SMP techniqu Figure 6. Distribution of the SMP techniques. Figure 6. Distribution of the SMP techniqu Figure 7. Comparison of the accuracies with different types of dat 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 Market Data Market Data+ Market Indicators Textual Data Market Data + Textual Data +Market Indicators Accuracy Figure 7 Comparison of the accuracies with different types of dat 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 Market Data Market Data+ Market Indicators Textual Data Market Data + Textual Data +Market Indicators Accuracy Fi 7 C i f th i ith diff t t f d t 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 Market Data Market Data+ Market Indicators Textual Data Market Data + Textual Data +Market Indicators Accuracy Fi 7 C i f th i ith diff t t f d t Figure 7. Comparison of the accuracies with different types of Figure 7. Comparison of the accuracies with different types of Figure 7. Comparison of the accuracies with different types of Figure 7. Comparison of the accuracies with different types of data. 9. Comparative Analysis However, if data from sources is combined with them, then the model performance increases. textual sources is combined with them, then the model performanc sources is combined with them, then the model performance Figure 5. Number of publications per year. Figure 6. Distribution of the SMP techniques. Figure 7. Comparison of the accuracies with different types of data 0 5 10 15 20 Number of publications Year 14% 16% 8% 9% 9% 11% 9% 10% ANN SVM NB GA FL DNN RA HA 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 Market Data Market Data+ Market Indicators Textual Data Market Data + Textual Data +Market Indicators Accuracy Figure 5. Number of publications per year. g sources is combined with them, then the model performance Figure 5. Number of publications per year. Figure 6. Distribution of the SMP techniques. Figure 7. Comparison of the accuracies with different types of data 0 5 10 15 20 Number of publications Year 14% 16% 8% 9% 9% 11% 9% 10% ANN SVM NB GA FL DNN RA HA 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 Market Data Market Data+ Market Indicators Textual Data Market Data + Textual Data +Market Indicators Accuracy Figure 6. Distribution of the SMP techniques. o e e a u i g a e a a a e i a i i a o sources is combined with them, then the model performance Figure 5. Number of publications per year. Figure 6. Distribution of the SMP techniques. Figure 7. Comparison of the accuracies with different types of data 0 5 10 15 20 Number of publications Year 14% 16% 8% 9% 9% 11% 9% 10% ANN SVM NB GA FL DNN RA HA 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 Market Data Market Data+ Market Indicators Textual Data Market Data + Textual Data +Market Indicators Accuracy Figure 7. Comparison of the accuracies with different types of data. Figure 5. Number of publications per year. 0 5 10 15 20 Number of publications Year Figure 5. Number of publications per year. Figure 5. Number of publications per year. 0 5 10 15 20 Number of publications Year Figure 5. Number of publications per year. 0 5 10 15 20 Number of publications Year Figure 5. Number of publications per ye Figure 5. Number of publications per year. Figure 5. 10. Challenges and Open Issu 10 Challenges and Open Issu 10 Challenges and Open Issu 10. Challenges and Open Issues International politics also plays a dramatic role in stock market volatility [159]. Events in which panic selling is triggered are nowadays becoming more common, and they result in market overreaction. Panic selling is the reaction to fear and loss, which leads to the wide-scale selling of investments. The leading causes which result in panic selling are high speculation in the market, political issues, and economic instability [46,160]. It becomes more difﬁcult for a researcher to evaluate market behavior in such situations. New algorithms are proceeding to ﬂood the markets consistently at a pace, and it is challenging to compare the adequacy and exactness of these algorithms. A fascinating part of this research area is its self-defeating nature. In simple terms, sharing the methodologies that generate high proﬁts with market competitors will render the methodologies useless. In this way, best-class algorithm exchanging in the markets is restricted, and is private. The procedure or strategy behind such algorithms is never published. The data on social media platforms can either be generated by humans or bots. The sentiments of bots can sometimes result in inaccurate predictions. As such, there arises a need for social bot detection to obtain better predictions [161]. Investigators, analysts, and researchers are continuously reporting the potential dangers brought about by social bots. Market investors actively participate in and react to social media sentiments. As such, it can be said that the data from social platforms play a signiﬁcant role in stock prediction. One example is of 23 April 2013, when the Syrian Electronic Army hacked the Twitter account of the Associated Press, and they posted fake news of a terror attack on the White House in which President Obama was allegedly injured. This provoked an immediate crash in the stock markets [162–164]. Due to the rising impact of online networking on numerous aspects of our lives, more attention is paid to sentiment analysis based on data generated from social media. This data can be temperamental and hard to process due to various factors, such as fake news and the bot data published on the web by numerous sources. It is challenging to identify the quality data and draw valuable insights from it. A decent option or an extra asset that can be used is quarterly or yearly reports documented by the organizations for the prediction of stocks. 10. Challenges and Open Issu 10 Challenges and Open Issu 10 Challenges and Open Issu 10. Challenges and Open Issues g p Financial market analysis and prediction continues to be a fascinating and ch ing problem. Nowadays, data access is becoming easier, but difficulties are increa the acquisition and processing of data to extract valuable insights and analyze their 10. Challenges and Open Issues Financial market analysis and prediction continues to be a fascinating and ch ing problem. Nowadays, data access is becoming easier, but difficulties are increa the acquisition and processing of data to extract valuable insights and analyze their 10. Challenges and Open Issues Financial market analysis and prediction continues to be a fascinating and ch ing problem. Nowadays, data access is becoming easier, but difficulties are increa the a ui itio a d o e i of data to e t a t aluable i i ht a d a aly e thei Financial market analysis and prediction continues to be a fascinating and challenging problem. Nowadays, data access is becoming easier, but difﬁculties are increasing in the acquisition and processing of data to extract valuable insights and analyze their impact on Electronics 2021, 10, 2717 17 of 25 17 of 25 stock prices. Feature extraction from the ﬁnancial data is a challenging task, as it is essential to observe the diversity of the variables that are used for the prediction. The Financial datasets are usually noisy [156]. The quality of the data signiﬁcantly affects SMPs. Most literature on stock prediction regarding live testing afﬁrms that the previously proposed methodologies can be utilized in real time. However, these methods may work in controlled circumstances. Still, a big challenge will be the live testing for the prediction. The live testing comes up with challenging factors, such as variations in prices, noise, and unpredicted events. One such example is the Knight Capital Tragedy, in which the loss of 440 million dollars was endured by the company [157]. y p y Market volatility is the severity with which the market price of an investment ﬂuctu- ates. The main reasons for the volatility are uncertainty and inﬂation, and the risk increases when the market is volatile. The inﬂuence of volatility on our emotions is ceaseless. The prediction of stock prices is challenging when the market is volatile. One of the reasons for market volatility is algorithmic trading. One such example is the ﬂash crash, which expunged $860 billion within 30 min from US stock markets [158]. 10. Challenges and Open Issu 10 Challenges and Open Issu 10 Challenges and Open Issu 10. Challenges and Open Issues These records, when decoded accurately, give a signiﬁcant knowledge of an organization’s status, which can help with the understanding of the future stock trend. 11. Conclusions Financial markets provide an excellent platform for investors and traders, who can trade from any gadget that connects to the internet. Over the last few years, people have become more attracted to stock trading. Like any other walk of life, the stock market has also changed due to the advent of technology. Now, people can make their investments grow. Online trading has only changed the way individuals purchase and sell stocks. The budgetary markets have advanced rapidly, and have formed an interconnected global marketplace. These advancements pave the way to new opportunities. Electronics 2021, 10, 2717 18 of 25 18 of 25 In contrast to conventional frameworks, SMP is currently performed using machine learning, big data analytics, and deep learning, which provide more optimal decision making. Stock markets, nowadays, are vulnerable to social media sentiments and cyber- attacks. Researchers can play a signiﬁcant role and ﬂourish in these areas by developing the frameworks for better and more secure trading. This article reviewed studies based on a generic framework of SMP, as presented in Figure 2. It mainly focused on the studies from last decade (2011–2021). The studies were analyzed and compared based on the type of data used as the input, the data pre-processing approaches, and the machine learning techniques used for the predictions. Furthermore, it reviewed the different evaluation metrics used for performance measurement by different studies, as presented in Section 7. Moreover, an extensive comparative analysis was performed, and it was concluded that SVM is the most popular technique used for SMP. However, techniques like ANN and DNN are mostly used, as they provide more accurate and faster predictions. Furthermore, the inclusion of both market data and textual data from online sources improve the prediction accuracies. Section 9 discussed the generic challenges and open issues in SMP systems. Author Contributions: Conceptualization, N.R., M.B.M., T.A., S.S. (Sparsh Sharma) and S.S. (Saurabh Singh); methodology, N.R., M.B.M. and T.A.; validation, S.A., S.S. (Sparsh Sharma) and H.-C.K.; formal analysis, S.A. and S.S. (Saurabh Singh); data curation, N.R., M.B.M. and T.A.; writing—original draft preparation, N.R., M.B.M. and T.A.; writing—review and editing, N.R., M.B.M., T.A. and S.S. (Sparsh Sharma); supervision, S.A. and H.-C.K.; project administration, S.A. and H.-C.K.; funding acquisition, H.-C.K. All authors have read and agreed to the published version of the manuscript. 11. Conclusions Funding: This work was supported by the Commercializations Promotion Agency for R&D Out- comes (COMPA) grant funded by the Korean Government (Ministry of Science and ICT) (R&Dproject No.1711139492). Data Availability Statement: No new data were created or analyzed in this study. Data sharing is not applicable to this article. Conﬂicts of Interest: The authors declare no conﬂict of interest. Abbreviations SMP Stock Market Prediction SVM Support Vector Machine ANN Artiﬁcial Neural Network DNN Deep Neural Network RA Regression Analysis FA Fuzzy Algorithm NB Naïve Bayes GA Genetic Algorithm HA Hybrid Approach kNN k- Nearest Neighbors LDA Latent Dirichlet Allocation PCA Principle Component Analysis XGB eXtreme Gradient Boost FO Fireﬂy Optimization TF-IDF Term Frequency- Inverse Document Frequency GARCH Generalized Auto-regressive Conditional Heteroskedasticity DAN Deep Attention Neural Network MLP Multi-linear Perceptron GFF Generalized Feed Forward NARX Non-linear Auto-regressive Network with exogenous inputs RBF Radial Basis MA Moving Average LPP Locality Preserving Projection 19 of 25 Electronics 2021, 10, 2717 FRPCA Fast Robust Principle Component Analysis KPCA Kernel Principle Component Analysis GRU Gated Recurrent Unit LSTM Long Short Term Memory ANFIS Adaptive Neuro-Fuzzy Inference System ABC Ant Bee Colony RNN Recurrent Neural Network RMSE Root Mean Square Error SVR Support Vector Regression CNN Convolution Neural Network DBN Deep Belief Network ARIMA Auto Regressive Integrated Moving Average VAR Vector Auto-regression AUC Area Under Curve MSE Mean Square Error MAE Mean Absolute Error R2 R-Squared MAPE Mean Absolute Percentage Error POCID Prediction of Change in Direction DJIA Dow Jones Industrial Average S&P Standard and Poor’s GDP Gross Domestic Product NASDAQ National Association of Securities Dealers Automated Quotation DAX Deutscher Aktien Index KSE Karachi Stock Exchange LSE London Stock Exchange NYSE New York Stock Exchange BSE Bombay Stock Exchange AIC Akaike Information Criterion References 1. 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https://openalex.org/W4385298033	https://www.nature.com/articles/s41420-023-01572-1.pdf	English	null	PME-1 sensitizes glioblastoma cells to oxidative stress-induced cell death by attenuating PP2A-B55α-mediated inactivation of MAPKAPK2-RIPK1 signaling	Cell death discovery	2,023	cc-by	10,552	www.nature.com/cddiscovery 1Laboratory of Protein Phosphorylation & Proteomics, Dept. Cellular & Molecular Medicine, KU Leuven, B-3000 Leuven, Belgium. 2KU Leuven Cancer Institute (LKI), B-3000 Leuven, Belgium. 3SyBioMa, KU Leuven, B-3000 Leuven, Belgium. ✉email: veerle.janssens@kuleuven.be ARTICLE OPEN PME-1 sensitizes glioblastoma cells to oxidative stress-induced cell death by attenuating PP2A-B55α-mediated inactivation of MAPKAPK2-RIPK1 signaling Liesbeth Guffens 1,2, Rita Derua1,3 and Veerle Janssens 1,2✉ © The Author(s) 2023 Glioblastoma (GBM) is the most common primary brain tumor in adults. Current standard therapy is surgery followed by radiotherapy, with concurrent and adjuvant temozolomide chemotherapy. GBM is characterized by almost uniformly fatal outcomes, highlighting the unmet clinical need for more efﬁcient, biomarker-guided treatments. Protein phosphatase methylesterase-1 (PME-1), a regulator of the tumor suppressive phosphatase PP2A, promotes PP2A demethylation and inactivation, and is overexpressed in 44% of GBM, associated with increased tumor grade and cellular proliferation. Here, we aimed to investigate how reactive oxygen species (ROS), a frequent by-product of radiotherapy and temozolomide chemotherapy, regulate PP2A function via its methylesterase PME-1, and how PME-1 overexpression impacts the response of GBM cells to oxidative stress. We found that in two glioblastoma cell lines, U87MG and U251MG, expression of PME-1 is positively correlated with the sensitivity of the cells to H2O2 or t-BHP-induced oxidative stress. Experiments using the irreversible pharmacologic PME-1 inhibitor, AMZ30, and different PME-1 mutants, revealed that the methylesterase function, the PP2A binding capacity, and the nuclear localization of PME-1 are all important for the sensitizing effect of PME-1 expression. Furthermore, we identiﬁed increased nuclear localization of the PP2A-B55α subunit, increased binding of PP2A-B55α to PME-1, and increased B55α-bound PP2A-C demethylation upon oxidative stress. Lastly, we uncovered increased stress-induced phosphorylation and activity of MAPKAPK2 and RIPK1 in PME-1 overexpressing U87MG cells, which caused the observed sensitization to t-BHP treatment. Our data reveal a novel role for PME-1 in oxidative stress-induced GBM cell death, regulating nuclear PP2A-B55α activity and MAPKAPK2-RIPK1 signaling. Patients with GBM tumors overexpressing PME-1, although having a worse prognosis due to increased cellular proliferation of the tumor, could actually be more responsive to oxidative stress-inducing therapies. Cell Death Discovery (2023) 9:265 ; https://doi.org/10.1038/s41420-023-01572-1 Cell Death Discovery (2023) 9:265 ; https://doi.org/10.1038/s41420-023-01572-1 Received: 22 June 2023 Revised: 13 July 2023 Accepted: 20 July 2023 1Laboratory of Protein Phosphorylation & Proteomics, Dept. Cellular & Molecular Medicine, KU Leuven, B-3000 Leuven, Belgium. 2KU Le Belgium. 3SyBioMa, KU Leuven, B-3000 Leuven, Belgium. ✉email: veerle.janssens@kuleuven.be INTRODUCTION R ibl i In GBM stem cells, PME-1 knockdown inhibited hypoxia-induced cell death [35], and glucose deprivation-mediated cell death in U251MG cells was shown to be dependent on PME-1 and on stress-induced PP2A demethylation and inactivation [36]. Thus, a picture emerges, in which PP2A and its cellular modulator PME-1 are not only in control of GBM growth, but also of the speciﬁc response of GBM cells to a large variety of cytotoxic stress factors. However, the molecular mechanisms mediating this PME-1-mediated regulation of PP2A in stressed cells, and regulation of PME-1 itself, under these circumstances, have mostly remained unclear. Methylesterase activity, PP2A-C binding capacity, and nuclear localization of PME-1 all contribute to the sensitizing effect of PME-1 overexpression to oxidative stress As explained, PME-1 plays a dual role in PP2A regulation (Fig. 2A): as the enzyme responsible for PP2A-C demethylation, and as a PP2A-C stabilizer/inhibitor during PP2A holoenzyme biogenesis or following holoenzyme disassembly [20]. Another characteristic of Methylesterase activity, PP2A-C binding capacity, and nuclear localization of PME-1 all contribute to the sensitizing effect of PME-1 overexpression to oxidative stress p As explained, PME-1 plays a dual role in PP2A regulation (Fig. 2A): as the enzyme responsible for PP2A-C demethylation, and as a PP2A-C stabilizer/inhibitor during PP2A holoenzyme biogenesis or following holoenzyme disassembly [20]. Another characteristic of y Here, we investigated the role of differential PME-1 expression on the sensitivity of GBM cells to oxidative stress, a stress that is often associated with radiotherapy and TMZ chemotherapy, the current standard treatments for GBM. Fig. 1 PME-1 expression sensitizes GBM cells to oxidative stress. A Endogenous PME-1 levels in U87MG and U251MG cells were determined and quantiﬁed using Western blotting (N = 3, two-tailed t-test); a representative blot is shown. B Sensitivity of U87MG and U251MG cells to 16 h t-BHP treatment (different concentrations) was determined using MTT (N = 3, two-way ANOVA). C Validation of stable PME-1-GFP overexpression in U87MG cells using Western blotting (left). Quantiﬁcation below the blots refers to ‘total’ PME-1 levels ( = endogenous PME- 1 + PME-1-GFP). The sensitivity of U87MG PME-1-GFP cells to 16 h treatment with indicated concentrations of t-BHP was determined using MTT (N = 4, two-way ANOVA) (right). D Validation of stable PME-1 knockdown in U251MG cells using Western blotting (left). The sensitivity of U251MG shPME-1 cells to 16 h treatment with indicated concentrations of t-BHP was determined using MTT (N = 5, two-way ANOVA) (right). INTRODUCTION R ibl i However, another, non-exclusive view inferred a role for PME-1 in stabilizing already inactive PP2A complexes that arise during PP2A holoenzyme biogenesis [3, 29] or immediately following its de novo biosynthesis. This function is independent on its esterase activity, but dependent on its high binding afﬁnity for inactive PP2A-C [22, 30]. One report also suggested the importance of PME-1 methylesterase activity to protect PP2A-C from ubiquitin/proteasome degradation [31]. The stabilizing role of PME-1 may be important, as, during times of stress, the PP2A holoenzyme can disassemble [32], increasing the risk of uncon- trolled activity of free PP2A-C. This infers the existence of potential PME-1 regulation mechanisms under cellular stress conditions. PME-1 expression sensitizes GBM cells to oxidative stress It is currently not known how PME-1 expression affects the response of GBM cells to oxidative stress. Therefore, we ﬁrst assessed endogenous PME-1 expression in two glioblastoma cell lines, U87MG and U251MG, and checked their sensitivity to oxidative stress (Fig. 1A, B). We induced oxidative stress by treating cells with H2O2, or for phenotypic experiments, with the more stable H2O2 homolog t-BHP. After 16 h of treatment with t- BHP, U251MG cells, which express more PME-1 than U87MG cells (Fig. 1A), appear to be signiﬁcantly more sensitive to t-BHP- induced cell death, as measured in MTT assay (Fig. 1B). To further elucidate the importance of PME-1 in the oxidative stress response in an isogenic cell context, we chose to increase PME-1 expression in the PME-1 low-expressing cell line and to reduce PME-1 expression in the PME-1 high-expressing cell line. We generated a stable PME-1-GFP overexpressing U87MG cell line and a PME-1 depleted U251MG cell line using a PME-1 3’UTR- targeting shRNA. The resulting PME-1 expression levels were veriﬁed by immunoblotting (Fig. 1C, D). Again, we observed increased sensitivity of the U87MG PME-1-GFP overexpressing cells compared to the GFP overexpressing control (Fig. 1C), and conversely, found increased resistance of U251MG shPME-1 cells compared to the shGFP control (Fig. 1D). These data underscore the sensitizing role of PME-1 in the oxidative stress response of two independent GBM cell lines. g Speciﬁcally, in GBM, PME-1 expression or inhibition of PP2A can indeed sensitize cells to different stresses. For instance, pharma- cologic inhibition of PP2A with LB100 enhanced the effects of DNA-damaging agents TMZ and doxorubicin [33] and increased the radiosensitivity of GBM cells [34]. INTRODUCTION R ibl i survival (<10% 5-year survival) and near uniformly fatal outcome [17]. The current standard treatment is maximal surgical resection, followed by irradiation, with concurrent and adjuvant chemotherapy using temozolomide (TMZ) [18]. Due to the ineffective treatment, there is a major unmet clinical need for more efﬁcient GBM (targeted) therapies and associated biomarkers. Reversible protein phosphorylation, catalyzed by antagonistic kinases and phosphatases, is of major importance for the regulation of cellular signaling. One of the main Ser/Thr phosphatases in the cell is protein phosphatase 2A (PP2A) [1]. PP2A represents a family of holoenzymes consisting of a core complex, composed of a catalytic subunit (PP2A-C) and a structural subunit (PP2A-A), that can be associated with one of many regulatory B subunits responsible for substrate speciﬁcity of the phosphatase [2, 3]. PP2A is involved in multiple cellular processes, including cell division and cell death [4, 5]. Recognized as a tumor suppressor [1, 6], PP2A is dysfunctional in various cancer types [7, 8]. In most cases, PP2A inactivation in cancer is achieved by non-genomic mechanisms involving overexpression of endogen- ous PP2A inhibitory proteins, such as cancerous inhibitor of PP2A (CIP2A) and PP2A methylesterase-1 (PME-1) [9–12]. In particular, PME-1 is frequently overexpressed in endometrial, gastric and lung cancer, hepatocellular carcinoma, and glioblastoma [13–16]. In GBM, PME-1 overexpression is found in 44% of cases (n = 222), positively correlating with malignancy (stage), increased Ki-67 proliferation index, and increased ERK and MEK phosphor- ylation [16], while PME-1 inhibition restricts GBM growth [16, 19]. Moreover, PME-1 expression causes widespread kinase inhibitor resistance in GBM [19]. PME-1 has a dual role in PP2A regulation [20]. First, it mediates the reversible carboxymethylation of PP2A-C. This modiﬁcation is catalyzed by S-adenosylmethionine-dependent leucine carboxyl methyltransferase 1 (LCMT1) [21] and reversed by PME-1 [22]. It differentially regulates PP2A-C assembly with the plethora of regulatory subunits that determine PP2A speciﬁcity and activity [23–25]. Enzymatic PP2A-C demethylation is dependent on a g Glioblastoma (GBM) is the most common primary brain tumor in adults. It is a very aggressive cancer, associated with short Ofﬁcial journal of CDDpress L. Guffens et al. 2 catalytic Ser residue in PME-1 (Ser156), while stable PME-1 binding to PP2A-C is not required [26, 27]. Second, PME-1 has been described to inactivate PP2A through metal eviction from the active site [28]. INTRODUCTION R ibl i Cell Death Discovery (2023)9:265 Fig. 1 PME-1 expression sensitizes GBM cells to oxidative stress. A Endogenous PME-1 levels in U87MG and U251MG cells were determined and quantiﬁed using Western blotting (N = 3, two-tailed t-test); a representative blot is shown. B Sensitivity of U87MG and U251MG cells to 16 h t-BHP treatment (different concentrations) was determined using MTT (N = 3, two-way ANOVA). C Validation of stable PME-1-GFP overexpression in U87MG cells using Western blotting (left). Quantiﬁcation below the blots refers to ‘total’ PME-1 levels ( = endogenous PME- 1 + PME-1-GFP). The sensitivity of U87MG PME-1-GFP cells to 16 h treatment with indicated concentrations of t-BHP was determined using MTT (N = 4, two-way ANOVA) (right). D Validation of stable PME-1 knockdown in U251MG cells using Western blotting (left). The sensitivity of U251MG shPME-1 cells to 16 h treatment with indicated concentrations of t-BHP was determined using MTT (N = 5, two-way ANOVA) (right). Cell Death Discovery (2023) 9:265 Fig. 2 Methylesterase activity, PP2A-C binding capacity, and nuclear localization of PME-1 all contribute to the PME-1-dependent decrease in cell viability upon oxidative stress. A Schematic representation of both possibly interconnected functions of PME-1 as PP2A methylesterase and PP2A inhibitor (created with BioRender.com). B Sensitivity of U87MG and U251MG cells to 16 h t-BHP treatment (different concentrations) with or without 1 h pretreatment with 20 μM AMZ30 was determined using MTT (N = 4, two-way ANOVA). C Demethylated PP2A-C and total PP2A-C levels in U87MG and U251MG cells were determined after 1 h treatment with 1 mM H2O2 using Western blotting (representative blots). D Quantiﬁcation of demethylated PP2A-C shown in (C) (normalized to total PP2A-C) (N = 5 (U87MG), N = 4 (U251MG), two-tailed t-test). E Sensitivity of U87MG PME-1-GFP and U251MG shPME-1 cells to 16 h t-BHP treatment (different concentrations) with or without pretreatment with 20 μM AMZ30 was determined using MTT (N = 4 (U87MG), N = 6 (U251MG), statistical signiﬁcance is visualized in Fig. S1A). F Sensitivity of U87MG cells expressing PME-1-GFP (WT), PME-1 R271A-GFP (NLS mutant), or PME-1 R369D-GFP (PP2A-C binding mutant) to 16 h treatment with different concentrations of t-BHP was determined using MTT (N = 3, two-way ANOVA). L. Guffens et al. L. Guffens et al. 3 3 3 Fig. 2 Methylesterase activity, PP2A-C binding capacity, and nuclear localization of PME-1 all contribute to the PME-1-dependent decrease in cell viability upon oxidative stress. A Schematic representation of both possibly interconnected functions of PME-1 as PP2A methylesterase and PP2A inhibitor (created with BioRender.com). B Sensitivity of U87MG and U251MG cells to 16 h t-BHP treatment (different concentrations) with or without 1 h pretreatment with 20 μM AMZ30 was determined using MTT (N = 4, two-way ANOVA). C Demethylated PP2A-C and total PP2A-C levels in U87MG and U251MG cells were determined after 1 h treatment with 1 mM H2O2 using Western blotting (representative blots). D Quantiﬁcation of demethylated PP2A-C shown in (C) (normalized to total PP2A-C) (N = 5 (U87MG), N = 4 (U251MG), two-tailed t-test). E Sensitivity of U87MG PME-1-GFP and U251MG shPME-1 cells to 16 h t-BHP treatment (different concentrations) with or without pretreatment with 20 μM AMZ30 was determined using MTT (N = 4 (U87MG), N = 6 (U251MG), statistical signiﬁcance is visualized in Fig. S1A). Cell Death Discovery (2023) 9:265 F Sensitivity of U87MG cells expressing PME-1-GFP (WT), PME-1 R271A-GFP (NLS mutant), or PME-1 R369D-GFP (PP2A-C binding mutant) to 16 h treatment with different concentrations of t-BHP was determined using MTT (N = 3, two-way ANOVA). PME-1 is its predominant nuclear localization due to the presence of a functional nuclear localization signal (NLS) [26]. PME-1 is its predominant nuclear localization due to the presence of a functional nuclear localization signal (NLS) [26]. important role in the oxidative stress response and may contribute to the sensitizing effect of PME-1 overexpression. To further strengthen this hypothesis, we pretreated U87MG PME-1-GFP and U251MG shPME-1 cells with AMZ30 and measured their sensitivity to t-BHP treatment (Fig. 2E and Fig. S1A (statistics)). We found that the sensitizing effect of PME-1 overexpression in the U87MG cells is abolished by pretreatment with AMZ30. Similar results were seen in U251MG cells, in which the increased sensitivity of the shGFP cells disappeared after pretreatment with AMZ30 (Fig. 2E). Thus, the demethylating activity of PME-1 is crucial for the sensitizing effect of PME-1 overexpression in response to oxidative stress. To assess whether the methylesterase activity of PME-1 contributed to the sensitizing effect of PME-1 to oxidative stress, we used the catalytic PME-1 inhibitor AMZ30, which irreversibly inhibits PME-1 demethylating activity [37]. Interestingly, in the presence of AMZ30, the resistance of both U87MG and U251MG cells to t-BHP increased (Fig. 2B). Furthermore, in the absence of AMZ30, demethylated PP2A-C levels increased in response to H2O2 treatment in both cell lines (Fig. 2C, D), suggesting increased methylesterase activity of PME-1 under these conditions. These results indicate that the demethylating activity of PME-1 plays an Cell Death Discovery (2023) 9:265 L. Guffens et al. 4 PME-1 overexpression induces increased MAPKAPK2 phosphorylation/activity during oxidative stress, which subsequently causes the observed cell death sensitization To assess the importance of the PP2A binding capacity and the nuclear localization of PME-1, we generated two PME-1 mutants devoid of either of these characteristics and (over)expressed them in the PME-1-low U87MG cell line, in order to see if they could sensitize the cells to the same extent as wild type PME-1 to t-BHP- induced oxidative stress. Neither the PP2A-C non-binding mutant (PME-1 R369D) [28, 38], nor the PME-1 NLS mutant (PME-1 R271A) [26] could mimic the sensitizing effect of PME-1 WT over- expression (Fig. 2F). Oxidative stress promotes B55α nuclear localization, PP2A-B55α binding to PME-1 and B55α-bound PP2A-C demethylation To assess whether increased phosphorylation, and thus kinase activity, of MAPKAPK2 is causally related to decreased viability upon oxidative stress in our GBM cell models, we next pretreated U87MG and U251MG cells with 10 μM PF-364402 (MAPKAPK2 inhibitor, MK2i) before exposing them to t-BHP (Fig. 4E). In both cell lines, we found that inhibition of MAPKAPK2 activity with MK2i indeed resulted in increased viability upon exposure to t-BHP. More importantly, we repeated this experiment in U87MG PME-1- GFP and U251MG shPME-1 cells (Fig. 4F and Fig. S1B (statistics)), and found that the increased sensitivity of the U87MG PME-1-GFP cells to oxidative stress was entirely abolished by pretreatment with MK2i, while the increased sensitivity of the U251MG shGFP cells compared to the shPME-1 cells likewise disappeared in the presence of MK2i. We conclude that the increased phosphoryla- tion/activity of MAPKAPK2 observed in PME-1-high-expressing GBM cells is, therefore, causally linked to the increased sensitivity of these cells to oxidative stress. As Tang et al. recently described a redistribution of PME-1 to the cytoplasm in H2O2-treated human primary dermal ﬁbroblasts [39], we prepared nuclear and cytoplasmic extracts of stressed (1 mM H2O2) and unstressed U87MG cells to examine potential changes in the subcellular localization of PME-1 upon oxidative stress. Interestingly, we observed no redistribution of PME-1 to the cytoplasm, but found an increase in the level of the PP2A regulatory B55α subunit in the nucleus during oxidative stress treatment (Fig. 3A, B). PP2A-C and -A subunits did not show an obvious translocation (Fig. 3A, B). g Furthermore, when we assessed potential, oxidative stress- induced changes in the PME-1 interactome by an afﬁnity puriﬁcation (AP)-LC-MS/MS-based approach in PME-1-GFP over- expressing cells, we found an approximately ﬁvefold and fourfold increased binding of B55 relative to PME-1-GFP in stressed versus untreated U87MG and U251MG cells, respectively (Fig. 3C). Two different B55 peptides were identiﬁed: one unique for the B55α isoform and one common to B55α and B55γ. The B55 family of PP2A B subunits consists of four isoforms (α, β, γ, δ) that rely on PP2A-C methylation for their assembly into trimeric PP2A-B55 holoenzymes [24, 40] and regulate different cellular signaling cascades [41]. We further focused on the increased interaction of B55α with PME-1, although we cannot exclude that B55γ may also be involved in the oxidative stress response in GBM cells. On the contrary: the PME-1 R369D mutant even made the cells more resistant to oxidative stress compared to the control cell lines (Fig. 2F), suggestive for a dominant- negative effect. subsequently causes the observed cell death sensitization The p38 mitogen-activated protein kinase (p38/MAPK) pathway is known to be involved in many cellular stress responses [42]. Interestingly, we observed a clear increase in the phosphorylation of MAPKAPK2 (Thr334), a downstream substrate of p38, in H2O2- treated U87MG PME-1-GFP cells, while phosphorylation of MAPKAPK2 was decreased in H2O2-treated U251MG shPME-1 cells compared to their respective controls (Fig. 4A–D). Phospho-p38 and phospho-MAPKAPK2 form a productive signaling complex in the nucleus that quickly translocates to the cytoplasm upon MAPKAPK2 activation [43]. Although the inactivation of MAP- KAPK2 by speciﬁc Ser/Thr phosphatases has remained largely elusive [43], MAPKAPK2 has been suggested as a direct PP2A substrate [44]. As Thr334 resides in a TP phosphorylation motif (PQT334P), it would be an excellent substrate for dephosphoryla- tion by PP2A-B55 holoenzymes [45, 46]. We conclude that PME-1’s role in response to oxidative stress is multifaceted and that its methylesterase activity, nuclear localiza- tion, and capacity to bind to PP2A-C are all necessary factors contributing to its stress sensitizing effect. Oxidative stress promotes B55α nuclear localization, PP2A-B55α binding to PME-1 and B55α-bound PP2A-C demethylation PME-1 overexpression induces increased RIPK1 phosphorylation/activity during oxidative stress, which subsequently causes the observed cell death sensitization The necroptosis-inducing kinase RIPK1 is a known phosphoryla- tion target of MAPKAPK2 upon TNFR activation [47]. To uncover if MAPKAPK2 also regulates RIPK1 upon oxidative stress in our GBM cell lines, we assessed potential changes in RIPK1 Ser320 phosphorylation upon treatment with H2O2. Not only did we observe RIPK1 phosphorylation of Ser320 upon H2O2 treatment, we also found increased phospho-RIPK1 levels in U87MG PME-1- GFP cells as opposed to the U87MG GFP control cells (Fig. 5A, B). Moreover, pretreatment of the cells with 100 μM necrostatin-1 (a RIPK1 inhibitor) [48] selectively abolished the sensitizing effect of increased PME-1 expression (Fig. 5C and Fig. S3A (statistics)), while pretreatment with 100 μM Z-VAD-FMK (a pan-caspase inhibitor) had no effect on t-BHP-induced cell death (Fig. 5D and Fig. S3B (statistics)). Thus, upon oxidative stress, PME-1 overexpression facilitates a form of cell death that is RIPK1 activity-dependent and does not rely on caspase activity (i.e., “necroptosis”). In a reciprocal AP-LC-MS/MS experiment executed in B55α-ﬂag overexpressing U251MG cells (Fig. 3D), PME-1 showed approxi- mately eightfold increased binding relative to B55α-ﬂag after H2O2-induced oxidative stress (Fig. 3E). To further validate and extend these data, we performed immunoblot experiments using PME-1-GFP pull-downs isolated from H2O2-treated U87MG cells, and did not only ﬁnd a clear increase in B55α, but also in PP2A-C and a trend towards increased PP2A-A binding to PME-1 (Fig. 3F, G). Similar results were seen upon treatment with t-BHP (Fig. S2B, C). Additionally, we performed GFP pull-downs on lysates isolated from H2O2-treated U87MG cells, transiently transfected with B55α- GFP (Fig. 3H, I). Although binding of PP2A-C and PP2A-A subunits to B55α was not altered upon H2O2 treatment, bound PP2A-C appeared more demethylated in stressed conditions, consistent with the observed increase in PME-1 binding to B55α by the MS approach (Fig. 3E). The latter could however only be occasionally reproduced by Western blotting (Fig. S2D, E), as in most experiments, PME-1 appeared below the detection limit in B55α- GFP pull-downs (Fig. 3H). y y We conclude that, upon oxidative stress, PME-1-mediated inhibition of nuclear PP2A-B55α promotes increased MAPKAPK2 Thr334 phosphorylation and activity in PME-1-high-expressing GBM cells. This increased MAPKAPK2 activity subsequently induces higher levels of RIPK1 Ser320 phosphorylation and activity, which eventually causes increased GBM cell death (Fig. 6). DISCUSSION PP2A d PP2A dysregulation and the emerging therapeutic options of pharmacologic PP2A modulation have recently received increas- ing attention from the brain oncology community [49, 50]. Speciﬁcally in GBM, overexpression of cellular PP2A inhibitors, Together, our data suggest increased oxidative stress-induced nuclear localization of PP2A-B55α, followed by an increased interaction with PME-1, and increased PP2A-C demethylation within the PME-1-PP2A-B55α complex. Cell Death Discovery (2023) 9:265 L. Guffens et al. 5 Fig. 3 Oxidative stress promotes B55α nuclear localization, PP2A-B55α binding to PME-1, and B55α-bound PP2A-C demethylation A Western blot of cytoplasmic (Hsp90 marker) and nuclear (SP1 marker) lysates of U87MG cells were treated with 1 mM H2O2 for the indicated duration. B Quantiﬁcation of nuclear and cytoplasmic B55α and PME-1 levels (relative to ponceau) shown in A (N = 5 or 6, one-way ANOVA C B55 protein abundance ratio in the PME-1-GFP interactome as determined by LC-MS/MS with or without a 10 min treatment with 10 mM H2O2. D Validation of stable B55α-ﬂag expressing U251MG cells using Western blotting. E PME-1 protein abundance ratio in the B55α-ﬂag interactome as determined by LC-MS/MS with or without a 10 min treatment with 2.5 mM H2O2. F B55α, PP2A-C, and PP2A-A subunit binding to PME-1-GFP in U87MG cells was determined after 1 h treatment with 1 mM H2O2 by GFP pull-down followed by Western blotting (non cropped blots are shown in Fig. S2A). G Quantiﬁcation of B55α, PP2A-C, and PP2A-A subunit binding to PME-1-GFP shown in F (N = 3 or 4 one-way ANOVA). H PP2A-C, demethylated PP2A-C and PP2A-A binding to B55α-GFP in U87MG cells was determined after 1 h treatment wit 1 mM H2O2 by GFP pull-down followed by Western blotting. I Quantiﬁcation of PP2A-C, demethylated PP2A-C, demethylated PP2A-C/tota PP2A-C, and PP2A-A binding to B55α-GFP shown in H (N = 3 or 5, one-way ANOVA). Cell Death Discovery (2023) 9:265 Fig. 3 Oxidative stress promotes B55α nuclear localization, PP2A-B55α binding to PME-1, and B55α-bound PP2A-C demethylation. A Western blot of cytoplasmic (Hsp90 marker) and nuclear (SP1 marker) lysates of U87MG cells were treated with 1 mM H2O2 for the indicated duration. B Quantiﬁcation of nuclear and cytoplasmic B55α and PME-1 levels (relative to ponceau) shown in A (N = 5 or 6, one-way ANOVA). C B55 protein abundance ratio in the PME-1-GFP interactome as determined by LC-MS/MS with or without a 10 min treatment with 10 mM H2O2. DISCUSSION PP2A d A Representative Western blots of phospho-MAPKAPK2 and total MAPKAPK2 levels in U87MG PME-1-GFP cells after treatment with 2.5 mM H2O2 for the indicated duration. B Quantiﬁcation of phospho-MAPKAPK2/total MAPKAPK2 shown in panel A (N = 3, two-way ANOVA). C Representative Western blots of phospho-MAPKAPK2 and total MAPKAPK2 levels in U251MG shPME-1 cells after treatment with 2.5 mM H2O2 for the indicated duration. D Quantiﬁcation of phospho-MAPKAPK2/total MAPKAPK2 shown in panel C (N = 3, two-way ANOVA). E Sensitivity of U87MG and U251MG cells to 16 h t-BHP treatment (different concentrations) with or without 1 h pretreatment with 10 μM MK2i was determined using MTT (N = 3 (U87MG), N = 4 (U251MG), two-way ANOVA). F Sensitivity of U87MG PME-1- GFP and U251MG shPME-1 to 16 h t-BHP treatment (different concentrations) with or without 1 h pretreatment with 10 μM MK2i was determined using MTT (N = 4 (U87MG), N = 6 (U251MG), statistical signiﬁcance is shown in Fig. S1B). L. Guffens et al. L. Guffens et al. 7 Fig. 4 PME-1 overexpression promotes increased MAPKAPK2 phosphorylation during oxidative stress, which is causally involved in the observed increase in cell death. A Representative Western blots of phospho-MAPKAPK2 and total MAPKAPK2 levels in U87MG PME-1-GFP cells after treatment with 2.5 mM H2O2 for the indicated duration. B Quantiﬁcation of phospho-MAPKAPK2/total MAPKAPK2 shown in panel A (N = 3, two-way ANOVA). C Representative Western blots of phospho-MAPKAPK2 and total MAPKAPK2 levels in U251MG shPME-1 cells after treatment with 2.5 mM H2O2 for the indicated duration. D Quantiﬁcation of phospho-MAPKAPK2/total MAPKAPK2 shown in panel C (N = 3, two-way ANOVA). E Sensitivity of U87MG and U251MG cells to 16 h t-BHP treatment (different concentrations) with or without 1 h pretreatment with 10 μM MK2i was determined using MTT (N = 3 (U87MG), N = 4 (U251MG), two-way ANOVA). F Sensitivity of U87MG PME-1- GFP and U251MG shPME-1 to 16 h t-BHP treatment (different concentrations) with or without 1 h pretreatment with 10 μM MK2i was determined using MTT (N = 4 (U87MG), N = 6 (U251MG), statistical signiﬁcance is shown in Fig. S1B). Fig. 5 Increased sensitivity of PME-1 overexpressing GBM cells to oxidative stress correlates with increased RIPK1 phosphorylation, and is inhibited by necrostatin-1 but not by a pan-caspase inhibitor. DISCUSSION PP2A d A Representative Western blots of phospho-RIPK1 and total RIPK1 levels in U87MG PME-1-GFP cells after treatment with 2.5 mM H2O2 for the indicated duration. B Quantiﬁcation of phospho-RIPK1/total RIPK1 shown in panel A (N = 3, two-way ANOVA). C Sensitivity of U87MG PME-1-GFP cells to 16 h t-BHP treatment (different concentrations) with or without 8 h pretreatment with 100 μM necrostatin-1, as determined by MTT (N = 4, statistical signiﬁcance is shown in Fig. S3A). D Sensitivity of U87MG PME-1-GFP cells to 16 h t-BHP treatment (different concentrations) with or without 8 h pretreatment with 100 μM Z-VAD-FMK, as determined by MTT (N = 4, statistical signiﬁcance is shown in Fig. S3B). Fig. 5 Increased sensitivity of PME-1 overexpressing GBM cells to oxidative stress correlates with increased RIPK1 phosphorylation, and is inhibited by necrostatin-1 but not by a pan-caspase inhibitor. A Representative Western blots of phospho-RIPK1 and total RIPK1 levels in U87MG PME-1-GFP cells after treatment with 2.5 mM H2O2 for the indicated duration. B Quantiﬁcation of phospho-RIPK1/total RIPK1 shown in panel A (N = 3, two-way ANOVA). C Sensitivity of U87MG PME-1-GFP cells to 16 h t-BHP treatment (different concentrations) with or without 8 h pretreatment with 100 μM necrostatin-1, as determined by MTT (N = 4, statistical signiﬁcance is shown in Fig. S3A). D Sensitivity of U87MG PME-1-GFP cells to 16 h t-BHP treatment (different concentrations) with or without 8 h pretreatment with 100 μM Z-VAD-FMK, as determined by MTT (N = 4, statistical signiﬁcance is shown in Fig. S3B). effect on MAPKAPK2-RIPK1 signaling, via the regulation of nuclear PP2A-B55α holoenzymes. which both induce oxidative stress as well [57]. However, it remains to be formally determined whether PME-1 expression will indeed affect the outcome of these DNA-damaging therapies, as predicted by our current study. In cells, reactive oxygen species (ROS), such as superoxide anions, hydrogen peroxide, and hydroxyl radicals, can be formed during the metabolism of oxygen and in response to different xenobiotics or chemicals. In addition, reactive nitrogen species (RNS) can be formed through chemical reactions with NO. Oxidative stress occurs when there is an imbalance between ROS production and ROS removal by antioxidants [54]. Cancer cells are, in general, more sensitive to oxidative stress due to their higher metabolic rate [55]. This initiated an interest in oxidative therapies, which are aiming at further increasing ROS levels to induce cancer cell death [56]. DISCUSSION PP2A d D Validation of stable B55α-ﬂag expressing U251MG cells using Western blotting. E PME-1 protein abundance ratio in the B55α-ﬂag interactome as determined by LC-MS/MS with or without a 10 min treatment with 2.5 mM H2O2. F B55α, PP2A-C, and PP2A-A subunit binding to PME-1-GFP in U87MG cells was determined after 1 h treatment with 1 mM H2O2 by GFP pull-down followed by Western blotting (non- cropped blots are shown in Fig. S2A). G Quantiﬁcation of B55α, PP2A-C, and PP2A-A subunit binding to PME-1-GFP shown in F (N = 3 or 4, one-way ANOVA). H PP2A-C, demethylated PP2A-C and PP2A-A binding to B55α-GFP in U87MG cells was determined after 1 h treatment with 1 mM H2O2 by GFP pull-down followed by Western blotting. I Quantiﬁcation of PP2A-C, demethylated PP2A-C, demethylated PP2A-C/total PP2A-C, and PP2A-A binding to B55α-GFP shown in H (N = 3 or 5, one-way ANOVA). Cell Death Discovery (2023) 9:265 L. Guffens et al. L. Guffens et al. 6 such as CIP2A [51–53], ARPP-19 [9], or PME-1 [16, 19], constitutes one of the major PP2A inhibitory mechanisms. PME-1 was found to be overexpressed in 44% of GBM, correlating with increased malignancy and proliferation [16]. Although patients with PME-1- high tumors were reported to be more resistant to kinase inhibitor therapies [19], the current study indicates these patients might be more responsive to oxidative stress-inducing therapies. While the oncogenic role of PME-1 was explained by its activating effect on MEK-ERK signaling [16], the sensitizing role of PME-1 to oxidative stress-induced cell death could be attributed to its activating Cell Death Discovery (2023)9:265 therapies [19], the current study indicates these patients might be more responsive to oxidative stress-inducing therapies. While the oncogenic role of PME-1 was explained by its activating effect on MEK-ERK signaling [16], the sensitizing role of PME-1 to oxidative stress-induced cell death could be attributed to its activating such as CIP2A [51–53], ARPP-19 [9], or PME-1 [16, 19], constitutes one of the major PP2A inhibitory mechanisms. PME-1 was found to be overexpressed in 44% of GBM, correlating with increased malignancy and proliferation [16]. Although patients with PME-1- high tumors were reported to be more resistant to kinase inhibitor Cell Death Discovery (2023) 9:265 Fig. 4 PME-1 overexpression promotes increased MAPKAPK2 phosphorylation during oxidative stress, which is causally involved in the observed increase in cell death. Cell Death Discovery (2023) 9:265 DISCUSSION PP2A d Here, we observed an increased sensitivity of PME-1 overexpressing GBM cells to t-BHP or H2O2-induced oxidative stress. Therefore, PME-1 expression could potentially serve as a marker to stratify GBM patients when testing oxidative therapies, or to stratify patients in non/semi/good-response groups for current standard therapies (radiotherapy and TMZ chemotherapy), The impact of our ﬁndings on human disease may be broader than just the treatment of GBM or other cancer types in which PME-1 is frequently overexpressed [13–16]. Indeed, a similar response might be triggered in normal brain cells upon oxidative stress. This should be studied more in detail and could potentially broaden the therapeutic relevance of our ﬁndings. For example, PME-1 overexpression is commonly observed in brain samples from patients with tauopathies, such as progres- sive supranuclear palsy (PSP) and Alzheimer’s disease (AD) [58]. Oxidative stress generally increases upon aging, and high levels of ROS are commonly observed in patients with neurodegen- erative diseases [59]. Therefore, inhibiting PME-1 by AMZ30, or inhibiting PME-1-controlled MAPKAPK2-RIPK1 signaling by MK2i or necrostatin-1, could potentially limit an increase in neural cell Cell Death Discovery (2023) 9:265 L. Guffens et al. 8 Fig. 6 Model of the sensitizing effect of PME-1 to oxidative stress-induced cell death in GBM. A Visual representation of oxidative stress- induced PP2A-B55α binding to PME-1 and subsequent PP2A-C demethylation and inactivation in the nucleus (created with BioRender.com). B Model of the PP2A-B55α-inactivating function of PME-1 that subsequently controls activation of the MAPKAPK2-RIPK1 pathway and induction of RIPK1-dependent cell death (necroptosis) (created with BioRender.com). Fig. 6 Model of the sensitizing effect of PME-1 to oxidative stress-induced cell death in GBM. A Visual representation of oxidative stres induced PP2A-B55α binding to PME-1 and subsequent PP2A-C demethylation and inactivation in the nucleus (created with BioRender.com B Model of the PP2A-B55α-inactivating function of PME-1 that subsequently controls activation of the MAPKAPK2-RIPK1 pathway an induction of RIPK1-dependent cell death (necroptosis) (created with BioRender.com). the sensitizing effect of PME-1 to oxidative stress-induced cell death in GBM. A Visual representation of ox 55α binding to PME-1 and subsequent PP2A-C demethylation and inactivation in the nucleus (created with Bio Fig. 6 Model of the sensitizing effect of PME-1 to oxidative stress-induced cell death in GBM. A Visual representation of oxidative stress- induced PP2A-B55α binding to PME-1 and subsequent PP2A-C demethylation and inactivation in the nucleus (created with BioRender.com). DISCUSSION PP2A d B Model of the PP2A-B55α-inactivating function of PME-1 that subsequently controls activation of the MAPKAPK2-RIPK1 pathway and induction of RIPK1-dependent cell death (necroptosis) (created with BioRender.com). death caused by high oxidative stress levels in patients with PSP or AD. Moreover, AMZ30 may not only prevent neural cell death, it might also limit the formation of tau aggregates by preventing PME-1-mediated inhibition of PP2A-B55α–catalyzed tau depho- sphorylation [60]. inhibition of PP2A has been shown to occur by direct tyrosine nitration of PP2A-C [69], or of the B56δ subunit—in the latter case, resulting in its failure to be incorporated into a PP2A holoenzyme [67]. More relevant to our current study, ROS-induced PP2A inhibition has frequently been correlated with PP2A-C demethyla- tion and increased apoptosis, e.g., in neurons [66], embryonic kidney [70], and cancer cells [65]. Here, we mechanistically extended these ﬁndings by showing for the ﬁrst time that oxidative stress cannot only lead to PP2A-C demethylation in general, but to a speciﬁc demethylation (and inactivation) of the PP2A-B55α holoenzyme in the nucleus, and to a speciﬁc form of cell death dependent on RIPK1 activity (necroptosis). Although the observed increase in PME-1-PP2A-B55α binding is likely attributable to an increased presence of PP2A-B55α in the nucleus (Fig. 3A, B), we cannot entirely exclude that potential y PP2A regulation during oxidative stress has been repeatedly demonstrated, although results from published studies were not always consistent, and could be explained by context- and holoenzyme-dependent differences [61, 62]. However, most studies demonstrated PP2A inhibition by ROS or RNS in diverse cell contexts and through multiple mechanisms [63–67]. ROS can result in direct oxidation of at least two cysteine residues in PP2A- C [64], as well as in direct glutathionylation of PP2A-C [63]. Both modiﬁcations are reversible by the addition of reducing agents, and inhibit PP2A activity in vitro and in cells [68]. RNS-mediated Cell Death Discovery (2023) 9:265 Cell culture and transfection Human GBM cell lines U87MG (ATCC, #HTB-14) and U251MG (ECACC, #09063001) were cultured at 37 °C and 5% CO2 in DMEM (Sigma, #D6546) supplemented with 10% heat-inactivated fetal bovine serum (Sigma, #F7524), 2 mM L-glutamine (Sigma, #G7513), and 100 units/mL penicillin and 100 mg/mL streptomycin (Sigma, #P0781). Transfections were performed with lipofectamine 3000 (Thermo Fisher, #L3000001). All cell experiments were performed at passage number <30. Cultures were regularly tested for Mycoplasma contamination (Venor™GeM, Minerva Biolabs, #11-1050). While PME-1 relocalization to the cytoplasm after H2O2 treatment has been described in primary dermal ﬁbroblasts [39], we could not observe a redistribution of PME-1 to the cytoplasm in GBM cells. Also, the PME-1 NLS mutant, which localizes to the cytoplasm [26], failed to sensitize GBM cells to oxidative stress- induced cell death, further underscoring that the site of PME-1 function and regulation of PP2A-B55α is not the cytoplasm, but the nucleus. Although we could not detect a clear concomitant stress-induced increase of PP2A-C or -A in the nucleus, we presume that this is still the case, since B55α is not stable as a monomer [24, 41] and the translocating (B55α-bound) fraction of PP2A-C and -A might just be too small to be discriminated from the non-translocating PP2A-C and -A pool (bound to other B-type subunits) in a Western blot assay. In any case, the increased nuclear B55α presence upon oxidative stress could serve as a physiologic way to restrict excessive stress- induced signaling pathways in the nucleus, and, thereby, to prevent induction of cell death. Only in the pathologic case of PME-1 overexpression, this nuclear function of PP2A-B55α would speciﬁcally be attenuated, and stress-induced cell death would be promoted. As explained before, this is obviously of interest in cancer therapy, but is not desirable in the aging brain. effect of RIPK1 Ser320 phosphorylation downstream of death receptor engagement [47, 77, 78], others have shown that RIPK1 Ser320 and Ser166 phosphorylation mediate necroptosis under oxidative stress conditions [79], very much in line with our current ﬁndings. g We anticipate that the PP2A-modulating role of PME-1 in different forms of stress-induced cell death may be much broader than currently anticipated, and certainly deserves to be further investigated in other cell and stress contexts. Chemical inhibitors and compounds y , y The p38 pathway is known to be important in transducing oxidative stress signals [42]. MAPKAPK2 is a direct downstream target of p38 in the nucleus, which, on its turn, can phosphorylate several cellular substrates, including RIPK1 [47, 77, 78]. In our GBM models, we found that in response to oxidative stress PME-1 overexpression, by inhibiting nuclear PP2A-B55α activity, increased the phosphorylation (and activation) of MAPKAPK2 Thr334, to promote subsequent RIPK1 Ser320 phosphorylation, and ﬁnally, cell death. As PP2A-B55 trimers preferentially depho- sphorylate proline-directed phospho-threonines [46], we spec- ulate that the p38 phosphorylation site of MAPKAPK2 (Thr344) is the likely target of PP2A-B55α. Although several cellular substrates of PP2A-B55α have already been identiﬁed [41], this would be the ﬁrst time MAPKAPK2 is suggested as a direct PP2A-B55α target, whose dephosphorylation is moreover controlled by PME-1. In addition, PP2A has been identiﬁed as a possible (direct or indirect) upstream phosphatase of RIPK1 in glucose-deprived conditions [36]. Although certain papers pointed to a cell death inhibiting p Tert-butyl hydroperoxide (t-BHP, #A13926) was purchased from Alfa Aesar, MAPKAPK2 inhibitor PF-364402 (#PZ0188) and H2O2 (#H1009) from Sigma, AMZ30 (#539695) from Millipore, Z-VAD-FMK (#S8102) from Bio-Connect and necrostatin-1 (#T1547) from Tebubio. Cloning and site-directed mutagenesis Cloning and site-directed mutagenesis Cloning and site-directed mutagenesis g g PME-1 (encoded by PPME1) was cloned into pEGFP-N1 (Clontech). B55α (encoded by PPP2R2A) was cloned into pEGFP-N1 and 3xFLAG CMV10 vector (Sigma). PME-1 mutants (in pEGFP-N1) were generated by PCR- based site-directed mutagenesis (primers in Table S1). Mutations were conﬁrmed by sequencing (LGC Genomics). Lentiviral expression vectors (pLKO.1) for PPME1 3’UTR targeting (shPME-1, target sequence: AACATC- GAGCTCTGTTGTAA) and control GFP-targeting shRNA (shGFP) were generated as described [80]. Generation of stable cell lines with altered PME-1 or B55α expression The binding of trimeric PP2A-B55α to PME-1 has been observed before by three independent groups [30, 38, 73], with Longin et al. initially reporting on an inactive, PME-1-bound PP2A-B55 holoen- zyme puriﬁed de novo from a porcine brain that served as a good substrate in an in vitro methylation turnover assay [30]. The latter implied that the PP2A-B55 holoenzyme is a good PME-1 substrate, although several crystallographic studies [28, 60, 74, 75] originally suggested signiﬁcant steric hindrance of PME-1 binding to the PP2A AC core by the regulatory B subunits. Recently, however, using a cryo-EM approach, Li et al. revealed that large structural shifts in both the holoenzyme and PME-1 do allow PME-1 to bind to PP2A-B56 trimers and block the substrate-binding groove [76]. They also discovered that two distinct disordered regions within PME-1 mediate its interaction with PP2A-B56 and with PP2A-B55 [76]. Thus, it is entirely conceivable that increased binding of PME- 1 to PP2A-B55α can indeed result in PP2A-B55α demethylation and catalytic inactivation, as observed in our study. p Stable PME-1-GFP and B55α-ﬂag overexpressing cells were generated by transfection of pEGFP-N1 (containing PPME1 cDNA) or 3xFLAG CMV10 vector (containing PPP2R2A cDNA) with lipofectamine 3000 (Thermo Fisher, #L3000001) and subsequent Geneticin (G418) selection at 400 μg/ ml. Polyclonal cell populations were frozen and used in all subsequent experiments. For the generation of lentiviral particles, TurboFect (Thermo Fisher, #R0533) was used to transfect 500 ng lentiviral expression vector (pLKO.1), 450 ng packaging vector (pCMV-deltaR 8.91), and 50 ng enveloping vector (pMD.G-VSVG) in HEK293T cells. Twenty-four hours and 48 h after transfection, lentiviral particles were harvested and ﬁltered through a 0.45 μm syringe ﬁlter. For transductions, U87MG and U251MG cells were incubated with different virus titers for 24 and 48 h, and selected with puromycin (1.25 μg/ml) for 1 week. Polyclonal cell populations were frozen for further use. L. Guffens et al. 9 post-translational modiﬁcations of B55α or PME-1, such as e.g., the previously described PME-1 Ser15 phosphorylation [71], might further facilitate their interaction. However, although we speciﬁ- cally investigated this possibility, we failed to identify any H2O2- induced changes in PME-1 or B55α phosphorylation in our MS- based assays (data not shown). It also remains to be determined how the increased nuclear presence of B55α in the stressed cells can be explained (translocation, stabilization), although a translo- cation of PP2A-B55 in the opposite direction (from the nucleus into the cytoplasm) has been observed by others in DNA- damaged cells [72]. Although we could not detect a clear concomitant stress-induced increase of PP2A-C or -A in the nucleus, we presume that this is still the case, since B55α is not stable as a monomer [24, 41] and the translocating (B55α-bound) fraction of PP2A-C and -A might just be too small to be discriminated from the non-translocating PP2A-C and -A pool (bound to other B-type subunits) in a Western blot assay. In any case, the increased nuclear B55α presence upon oxidative stress could serve as a physiologic way to restrict excessive stress- induced signaling pathways in the nucleus, and, thereby, to prevent induction of cell death. Only in the pathologic case of PME-1 overexpression, this nuclear function of PP2A-B55α would speciﬁcally be attenuated, and stress-induced cell death would be promoted. As explained before, this is obviously of interest in cancer therapy, but is not desirable in the aging brain. post-translational modiﬁcations of B55α or PME-1, such as e.g., the previously described PME-1 Ser15 phosphorylation [71], might further facilitate their interaction. However, although we speciﬁ- cally investigated this possibility, we failed to identify any H2O2- induced changes in PME-1 or B55α phosphorylation in our MS- based assays (data not shown). It also remains to be determined how the increased nuclear presence of B55α in the stressed cells can be explained (translocation, stabilization), although a translo- cation of PP2A-B55 in the opposite direction (from the nucleus into the cytoplasm) has been observed by others in DNA- damaged cells [72]. Antibodies 7. Sangodkar J, Farrington CC, McClinch K, Galsky MD, Kastrinsky DB, Narla G. All roads lead to PP2A: exploiting the therapeutic potential of this phosphatase. FEBS J. 2016;283:1004–24. Antibodies were obtained from the indicated suppliers: anti-PME-1 (Santa Cruz Biotechnology, #sc-25278), PP2A-C (Cell Signaling Technology, #2038), vinculin (Sigma-Aldrich, #V9131), B55α (Cell Signaling Technology, #5689), PP2A-A (mouse monoclonal, gift from Prof. S. Dilworth), MAPKAPK2 (Cell Signaling Technology, #3042), phospho-MAPKAPK2 (Cell Signaling Tech- nology, #3007), demethylated PP2A-C (Sigma-Aldrich, #05-577), SP1 (Cell Signaling Technology, #9389), Hsp90 (Cell Signaling Technology, #4877), RIPK1 (Cell Signaling Technology, #3493), and phospho-RIPK1 Ser320 (Cell Signaling Technology, #58274). 8. Ruvolo PP. The broken “Off” switch in cancer signaling: PP2A as a regulator of tumorigenesis, drug resistance, and immune surveillance. BBA Clin. 2016;6:87–99. 9. Jiang T, Zhao B, Li X, Wan J. ARPP-19 promotes proliferation and metastasis of human glioma. Neuroreport. 2016;27:960–6. 10. Kauko O, Westermarck J. Non-genomic mechanisms of protein phosphatase 2A (PP2A) regulation in cancer. Int J Biochem Cell Biol. 2018;96:157–64. 11. Dun MD, Mannan A, Rigby CJ, Butler S, Toop HD, Beck D, et al. Shwachman–Bodian–Diamond syndrome (SBDS) protein is a direct inhibitor of protein phosphatase 2A (PP2A) activity and overexpressed in acute myeloid leukaemia. Leukemia. 2020;34:3393–7. Protein extraction, immunoprecipitation, and Western blotting g Cells were rinsed with PBS, lysed with NET-lysis buffer (50 mM Tris-HCl, pH 7.4, 1% NP-40, 15 mM EDTA, and 150 mM NaCl) supplemented with protease inhibitor cocktail (Roche) and phosphatase inhibitors (PhosStop, Roche) and centrifuged for 15 min at 13,000 × g. For PME-1-GFP or B55α- ﬂag/pull-down followed by Western blot analysis, phosphatase inhibitors were omitted from the lysis buffer. For pull-down experiments, 300 μl lysate was incubated at 4 °C for 1 h with 500 μl NENT100 (20 mM Tris-HCl, pH 7.4, 1% NP-40, 15 mM EDTA, and 100 mM NaCl) containing 1 mg/mL bovine serum albumin (BSA), and 40 μl prewashed GFP-trap-A beads (ChromoTek) or anti-ﬂag mAb conjugated sepharose beads (FLAG M2 afﬁnity gel, Sigma) on a rotating wheel. Beads were washed three times with 0.5 mL NENT150 (containing 150 mM NaCl). Cell Death Discovery (2023) 9:265 L. Guffens et al. 10 2. Janssens V, Goris J. Protein phosphatase 2A: a highly regulated family of serine/ threonine phosphatases implicated in cell growth and signalling. Biochem J. 2001;353:417–39. Bound proteins were eluted in 2x NuPage sample buffer (Invitrogen) and boiled at 95 °C for 10 min, prior to loading on a denaturing SDS-PAGE gel (4–12% Bis-Tris Midi, Invitrogen). Proteins were transferred to a nitrocellu- lose membrane (GE Healthcare) by wet blotting at 100 V for 40 min. Membranes were blocked in 5% BSA in TBS/0. 1% Tween-20 for 1 h at room temperature and incubated with primary antibody overnight at 4 °C. Membranes were washed three times with TBS-T and incubated with HRP- conjugated secondary antibodies (Dako) for 1 h at room temperature. After a ﬁnal wash with TBS-T, membranes were developed using Western Bright ECL (Advansta) on the ImageQuant LAS 4000 scanner (GE Healthcare). Densitometric analysis was performed using Image Studio lite software. 3. Sents W, Ivanova E, Lambrecht C, Haesen D, Janssens V. The biogenesis of active protein phosphatase 2A holoenzymes: a tightly regulated process creating phosphatase speciﬁcity. FEBS J. 2013;280:644–61. 4. Narla G, Sangodkar J, Ryder CB. The impact of phosphatases on proliferative and survival signaling in cancer. Cell Mol Life Sci. 2018;75:2695–718. 5. Janssens V, Rebollo A. The role and therapeutic potential of ser/Thr phosphatase PP2A in apoptotic signalling networks in human cancer cells. Curr Mol Med. 2012;12:268–87. 6. Perrotti D, Neviani P. Protein phosphatase 2A: a target for anticancer therapy. Lancet Oncol. 2013;14:229–38. DATA AVAILABILITY 27. Yabe R, Tsuji S, Mochida S, Ikehara T, Usui T, Ohama T, et al. A stable association with PME‐1 may be dispensable for PP2A demethylation – implications for the detection of PP2A methylation and immunoprecipitation. FEBS Open Bio. 2018;8:1486–96. The data that support the ﬁndings of this study are available from the corresponding author upon reasonable request. 28. Xing Y, Li Z, Chen Y, Stock JB, Jeffrey PD, Shi Y. Structural mechanism of deme- thylation and inactivation of protein phosphatase 2A. Cell. 2008;133:154–63. Nuclear and cytoplasmic extraction y Subcellular fractionation was performed with NE-PER nuclear and cytoplasmic extraction kit (Thermo Fisher, #7833), according to the manufacturer’s instructions. 12. Mäkelä E, Pavic K, Varila T, Salmenniemi U, Löyttyniemi E, Nagelli SG, et al. Dis- covery of a Novel CIP2A Variant (NOCIVA) with clinical relevance in predicting TKI resistance in myeloid leukemias. Clin Cancer Res. 2021;27:2848–60. 13. Wandzioch E, Pusey M, Werda A, Bail S, Bhaskar A, Nestor M, et al. PME-1 modulates protein phosphatase 2A activity to promote the malignant phenotype of endometrial cancer cells. Cancer Res. 2014;74:4295–305. Statistics Data were represented as mean plus standard deviation (SD), unless described otherwise. Statistical tests, as indicated in ﬁgure legends, were performed using GraphPad Prism 8.0.1. Signiﬁcance was represented as , , , and ** representing p values of ≤0.05, ≤0.01, ≤0.001, and ≤0.0001, respectively. “N” denotes the number of biological replicates. 24. Longin S, Zwaenepoel K, Louis JV, Dilworth S, Goris J, Janssens V. Selection of protein phosphatase 2A regulatory subunits is mediated by the C terminus of the catalytic subunit. J Biol Chem. 2007;282:26971–80. 25. 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The resulting peptides were desalted by Micro Spin Tips (Harvard Apparatus) and subjected to high-resolution LC-MS/MS, using an Ultimate 3000 Nano Ultra High Pressure Chromatography system interfaced with an Orbitrap Elite Hybrid Ion Trap-Orbitrap (Q Exactive) mass spectrometer via an EASY-spray (C-18, 15 cm) column (Thermo Fisher Scientiﬁc). Peptides were identiﬁed by MASCOT (Matrix Science) using UniProt Homo sapiens (173,330 sequences) as a database. Progenesis software (Nonlinear Dynamics) was used for relative quantiﬁcation of peptides, and Proteome Discoverer 2.2 software (Thermo Fisher Scientiﬁc) for peptide validation (FDR <1%) using Percolator node. Protein abundances were normalized according to the protein abundance of the bait in different conditions. 17. Chandana SR, Movva S, Arora M, Singh T. Primary brain tumors in adults. Am. Fam. Physician. 2008;77:1423–30. 18. Stupp R, Hegi ME, Mason WP, van den Bent MJ, Taphoorn MJ, Janzer RC, et al. 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AUTHOR CONTRIBUTIONS Reprints and permission information is available at http://www.nature.com/reprints Reprints and permission information is available at http://www.nature.com/reprints All authors contributed to the design of the work, the acquisition of data, the interpretation of the results, and the writing of the manuscript. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. FUNDING This work was funded by a senior project grant from the Research Foundation- Flanders (FWO-Vlaanderen, G0B1719N). 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 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 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 license, visit http:// creativecommons.org/licenses/by/4.0/. ACKNOWLEDGEMENTS 56. Wondrak GT. Redox-directed cancer therapeutics: molecular mechanisms and opportunities. Antioxid Redox Signal. 2009;11:3013–69. We would like to thank Ms. Lisa Lenaerts (KU Leuven, Belgium) for helping with the preparation of MS samples. We would like to thank Ms. Lisa Lenaerts (KU Leuven, Belgium) for helping with the preparation of MS samples. Cell Death Discovery (2023) 9:265 L. Guffens et al. 12 Cell Death Discovery (2023) 9:265 COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41420-023-01572-1. Correspondence and requests for materials should be addressed to Veerle Janssens. Correspondence and requests for materials should be addressed to Veerle Janssens. © The Author(s) 2023 Cell Death Discovery (2023) 9:265 Cell Death Discovery (2023) 9:265
https://openalex.org/W2518119385	https://europepmc.org/articles/pmc4989153?pdf=render	English	null	Localization of Waves in Merged Lattices	Scientific reports	2,016	cc-by	7,756	Localization of Waves in Merged Lattices G. Alagappan & C. E. Png This article describes a new two–dimensional physical topology–merged lattice, that allows dense number of wave localization states. Merged lattices are obtained as a result of merging two lattices of scatters of the same space group, but with slightly different spatial resonances. Such merging creates two–dimensional scattering “beats” which are perfectly periodic on the longer spatial scale. On the shorter spatial scale, the systematic breakage of the translational symmetry leads to strong wave scattering, and this causes the occurrences of wave localization states. Merged Lattices promises variety of localization states including tightly confined, and ring type annular modes. The longer scale perfect periodicity of the merged lattice, enables complete prediction and full control over the density of the localization states and its’ quality factors. In addition, the longer scale periodicity, also allows design of integrated slow wave components. Merged lattices, thus, can be engineered easily to create technologically beneficial applications. received: 08 April 2016 accepted: 22 July 2016 Published: 18 August 2016 Localization is a key concept in wave physics that enables control and manipulation of wave propagations. A periodic lattice of scatters with a complete bandgap for wave propagations in general is able to localize wave cor- responding to bandgap frequencies if a systematic single or an extended disorder (defects) is introduced in the lattice1–6. There are also some special periodic geometries such as Lieb7–12 and Kagome13–17 lattices, which permit wave localizations without the presence of any defects. The unit cells of such lattices naturally allow destruc- tive waves interference, enabling localization. Apart from the periodic topologies, quasi–periodic structures18–22 and random structures23–33 have been intensively studied for wave localizations. Random dielectric structures exhibiting strong wave diffusion is able to localize wave if the mean free path of the diffusion is equal or smaller than λ/2π, where λ is the wavelength. Such transformation from a diffusion state to a localized state is the direct consequence of the wave interference, and it is well known in the name of Anderson localization. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Department of Electronics and Photonics, Institute of High Performance Computing, Agency for Science, Technology, and Research (A-STAR), 1 Fusionopolis Way, #16-16 Connexis, 138632 Singapore. Correspondence and requests for materials should be addressed to G.A. (email: gandhi@ihpc.a-star.edu.sg) Scientific Reports \| 6:31620 \| DOI: 10.1038/srep31620 Localization of Waves in Merged Lattices Though, Philips Anderson first predicted such localization for electronic wavefunctions23, now Anderson localization is an ubiq- uitous phenomenon in wave physics, and it has been demonstrated for various wave topologies such as light in semiconductor dielectric powders25, light in photonic crystals with random disorders24,26, light in complex optical communication cluster27, light in disorder fiber beams28, microwaves in random copper tubes filled with metallic and dielectric spheres29, Bose Einstein condensates in random optical lattices30, acoustic waves in glasses31, acous- tic waves in percolation systems32 water waves in random underwater structures33, etc. p y Apparently, new topologies lead to new physics, and paves the way for a new technological exploration. In this article, using light wave as an illustration, we introduce a new paradigm of wave localization topology that pos- sesses double spatial resonances. Doubly resonant systems such as mediums with electromagnetic induced trans- parencies34–36, and their optical37–40, and plasmonic41,42 analogues are typically made of two physical sub-systems (usually resonators or atomic states) of slightly different resonant frequencies. These systems are known for their coherent interference effects, especially they can induce transmission in the originally opaque or reflective state. We borrow this idea to create majestic, large number of light localization states in two dimensional (2D) periodic lattices that has slightly different Bragg resonances.f g yf gg Two lattices of slightly different periodicities but with the same space group can be merged together to pro- duce a new lattice of the same space group but periodic on the longer spatial scale. For an example, consider two square lattices of periods a and ra (r > 1, and is a rational number close to 1). These two square lattices can be merged together to generate a merged lattice (ML) that is also a square lattice, but periodic on the longer spatial scale with a period Ra. Here, R is the least integer multiple of r. The square lattice dielectric functions with periods a and ra, have fundamental spatial resonances when the absolute value of the reciprocal lattice vectors equal 2π/a and 2π/ra, respectively. The closer these spatial resonances are, the longer the resulting 2D spatial dielectric beat. Department of Electronics and Photonics, Institute of High Performance Computing, Agency for Science, Technology, and Research (A-STAR), 1 Fusionopolis Way, #16-16 Connexis, 138632 Singapore. Correspondence and requests for materials should be addressed to G.A. Localization of Waves in Merged Lattices This requirement constraints r as r = R/(R−1) {or equivalently R = r/(r−1)}. Figure 1(a) to 1(h) illustrates the basic principles of creating MLs. Figure 1(c,g) show the primitive unit cells ( h D d l b ) f ML h R d l Th ll f h ML b d The symmetrically allowed closest proximity between the spatial resonances is 2π/Ra [i.e., absolute value of one reciprocal lattice vector of the ML]. This requirement constraints r as r = R/(R−1) {or equivalently R = r/(r−1)}. The symmetrically allowed closest proximity between the spatial resonances is 2π/Ra [i.e., absolute value of one reciprocal lattice vector of the ML]. This requirement constraints r as r = R/(R−1) {or equivalently R = r/(r−1)}. Figure 1(a) to 1(h) illustrates the basic principles of creating MLs. Figure 1(c,g) show the primitive unit cells (i.e., the 2D dielectric beats) of MLs with R = 3 and 5, respectively. These primitive unit cell of the ML is obtained by combining R primitive unit cells of the lattice with period a {Fig. 1(a) [R = 3] and 1(e) [R = 5]}, with R−1 primitive unit cells of the lattice with period ra {Fig. 1(b) [R = 3] and 1(f) [R = 5]}. Note that (R−1) × ra = Ra, and when merging two lattices, the motifs at each lattice site is also merged [see Fig. 1(g,h)]. The primitive unit cells of MLs shown in Fig. 1(c,g) are not unique. Another set of primitive unit cells are shown in Fig. 1(d,h) for R = 3 and 5, respectively. The positions of these two different primitive unit cells [Fig. 1(c,d)] are illustrated in the Fig. 2 for R = 3.h ph q q y Figure 1(a) to 1(h) illustrates the basic principles of creating MLs. Figure 1(c,g) show the primitive unit cells (i.e., the 2D dielectric beats) of MLs with R = 3 and 5, respectively. These primitive unit cell of the ML is obtained by combining R primitive unit cells of the lattice with period a {Fig. 1(a) [R = 3] and 1(e) [R = 5]}, with R−1 primitive unit cells of the lattice with period ra {Fig. 1(b) [R = 3] and 1(f) [R = 5]}. Note that (R−1) × ra = Ra, and when merging two lattices, the motifs at each lattice site is also merged [see Fig. 1(g,h)]. Localization of Waves in Merged Lattices The primitive unit cells of MLs shown in Fig. 1(c,g) are not unique. Another set of primitive unit cells are shown in Fig. 1(d,h) for R = 3 and 5, respectively. The positions of these two different primitive unit cells [Fig. 1(c,d)] are illustrated in the Fig. 2 for R = 3.h g The single 2D dielectric beat [i.e., the primitive unit cell of the ML] itself has no translational symmetries within, and hence it enables creation of large number of scattering loop that facilitates polychromatic, light local- ization. Unlike quasi–periodic dielectric structures18,19, or random dielectric structures25–28, such light localiza- tions in dielectric MLs are completely predictable (and therefore controllable) using photonic band structures. The density of localization states and their quality factors scale as a function of R. Merged lattices (MLs) lie in between the two extremes of completely random system (where Anderson localization23–28 prevails), and the con- ventional periodic system of photonic crystals4,5. In the MLs, the periodicity prevails on the longer spatial scale (spatial distance >Ra). The translational symmetry is completely broken on the spatial scale <Ra. Localization in ML takes place within this shorter spatial scale, for wavelengths on the order of fractions of a.fifi p p g MLs for light wave will be useful to create multimode miniature lasers43, efficient quantum computing44, effi- cient solar energy harvesting45, and enhanced non-linear optical interactions46. In addition to that, as MLs are perfectly periodic with the period Ra, thus they can be engineered easily to create defect paths enabling integrated slow light components, and photonic chips. Localization of Waves in Merged Lattices (email: gandhi@ihpc.a-star.edu.sg) 1 Scientific Reports \| 6:31620 \| DOI: 10.1038/srep31620 www.nature.com/scientificreports/ Figure 1. Merging of two square lattices of periods a and ra. Here, r = R/(R-1), with R is a integer greater than 2. (a–d) Merging for R = 3, (e–h) Merging for R = 5. (a,e) R periods of square lattice with period a. (b,f) (R-1) periods of square lattice with period ra. (c) One primitive unit cell of the lattice obtained from merging lattice in (a) with lattice in (b). (g) One primitive unit cell of the lattice obtained from merging lattice in (e) with lattice in (f). (d,h) Alternative primitive unit cells of the merged lattices. a and ra. Here, r = R/(R-1), with R is a integer greater than Figure 1. Merging of two square lattices of periods a and ra. Here, r = R/(R-1), with R is a integer greater than 2. (a–d) Merging for R = 3, (e–h) Merging for R = 5. (a,e) R periods of square lattice with period a. (b,f) (R-1) periods of square lattice with period ra. (c) One primitive unit cell of the lattice obtained from merging lattice in (a) with lattice in (b). (g) One primitive unit cell of the lattice obtained from merging lattice in (e) with lattice in (f). (d,h) Alternative primitive unit cells of the merged lattices. The symmetrically allowed closest proximity between the spatial resonances is 2π/Ra [i.e., absolute value of one reciprocal lattice vector of the ML]. This requirement constraints r as r = R/(R−1) {or equivalently R = r/(r−1)}. Figure 1(a) to 1(h) illustrates the basic principles of creating MLs. Figure 1(c,g) show the primitive unit cells (i.e., the 2D dielectric beats) of MLs with R = 3 and 5, respectively. These primitive unit cell of the ML is obtained by combining R primitive unit cells of the lattice with period a {Fig. 1(a) [R = 3] and 1(e) [R = 5]}, with R−1 primitive unit cells of the lattice with period ra {Fig. 1(b) [R = 3] and 1(f) [R = 5]}. Note that (R−1) × ra = Ra, and when merging two lattices, the motifs at each lattice site is also merged [see Fig. 1(g,h)]. The primitive unit The symmetrically allowed closest proximity between the spatial resonances is 2π/Ra [i.e., absolute value of one reciprocal lattice vector of the ML]. Results f 3(d–f)], possess many degenerate point and degenerate bands. Such degeneracies accounts for the finite translational symmetries within the corre- sponding non-primitive unit cells. These degeneracies will be lifted up, when the translational symmetries within the unit cell becomes broken. As we shall see, this is the exactly the case for the MLs, which breaks the transla- tional symmetries of the original lattice before the merging. Now let us analyze the photonic band structures of MLs. Let assume MLs are created by merging the dielectric functions of two square lattice PCs [silicon rods in air ambience] with periods a and ra as described in Fig. 1. For the purpose of comparison with the band structures in Fig. 3, the radii of the rods are taken as 0.15a in both PCs. Figure 4 exhibits photonic band structures of the MLs for R = 3, 5, 7 and 9, respectively [see the methods sections, for the details of the calculation]. In the same diagram, we have also plotted the folded band structure of the conventional PC [i.e., lattice with period a; the corresponding folded band structures are also shown in Fig. 3(d–f)] with non-primitive unit cells of the length Ra. As we can from these figures, in the long wavelength limit, the bands of the ML look similar to the folded bands of the conventional PC. For this spectral region, the wavelengths are much larger than a, and as both ML and the conventional PC exhibit similar long range transla- tional symmetries, it is not surprising to find their bands are similar. On the other hand, for the spectral region closer to the bandgap of the conventional PC, for which the wavelengths are on the order of fractions of a, the original translational symmetries in the non-primitive unit cells are completely broken. This induces coupling between the various folded bands of the conventional PC [Fig. 3(d–f)]. The coupling splits and flattens the folded bands, lifts–up the degeneracies, and pushes them into the bandgap region [Fig. 4]. From Fig. 4, we can clearly see that, MLs have dense number of flat bands in their band structure right at the vicinity of the bandgap region of the conventional PC. These flat bands occur for wavelengths (λ) on the order of fractions of a [see Fig. 4 for the normalized frequencies (a/λ) of the flat bands]. Results f In other words, each band in the band structure of the primitive unit cell, becomes R2 folded bands in the band structure of the non-primitive unit cell. For a conventional PC, it is well known that the group velocities for the band edge modes are small, and the bands flatten towards the photonic bandgap frequencies. Therefore, the folded bands of the conventional PC in the proximity of photonic bandgaps are flatter [for example, see the folded band diagrams of Fig. 3(d–f)]. The mode pattern corresponding to the band edge mode is basically a standing wave that extends through the entire 2D lattice5. Thus, the band edge modes of the conventional PC are not spatially localized. sections, for the details of the calculation). Figure 3(c) shows the result of calculation for a primitive unit cell (R = 1), and as we can see clearly see, the PC exhibits a large bandgap between the bands 1 and 2 for the TM polarized light. Figure 3(d–f) display photonic band structures for the non-primitive unit cells. The vertical axes in the figures represent normalized frequencies (a/λ). As all unit cells belong to the same 2D PC, the correspond- ing bandgaps remains exactly the same. The unit cell length of the non-primitive unit cell is Ra, and its’ Brillouin zone (BZ), as shown in Fig. 3(b), is shrunk R times in each direction. Therefore, the corresponding photonic band structures of the non-primitive unit cells [Fig. 3(d–f)] can be obtained by folding the original bands of the primitive cell [Fig. 3(c)], R2 times into the BZs of the non-primitive unit cells6,49. In other words, each band in the band structure of the primitive unit cell, becomes R2 folded bands in the band structure of the non-primitive unit cell. For a conventional PC, it is well known that the group velocities for the band edge modes are small, and the bands flatten towards the photonic bandgap frequencies. Therefore, the folded bands of the conventional PC in the proximity of photonic bandgaps are flatter [for example, see the folded band diagrams of Fig. 3(d–f)]. The mode pattern corresponding to the band edge mode is basically a standing wave that extends through the entire 2D lattice5. Thus, the band edge modes of the conventional PC are not spatially localized. h Note that the folded band structure of the conventional PC for R > 1 [Fig. Scientific Reports \| 6:31620 \| DOI: 10.1038/srep31620 Results f Before we discuss the optical properties of the 2D structures created from MLs, let us briefly review the band structure of the conventional 2D square lattice photonic crystal (PC) with the period a. For the sake of numerical illustration, assume the PC is made of circular silicon rods (refractive index, n = 3.4, radii of the rods equal to 0.15a) in an air ambience. Figure 3(a) shows the schematic of the non-primitive unit cell of the 2D PC with the length Ra. Rod PC with a square lattice is favorable for the transverse magnetic (TM) polarization [electric field along the axis of the rod]5. The photonic band structure of the 2D PC for light wave with the TM polarization, can be obtained by solving Maxwell’s equations using a plane wave expansion methodology47–49 (see the methods Scientific Reports \| 6:31620 \| DOI: 10.1038/srep31620 2 www.nature.com/scientificreports/ Figure 2. The relative positions of the two unique primitive unit cells for the case of R = 3 [primitive unit cell of Fig. 1(c) is shown in dark green color, and primitive unit cell of Fig. 1(d) is shown in purple color]. Figure 2. The relative positions of the two unique primitive unit cells for the case of R = 3 [primitive unit cell of Fig. 1(c) is shown in dark green color, and primitive unit cell of Fig. 1(d) is shown in purple color]. sections, for the details of the calculation). Figure 3(c) shows the result of calculation for a primitive unit cell (R = 1), and as we can see clearly see, the PC exhibits a large bandgap between the bands 1 and 2 for the TM polarized light. Figure 3(d–f) display photonic band structures for the non-primitive unit cells. The vertical axes in the figures represent normalized frequencies (a/λ). As all unit cells belong to the same 2D PC, the correspond- ing bandgaps remains exactly the same. The unit cell length of the non-primitive unit cell is Ra, and its’ Brillouin zone (BZ), as shown in Fig. 3(b), is shrunk R times in each direction. Therefore, the corresponding photonic band structures of the non-primitive unit cells [Fig. 3(d–f)] can be obtained by folding the original bands of the primitive cell [Fig. 3(c)], R2 times into the BZs of the non-primitive unit cells6,49. Results f The number of flat bands in ML increases as R increases, and the bandwidth (frequency span of the band) of each flat band decreases as R increases.hl l Flat band has a vanishing group velocity, and it is the key signature of a slow mode50–54. Thus, the dense bands shown in Fig. 4 guarantees a varieties of localized modes in MLs. Figure 5 illustrates the localized m Scientific Reports \| 6:31620 \| DOI: 10.1038/srep31620 3 www.nature.com/scientificreports/ Figure 3. (a) Non-primitive unit cell of the square lattice with a length Ra. (b) BZ (dashed line) of the square lattice. The dark solid line represents the irreducible part of the BZ. Photonic band structures [TM polarization] for the 2D square lattice PC made of silicon rods in air matrix, for various unit cell lengths (c) R = 1 [orange] (d) R = 3 [green] (e) R = 5 [red] (f) R = 7[blue]. In (d) to (f), the band structures in the orange color is for R = 1 [reproduced from (c) for a comparison]. Figure 3. (a) Non-primitive unit cell of the square lattice with a length Ra. (b) BZ (dashed line) of the square lattice. The dark solid line represents the irreducible part of the BZ. Photonic band structures [TM polarization] for the 2D square lattice PC made of silicon rods in air matrix, for various unit cell lengths (c) R = 1 [orange] (d) R = 3 [green] (e) R = 5 [red] (f) R = 7[blue]. In (d) to (f), the band structures in the orange color is for R = 1 [reproduced from (c) for a comparison]. Figure 4. Photonic band structures of the merged lattices [blue] for R = 3, 5, 7 and 9. The band structures in the orange color are the folded band structures of the conventional square lattice PC with a non-primitive unit cell of length Ra [see Fig. 3(c–f)]. Figure 4. Photonic band structures of the merged lattices [blue] for R = 3, 5, 7 and 9. The band structures in the orange color are the folded band structures of the conventional square lattice PC with a non-primitive unit cell of length Ra [see Fig. 3(c–f)]. Figure 4. Photonic band structures of the merged lattices [blue] for R = 3, 5, 7 and 9. Results f The band structure the orange color are the folded band structures of the conventional square lattice PC with a non-primitive un cell of length Ra [see Fig. 3(c–f)]. patterns [mode filed density] for R = 7 at the Γ point of the bands (R2 + 1) to (R2 + 42). As we can see from this figure, ML creates variety of configurations that enables localization of light. Quite clearly, the breaking of trans- lational symmetry in the ML (on the spatial scale <Ra), allows creation of multiple scattering loops within its primitive unit cell, and therefore realizes a large number of wave localization states. The density of the localized modes in ML scales as a function of R. As R increases, the spatial region of the broken translational symmetry, enlarges. This creates more opportunities for light localization. patterns [mode filed density] for R = 7 at the Γ point of the bands (R2 + 1) to (R2 + 42). As we can see from this figure, ML creates variety of configurations that enables localization of light. Quite clearly, the breaking of trans- lational symmetry in the ML (on the spatial scale <Ra), allows creation of multiple scattering loops within its primitive unit cell, and therefore realizes a large number of wave localization states. The density of the localized modes in ML scales as a function of R. As R increases, the spatial region of the broken translational symmetry, enlarges. This creates more opportunities for light localization. Scientific Reports \| 6:31620 \| DOI: 10.1038/srep31620 4 www.nature.com/scientificreports/ Figure 5. Mode field density (\|E\|2) at the Γ point of the merged square lattice with R = 7. The mode patterns correspond to the flat bands R2 + 1 to R2 + 42, and plotted for one primitive unit cell. The boxed pair of modes corresponds to the pair of bands with the degenerate frequencies. Figure 5. Mode field density (\|E\|2) at the Γ point of the merged square lattice with R = 7. The mode patterns correspond to the flat bands R2 + 1 to R2 + 42, and plotted for one primitive unit cell. The boxed pair of modes corresponds to the pair of bands with the degenerate frequencies. Using group theory, the eigenmode at every band, for any given every k vector, can be uniquely identified with an irreducible representation (IR) of the symmetry group of the k vector49,55–58. Scientific Reports \| 6:31620 \| DOI: 10.1038/srep31620 Results f In the reciprocal space, the high symmetrical k vectors of the square lattice are Γ Χ π( ) (0, 0), , 0 , Ra and π π ( ) M , Ra Ra [see Fig. 3(b)]. In Schoenflies notation55,56, the symmetry group for these k vectors are respectively, C4v, C2v, and C4v. The symmetry representa- tion for the group C4v consists four non-degenerate IRs, and one doubly degenerate IR. On the other hand, the symmetry representation for the group C2v consists four non-degenerate IRs. As any band is a continuous path (or a surface) in the reciprocal space, the adjacent bands are bound to touch each other if the bands possesses any degenerate IR at the symmetrical k vectors. In the square lattice, such touching will only occur at Γ and Μ points, because only these k vectors have doubly degenerate IRs. The degenerate modes at the Γ point of the discussed ML are boxed together in Fig. 5.hlh g g The knowledge on the degeneracy, allows us to categorize the flat bands into two important categories. The first category is flat bands with no degenerate points, and the second category is flat bands with at least one degen- erate point. These two genre of flat bands have distinct mode dispersion. In general, the first category of flat bands displays more symmetrical mode than the flat bands from the second category. In Fig. 6, we illustrate examples of mode profiles for the two genre of flat bands.lih pi gl For an example of flat band with no degenerate points [i.e., the first type], let us take the R2 + 1 –th band. The enlarged plot of the frequency dispersion for R = 7 is shown in Fig. 6(a) [green color], and the corresponding mode profiles at the symmetrical k vectors are shown in Fig. 6(b). As can be seen from Fig. 6(b), the first genre of Scientific Reports \| 6:31620 \| DOI: 10.1038/srep31620 5 reports/ gure 6. (a) Left: Enlarged version of the band structure shown in Fig. 4 for R = 7. Right: Two different genre flat bands. Results f The first genre is flat band with no degenerate points [for example, + R 1 2 –th band (shown in een)], and the second is flat band with at least one degenerate point [for example, the R2 + 8 –th and R2 + 9 –th nd (shown in red) touches each other at the Γ point]. (b) Mode pattern along the first genre of flat bands 2 + 1 –th band is shown for an illustration]. (c) Mode pattern along the second genre of flat bands [R2 + 8 –th nd R2 + 9 –th bands are shown for an illustration]. www.nature.com/scientificreports/ Figure 6. (a) Left: Enlarged version of the band structure shown in Fig. 4 for R = 7. Right: Two different genre of flat bands. The first genre is flat band with no degenerate points [for example, + R 1 2 –th band (shown in green)], and the second is flat band with at least one degenerate point [for example, the R2 + 8 –th and R2 + 9 –th band (shown in red) touches each other at the Γ point]. (b) Mode pattern along the first genre of flat bands [R2 + 1 –th band is shown for an illustration]. (c) Mode pattern along the second genre of flat bands [R2 + 8 –th and R2 + 9 –th bands are shown for an illustration]. flat bands exhibits a nearly same mode profile (i.e., a small dispersion) along the k vector path. The electric field of the R2 + 1 –th band is sharply confined in the center of the unit cell (almost entirely in one rod). Figure 7(a) shows the evolution of the mode profile [R2 + 1 –th band, Γ point] as a function of R. In Fig. 7(b), we show the horizontal (x) cross section of the mode field density for various R. Figure 7(b) shows that the mode radius remains the same for all R. The calculated mode area is 0.34 λ( ) n 2. As we have said earlier, the flatness of the band increases as R increases, and this is quantified in Fig. 7(c) for the R2 + 1 –th band. As we can clearly see from the Scientific Reports \| 6:31620 \| DOI: 10.1038/srep31620 6 www.nature.com/scientificreports/ Figure 7. (a) Mode field density of the + R 1 2 –th band (Γ point). Results f (b) The horizontal {x} cross section of the mode field density for various R. (c) The frequency span (band width) of the R2 + 1 –th band as a function of R. Figure 7. (a) Mode field density of the + R 1 2 –th band (Γ point). (b) The horizontal {x} cross section of the mode field density for various R. (c) The frequency span (band width) of the R2 + 1 –th band as a function of R. figure, the bandwidth (frequency span of the band) decreases almost one order, as R reduces from 3 to 9. As R increases, the length of the primitive unit cell increases, and therefore the evanescent coupling of localized to the adjacent unit cells decreases. This in turn results in a flatter dispersion50. A flatter band exhibits mode with higher quality factor. Given a fixed mode volume (area in 2D), and the higher quality factor as R increases, enhances Purcell factor and creates a favorable condition for variety of application in quantum computing and cavity quan- tum electrodynamics44,59. y Let us consider the R2 + 8 and R2 + 9 –th bands [Fig. 6(a) {red color}]. These pair of bands are degenerate at the Γ point, and hence belongs to the second genre of flat bands. The mode profiles of these bands at the Γ point consists of two independent eigenmodes as shown in the box A of Fig. 6(c). These degenerate mode splits along the path, Γ–X. The modes profiles at the X point are indicated as B’ and B for bands 8 and 9, respectively [see Fig. 6(a,c)]. As stated earlier, Γ point has C4v point group symmetry, and therefore the mode must be invar- iant with respect to all symmetry operations of the group C4v. However, the individual modes in box A, on its own, does not have the C4v symmetry [for example, the π/2 rotational symmetry is missing]. Thus, by symmetry requirement the two modes in box A must co-exist (i.e., degenerate). These individual mode possess a C2v symme- try, and when they split along the path Γ–X, this symmetry is retained. This splitting is perfectly consistent with the symmetry requirement at point X, which has the C2v symmetry.h y y q p 2v y y Figure 8 highlights a special group of modes in the ML that have ring shapes. Scientific Reports \| 6:31620 \| DOI: 10.1038/srep31620 Results f These modes are localized in the closed path shown in Fig. 8(a). As oppose to the usual ring modes which are trapped in the dielectric region, these ring modes [Fig. 8(b–d)] in the ML are trapped in the air region. Therefore, these ring modes are robust with respect to material dispersion, and hence suitable for variety of applications in non-linear optics. Furthermore, the periodic nature [on the longer spatial scale] of the ML, creates a two dimensional array of such ring modes, similar to a two dimensional array of ring resonators60.f y g Wave localization states in a ML are different from the band edge modes of a conventional PC [although both have vanishing group velocities]. Unlike the band edge modes of the conventional PC (for which the standing Scientific Reports \| 6:31620 \| DOI: 10.1038/srep31620 7 www.nature.com/scientificreports/ Figure 8. (a) The primitive unit cell for R = 7. The orange arrow shows the closed path for light localization. (c–d) The ring type of modes in the closed path of (a), correspond to the bands (b) 38 (c) 39, and (d) 42. Figure 8. (a) The primitive unit cell for R = 7. The orange arrow shows the closed path for light localization. (c–d) The ring type of modes in the closed path of (a), correspond to the bands (b) 38 (c) 39, and (d) 42. wave modes extend through the entire lattice), the modes of MLs are spatially localized with smaller modal volumes (areas in 2D). For the conventional PC, the band edge mode is a result of coherent interference effects arising from the periodic arrangement of the scatters (i.e., Bragg scattering), and hence, periodicity is an essen- tial prerequisite in observing the band edge modes. However, in the ML, flat band occur as a consequence wave localization due to the breaking in the translational symmetry, resulting from the merging. The periodicity Ra, is thus, not required to observe the localized modes of the ML (as they are not resulting from Bragg scattering).h q y g gg g The modes of ML are well localized within the primitive unit cell, and only the evanescent tail couples to the adjacent unit cell. Therefore, a single unit cell is sufficient to spatially confine light in ML. In fact, the mode pat- terns in Fig. Results f 5 can be reproduced using a single primitive unit cell of the ML, assuming a non-periodic boundary condition such as perfectly matching layer boundary condition61.h p y g y y The primitive unit cell of the ML also can be visualized as an optical resonator that supports large number of localized modes with finite and large lifetimes. The photonic band structure [Figs 4 and 6] is then, can be regarded as a display of the dispersion of these modes, when identical resonators arranged in a periodic fashion (i.e., the dispersion of the slow mode of the coupled optical resonators50). The photonic band structure also shows, how the degenerate mode of the optical resonator splits when identical resonators arranged in periodic fashion [for instance, see Fig. 6(c)]. Methodsh The photonic band structures [Figs 3, 4 and 6(a)], and the mode patterns [Figs 5, 6(b,c), 7(a) and 8(b–d)] are calculated using a plane wave expansion methodology. This method assumes a periodic boundary condition for the unit cell of the dielectric function, and Bloch theorem for the electric field. In the method, both electric field and the periodic dielectric functions are expanded in terms of plane waves, and substituted in the Maxwell’s equations. The resulting equations are combined as a matrix eigenvalue problem. In this paper, we used the freely available plane wave expansion solver, MPB48. The photonic band structure of the conventional lattice is obtained using 1024 plane waves. On the other hand, the photonic band structures, and the mode patterns of the MLs are obtained by employing (nreslR)2 plane waves, where nresl = 64 is the resolution parameter used in MPB. 8 Scientific Reports \| 6:31620 \| DOI: 10.1038/srep31620 www.nature.com/scientificreports/ Conclusion In this article, we have presented the principles of two dimensional merged lattice constructions. Using light wave in square lattice as an example, we have demonstrated the salient wave localization properties of the merged lattice. We show, how the photonic band structure of the merged lattices evolves from the corresponding folded band structure of the conventional photonic crystals. The destruction of the translational symmetry induces strong coupling between the folded bands, and results in dense number of flat bands with variety of localized mode profiles. The mode profiles include sharp localized modes with small mode volumes, and ring modes trapped in the annular air region. pp g Although, the periodicity of merged lattice on the longer spatial scale is not required to see the localized modes of the merged lattice, the natural periodicity that it has, is certainly an added advantage. 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https://openalex.org/W1764825378	https://bmcpulmmed.biomedcentral.com/counter/pdf/10.1186/1471-2466-14-202	English	null	Expiratory flow rate, breath hold and anatomic dead space influence electronic nose ability to detect lung cancer	BMC pulmonary medicine	2,014	cc-by	7,062	Keywords: Biomarkers, Breath test, Electronic nose, Lung cancer, Volatile organic compound Breath tests have unique advantages that they are completely non-invasive, well-tolerable, hold no risks for side effects and can be performed even in very sick pa- tients; therefore they pose an ideal tool for disease screening. This fact is particularly important in lung cancer, as early diagnosis is associated with significantly better prognosis [22]. © 2014 Bikov et al.; licensee BioMed Central. 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 credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Bikov et al. BMC Pulmonary Medicine 2014, 14:202 http://www.biomedcentral.com/1471-2466/14/202 Bikov et al. BMC Pulmonary Medicine 2014, 14:202 http://www.biomedcentral.com/1471-2466/14/202 Open Access Expiratory flow rate, breath hold and anatomic dead space influence electronic nose ability to detect lung cancer Andras Bikov1, Marton Hernadi2, Beata Zita Korosi1, Laszlo Kunos1, Gabriella Zsamboki1, Zoltan Sutto1, Adam Domonkos Tarnoki3, David Laszlo Tarnoki3, Gyorgy Losonczy1 and Ildiko Horvath1 Correspondence: andras.bikov@gmail.com 1Department of Pulmonology, Semmelweis University, 1/C Dios arok, Budapest 1125, Hungary Full list of author information is available at the end of the article Abstract Background: Electronic noses are composites of nanosensor arrays. Numerous studies showed their potential to detect lung cancer from breath samples by analysing exhaled volatile compound pattern (“breathprint”). Expiratory flow rate, breath hold and inclusion of anatomic dead space may influence the exhaled levels of some volatile compounds; however it has not been fully addressed how these factors affect electronic nose data. Therefore, the aim of the study was to investigate these effects. Methods: 37 healthy subjects (44 ± 14 years) and 27 patients with lung cancer (60 ± 10 years) participated in the study. After deep inhalation through a volatile organic compound filter, subjects exhaled at two different flow rates (50 ml/sec and 75 ml/sec) into Teflon-coated bags. The effect of breath hold was analysed after 10 seconds of deep inhalation. We also studied the effect of anatomic dead space by excluding this fraction and comparing alveolar air to mixed (alveolar + anatomic dead space) air samples. Exhaled air samples were processed with Cyranose 320 electronic nose. Results: Expiratory flow rate, breath hold and the inclusion of anatomic dead space significantly altered “breathprints” in healthy individuals (p < 0.05), but not in lung cancer (p > 0.05). These factors also influenced the discrimination ability of the electronic nose to detect lung cancer significantly. Conclusions: We have shown that expiratory flow, breath hold and dead space influence exhaled volatile compound pattern assessed with electronic nose. These findings suggest critical methodological recommendations to standardise sample collections for electronic nose measurements. Keywords: Biomarkers, Breath test, Electronic nose, Lung cancer, Volatile organic compounds Background Exhaled breath contains thousands of volatile molecules and their levels change with cellular metabolism and oxidative stress [1]. Therefore, it is not surprising that altered exhaled volatile compound levels were found in various disorders of the respiratory system such as lung cancer [2-9], malignant mesothelioma [10,11], obstruct- ive airway diseases [12-14], sarcoidosis [15], obstructive sleep apnoea [16-18], respiratory infections [19,20] and in patients with lung transplantation [21]. Gas-chromatography mass-spectometry (GC-MS) is the gold standard for exhaled volatile compound meas- urement. However, these machines are very expensive and need special skills and experience. Electronic noses, composites of nanosensor arrays and in-built processors, represent another, generally cheaper and easier tech- nique to measure exhaled volatile compounds [5]. These * Correspondence: andras.bikov@gmail.com 1Department of Pulmonology, Semmelweis University, 1/C Dios arok, Budapest 1125, Hungary Full list of author information is available at the end of the article Bikov et al. BMC Pulmonary Medicine 2014, 14:202 http://www.biomedcentral.com/1471-2466/14/202 Page 2 of 9 Page 2 of 9 disease. In smokers COPD has been excluded with lung function test. devices cannot identify and quantify the molecules in gas mixtures but are able to compare and discriminate gaseous samples based on the contour of volatile sub- stances (“breathprint”). In line with this, electronic noses could distinguish exhaled breath samples of patients with different respiratory disorders from those of healthy subjects [23-25]. All patients with lung cancer were current (n = 15) or ex-smokers (n = 12) with pack years of 60 ± 27. Lung cancer subjects were enrolled after the diagnosis has been established based on histology obtained either by endo- bronchial or transthoracic biopsy. Seventeen lung cancer patients had adenocarcinoma, 5 had squamous cell carcin- oma, 3 had small cell carcinoma, 1 large cell carcinoma, 1 carcinoid and 1 sarcoma. Fourteen subjects were diag- nosed newly and they did not receive any oncological treatment while 13 were receiving active chemotherapy. None of the lung cancer patients had asthma, allergy, renal of hepatic disorder, 12 patients were treated with COPD, and 4 with diabetes. All patients with COPD were using long-acting muscarin antagonists and 5 of them were on inhaled corticosteroids in combination with long- acting beta agonists. Lung cancer is probably the most frequently investi- gated respiratory disorder in electronic nose research. Numerous studies demonstrated that various electronic noses could identify lung cancer by analysing exhaled breath samples [5-9]. Study design All b All subjects were asked to perform four different breath collection procedures. Participants inhaled VOC-filtered room air with a deep inspiratory capacity manoeuvre then exhaled at a controlled flow rate (50 ml/sec) assessed with a flow-meter (VenThor, Thor Laboratories, Hungary) and, to close the soft palate, against resistance (15–20 cmH2O). The first 500 mL of exhaled air representing anatomic dead space was discarded using a small-resistance T-valve and the remaining air representing alveolar space was col- lected in a Teflon-coated Mylar bag. This method has originally been used for offline exhaled nitric oxide mea- surements (Ecomedics, Dürnten, Switzerland), but was standardised for VOC analyses by our workgroup and was shown to be reproducible within a day [40] and over 8 weeks [21]. The measurements with electronic nose were performed immediately after the collection. After at- tachment of a volatile molecule, sensors in Cyranose 320 respond with changes in resistance. After auto scale nor- malisation, sensor responses (dR) are calculated by the fol- lowing formula: dR = (Rs-R)/R, where Rs is the response to the sampled gas and R is the response to the baseline reading, the reference gas being the VOC-filtered ambient room air. Therefore, the aim of this study was to analyse the ef- fect of expiratory flow rate, breath hold and anatomic dead space on exhaled breath volatile compound pattern in lung cancer and health assessed with Cyranose 320. Background Cyranose 320 is one of the most widely used electronic noses and it applies a carbon black conducting polymer sensor array which is selective for polar compounds and its lower detection limit is around 0.1 ppm (particles per million) [26]. Numerous volatile substances were identified as being altered in lung cancer in this concentration range, such as acetone [3,4,27,28], isoprene [3,4,27,29], benzene [2], xylene [3], pentane [30], ethanol [28] and methanol [4]. Nonethe- less, methanol, isoprene [31] and benzene [3,30,31] were associated with smoking itself. Nevertheless, the discrim- ination potential of Cyranose 320 to detect lung cancer was reported to be good [6,7,9]. None of the volunteers had respiratory tract infection 4 weeks prior to the study. All subjects were instructed to avoid consuming food and beverages [38] and not to perform physical exercise [39] at least for 2 hours before sample collection. Current smokers were asked to re- frain from smoking for at least 6 hours prior to the measurements. Unfortunately, no international guidelines exist for sampling methodology for electronic nose analyses. Some of the collection-related factors, such as the effect of en- vironmental volatile substances [32], humidity [33,34] or sample storage [35] were addressed [24]. Alveolar gases may be influenced by breath hold (the period between in- halation and exhalation) and airway-borne substances are affected by expiratory flow rate [36]. In addition, in the in- vestigation of lower airway molecules, the exclusion of anatomical dead space should also be standardised. It is not surprising that expiratory flow rate, the presence of breath hold and the inclusion of anatomic dead space may affect the levels of exhaled volatile compounds [33,37,38], however in electronic nose research, only the effect of ex- piratory flow rate has been investigated, but only in a small group of healthy subjects [14]. Effects of expiratory flow, breath hold and dead space on “breathprint” Expiratory flow rate, breath hold and dead space signifi- cantly altered exhaled “breathprints” only in healthy in- dividuals (p < 0.05) but not in patients with lung cancer (p > 0.05, Figure 1). Analysing individual sensor responses in healthy par- ticipants, expiratory flow rate affected all but sensors 2, 3, 7, 9, 19, 23, 27 and 32, breath hold changed all sensor responses, while dead space altered all but sensors 1, 12 and 13 significantly (p < 0.05). In patients with lung can- cer, breath hold affected only sensor 29, while dead space altered responses of sensors 27 and 29 significantly (p < 0.05). Statistical analysis SPSS 15.0 (SPSS Inc., Chicago, IL, USA), GraphPad Prism 5.03 (GraphPad Software Inc., San Diego, CA, USA) and Statistica 8.0 software (Stat Soft, Inc., Tulsa, OK, USA) were used for statistical analysis. To avoid the confounding effect of water vapour the four water sensi- tive sensors (sensors 5, 6, 23, 31) were excluded, thus in total 28 sensors were analysed. To reduce data dimen- sionality principal component analysis (PCA) was per- formed on sensor responses, principal components (PCs) were sorted based on their initial Eigen value sizes and the highest three (PC1, PC2 and PC3) were used for further analysis. To classify cases into categorical divisions, linear canonical discriminant analysis was used following a step- wise approach, where Mahalanobis-distance was applied to exclude outliers [41]. Leave-one-out cross validation was also performed with Mahalanobis distance followed by lin- ear canonical discriminant analysis where the same dataset was used as the validation as well as the training groups. Pearson correlation, multivariate linear and logistic regres- sion analyses were performed to detect relationships be- tween clinical data and PCs. Methods Subjects Thirty-seven healthy individuals (44 ± 14 years, mean ± SD) and 27 patients with lung cancer (60 ± 10 years) par- ticipated in the study. Twenty-seven healthy subjects were never-smokers and 10 were active smokers (31 ± 17 pack years). None of them have any chronic disorder, in- cluding asthma, COPD, allergy, diabetes, renal or hepatic To study the effect of expiratory flow rate, breath hold and dead space the previously described procedure was altered. Assessing the effect of expiratory flow rate sub- jects exhaled at 75 ml/sec which was compared to Bikov et al. BMC Pulmonary Medicine 2014, 14:202 http://www.biomedcentral.com/1471-2466/14/202 Page 3 of 9 Page 3 of 9 breath samples obtained at 50 ml/sec. To study the effect of breath hold, after deep inhalation through a VOC-filter, subjects held their breath at total lung cap- acity for 10 seconds and exhaled at 50 ml/sec. Finally, subjects performed an expiratory manoeuvre similar to the first one, but this time the dead space air was not discarded and mixed air was collected. and a power of 0.80 the minimal estimated sample size was N = 44 [42]. However, because of the considerable within group variability of the clinical data in patients with lung cancer, to improve the power of our results, we decided to exceed the minimally required sample size by enrolling 20 additional subjects. Our secondary aim was to compare healthy and lung cancer groups when breath samples were collected with different collection setups. Post hoc power analysis based on a previous study [13] revealed that the stand- ard error for correct classification between the healthy and lung cancer groups was below 6% for all compari- sons. A p < 0.05 was considered significant. The study was approved by the University Ethics Committee (Semmelweis University, TUKEB 110/2007), and all subjects signed informed consent form prior to the measurements. The effect of sample collection on the ability of electronic nose to detect lung cancer We found a significant difference between exhaled “breathprints” in healthy subjects and patients with lung cancer when the previously standardised collection setup (50 ml/sec, no breath hold and the exclusion of dead space) was used (p = 0.02, Figure 2). The electronic nose could discriminate the two groups with a classification accuracy of 72%, cross-validated accuracy of 70%, 63% sensitivity, 78% specificity, 63% positive predictive value and 78% negative predictive value. When only healthy smokers were compared to patients with lung cancer, the difference was still significant (p = 0.01, classification accuracy of 81%, 96% sensitivity, 40% specificity, 81% positive predictive value, 80% negative predictive value). Similarly, the difference was significant (p < 0.001) also when healthy never-smokers were compared to patients (classification accuracy of 74%, 67% sensitivity, 81% spe- cificity, 78% positive predictive value, 71% negative pre- dictive value). The primary aim of this study was to investigate whether collection-related factors, including expiratory flow rate, breath hold and exclusion of anatomic dead space influ- ence the breathprints of healthy subjects and patients with lung cancer. Repeated-measures ANOVA on principal components followed by the Dunnett’s post hoc test was applied to analyse this aim in two groups with 4 subse- quent measurements. Principal components were derived from sensor responses, therefore a priori calculations on effect size were based on descriptive statistics of sensor re- sponses in our previous cohorts [18,40]. In these studies, the average standard deviation/mean ratio of the analysed (N = 28, after the exclusion of water-sensitive sensors) sen- sor responses was 0.35. In addition, we showed that changes in exhalation flow rate and breath hold caused a 12% change in exhaled VOC levels [38]. Together these two (standard devi- ation/mean ratio of 0.35 and a change of 0.12) result an effect size of 0.35. Using an effect size of 0.35 and inves- tigating two groups with 4 subsequent measurements When the healthy and lung cancer groups were com- pared at higher expiratory rate, the difference was signifi- cant with classification accuracy of 78%, cross-validated accuracy of 78%, 81% sensitivity, 76% specificity, 71% Bikov et al. BMC Pulmonary Medicine 2014, 14:202 http://www.biomedcentral.com/1471-2466/14/202 Page 4 of 9 Page 4 of 9 Figure 1 The effect of expiratory flow, breath hold and dead space on principal components. The effect of sample collection on the ability of electronic nose to detect lung cancer The collection-related factors caused significant differences only in healthy subjects (p < 0.05), the effect was not significant in patients with lung cancer (p > 0.05). Comparing to baseline measurements both higher expiratory flow and breath hold caused significant changes in PC1, PC2, PC3, while the inclusion of anatomic dead space affected only PC1. Data are expressed as mean ± SD. -p < 0.05, -p < 0.01, *-p < 0.001, NS-not significant. Figure 1 The effect of expiratory flow, breath hold and dead space on principal components. The collection-related factors caused significant differences only in healthy subjects (p < 0.05), the effect was not significant in patients with lung cancer (p > 0.05). Comparing to baseline measurements both higher expiratory flow and breath hold caused significant changes in PC1, PC2, PC3, while the inclusion of anatom dead space affected only PC1. Data are expressed as mean ± SD. -p < 0.05, -p < 0.01, -p < 0.001, NS-not significant. 64% positive predictive value and 75% negative predictive value. positive predictive value and 85% negative predictive value. The difference after 10 seconds of breath hold was also significant with classification accuracy of 70%, cross- validated accuracy of 70%, 78% sensitivity, 65% specificity, 62% positive predictive value and 80% negative predictive value. Although, the difference after inclusion of the dead space was still significant, the discrimination power was the poorest with classification accuracy of 70%, cross- validated accuracy of 69%, 67% sensitivity, 70% specificity, The effects of lung function, age and smoking history on “breathprint” The effects of lung function, age and smoking history on “breathprint” We found significant correlations between PC2 and FEV1 and PC2 and FEV1/FVC (r = 0.50 and r = 0.41, p < 0.05, Figure 3A) as well as PC3 and age (r = 0.56, p < 0.05, Figure 3B) in lung cancer subjects. Contrarily, there was no relationship between “breathprint” and lung function or “breathprint” and age in healthy subjects. Analysing individual sensors, we did not find significant relationship between sensor responses and lung function, age or smoking history in either group of subjects. there is still an unmet need for international guidelines for sampling methodology in electronic nose analyses. In this study we investigated collection-related methodo- logical factors in an attempt to improve the standardisa- tion of the sampling technique for electronic nose studies. The expiratory flow rate might influence the breath level of molecules originating from the conducting air- ways. On one hand, the levels of volatile molecules with a steady axial diffusion, such as nitric oxide, may decrease at higher expiratory flow rate [36,43]. On the other hand, if a VOC is taken up by the airway tissue, higher expira- tory flow rate does not allow rapid diffusion towards the airway vessels and it is associated with elevated breath VOC levels [37]. Of note, if a molecule is released in the conducting airways it does not rule out the possibility that it also originates from the more distant part of the lungs. Flow-dependence of different VOCs has already been demonstrated [33,37,38,44]. However, the airway kinetics of various volatile substances is not fully understood. It is known from experimental studies on nitric oxide that the To exclude the potential confounding effect of COPD, comparison between healthy and lung cancer groups was also performed when only non-COPD lung cancer subjects were included, yielding still significant differ- ences (p = 0.03, 60% sensitivity, 63% specificity). “Breath- prints” of healthy smokers were significantly different from healthy never smokers (p = 0.01), while pack years were not related to “breathprints” in any group of subjects. The effect of tumour histological type and active chemotherapeutic treatment on “breathprint” When comparing various histological subtypes, no dif- ference was observed in “breathprints” (p > 0.05). Simi- larly, there was no difference in “breathprints” between Page 5 of 9 Bikov et al. BMC Pulmonary Medicine 2014, 14:202 http://www.biomedcentral.com/1471-2466/14/202 Figure 2 Two dimensional PCA plot between healthy subjects (squares) and patients with lung cancer (circles). Electronic nose could discriminate the two groups with a classification accuracy of 72% when breath samples were collected with a previously standardised collection procedure. The difference was significant (p = 0.02). Figure 3 Relationships between “breathprint” and FEV1/FVC as well as age. Significant associations were found between PC2 and FEV1/FVC (p = 0.03, r = 0.41, Panel A) as well as between PC3 and age (p = 0.002, r = 0.56, Panel B). Figure 2 Two dimensional PCA plot between healthy subjects (squares) and patients with lung cancer (circles). Electronic nose could discriminate the two groups with a classification accuracy of 72% when breath samples were collected with a previously standardised collection procedure. The difference was significant (p = 0.02). patients receiving active chemotherapy and treatment- naive patients (p > 0.05). Figure 3 Relationships between “breathprint” and FEV1/FVC as well as age. Significant associations were found between PC2 and FEV1/FVC (p = 0.03, r = 0.41, Panel A) as well as between PC3 and age (p = 0.002, r = 0.56, Panel B). Discussion Of course, to understand the kinetics of VOC production in healthy and patho- logical airways more than two expiratory flow rates must be tested, and VOC levels should be measured with more precise instruments (i.e. GC-MS). The aim of the current study was only to demonstrate the importance of expira- tory flow rate control. Nevertheless, we found that collection-related factors influenced not only “breathprints” of healthy subjects but the differences between the two groups. Namely, the difference between healthy and lung cancer group wid- ened when breath samples were obtained at higher expiratory flow rates. This further highlights the import- ance of sampling standardization, but also suggests pos- sible opportunities to improve the classification accuracy of electronic nose. We used a leave-one out cross- validation method to test the discrimination ability of different collection setups. This technique has a certain limitation that the same dataset was used as the valid- ation as well as the training groups. Therefore, the im- proved discrimination ability has to be tested blindly in future studies enrolling external group of new patients. Unfortunately, there is no consensus which is the best statistical approach for the classification of electronic nose data. A recent study highlighted that linear discriminant analysis, used also in the current study has a superior pre- diction accuracy, sensitivity and specificity in comparison to the other techniques, including partial least squares- discriminant analysis and random forests [48]. In general, if subjects are not instructed properly, they may provide a breath sample after various times of breath hold [33]. The breath levels of molecules which are produced in the acinar airway/alveolar space or the ones which diffuse through the alveoli are influenced by breath hold. The longer the breath hold is, the more molecules accumulate in the airways resulting in higher breath levels [33,36-38,45]. The effect of dead space air on exhaled volatile com- pound concentrations is two-sided. Firstly, if a molecule is produced in the lower airways but not in the upper part, its concentrations may be diluted by the dead- space air. Secondly, some molecules may be produced in the upper airways influencing the results [46]. Therefore, comparison of alveolar (dead space excluded) and mixed air (dead space included) samples may reveal the origin of volatile molecules [27,47]. Discussion Electronic noses represent potential non-invasive tools for the early diagnosis of lung cancer. Unfortunately, Bikov et al. BMC Pulmonary Medicine 2014, 14:202 http://www.biomedcentral.com/1471-2466/14/202 Page 6 of 9 and acetone [38,44] concentrations and the inclusion of anatomic dead space may affect acetone levels [27]. Hence, it was not surprising that these factors altered “breathprints” significantly in the current study. Analysing the raw sensor data, collection-related factors altered dif- ferent sensor responses suggesting that they may affect various volatile molecules differently. Interestingly, these effects were present only in healthy individuals. Various studied concluded decreased VOC levels in lung cancer compared to non-lung cancer control groups [2,4]. This may imply that VOC levels in lung cancer were below the lower detection limit of electronic nose (0.1 ppm); there- fore the lack of changes in lung cancer group was not due to physiological but analytical reasons. On the other hand, we have previously shown that expiratory flow depend- ency of VOCs may not be present in all subjects. Exhaled ethanol concentration in healthy volunteers with very low baseline levels was not affected by higher exhalation flow rate [38]. In addition, as some of the lung cancer subjects had also COPD, because of the possible airflow limitation, this might have been biased the kinetics of VOC produc- tion. This could contribute to the negative results in lung cancer patients. relationship between expiratory flow rate and exhaled volatile compound levels may resemble a hyperbolic curve [43]. In line with this, proportionally higher expiratory flow rates may result only in marginal differences in ex- haled volatile levels which might not be detected with electronic nose. Therefore, we decided to use two rela- tively slow expiratory flow rates in this study. In addition, these two relatively slow flow rates could have been achieved by all subjects, while our experience is that expir- ation with constant high flow-rates (>200 ml/sec) could be very struggling for very sick patients. Flow-dependency of exhaled volatile compound pattern has been investigated only in a preliminary experimental setup. In that study 100 to 200 ml/sec and 300 to 500 ml/sec expiratory flow rates were compared in 10 healthy non-smoker subjects without any significant difference [14]. However, the lack of difference may have been due to the relatively high tar- get rates applied in that study. Discussion Two possible approaches are introduced to avoid the problem of dead space; by partitioning the first proportion of exhaled air via a T- valve (time or volume-controlled separation) [46] or by monitoring the exhaled CO2 concentration and selecting the period when CO2 reaches the alveolar plateau (CO2- plateau controlled separation) [47]. A study comparing the two methods showed that the CO2-controlled method provides more precise results [27], however the time or volume-controlled separation which was used in the current study is simpler and more feasible. Theoretically, expiratory flow-, breath hold-, ant dead space-dependency of volatile substances may draw a con- clusion about the anatomic site of production (i.e. con- ducting airways, alveoli or oral cavity, etc.). Electronic noses analyse a pattern of molecules, and it is plausible that volatile compounds contributing to “breathprint” ori- ginate from various sites. Unfortunately, the electronic nose used in this study is not able to separate exhaled volatile molecules; therefore, further studies coupling It was shown that expiratory flow rate may alter acetone [44], ethanol [38], isoprene [33,37] and pentane [37] levels, breath hold may increase isoprene [33,37], methanol [44] Bikov et al. BMC Pulmonary Medicine 2014, 14:202 http://www.biomedcentral.com/1471-2466/14/202 Page 7 of 9 Page 7 of 9 electronic noses with GC-MS are warranted to identify biomarkers associated with sampling-related changes. can modify exhaled “breathprint” itself [13]. Previous stud- ies showed that exhaled “breathprint” differs in lung can- cer when it was compared to COPD [5,6]. Of note, in this study, the difference in lung cancer was still significant when non-COPD lung cancer patients were analysed. However, one may argue that in fact the results of the dis- crimination between lung cancer and controls will im- prove when a more homogeneous group is studied. To explore the potential of electronic nose in lung cancer screening further studies are warranted to understand possible confounding clinical factors. Similarly to previous studies [6,7] we found significant differences in “breathprint” between healthy subjects and patients with lung cancer. However these differences might not be related to the disease itself but to some other factors. For example, age and smoking history may alter exhaled molecules [13,18,24,49]. We found a sig- nificant correlation between “breathprint” and age only in lung cancer subjects, and we cannot completely ex- clude the possibility that the difference in age between healthy and lung cancer groups might have biased our results. Competing interests h h d l h The authors declare that they have no competing interests. Authors’ contributions AB participated in exhaled breath analysis and drafted the manuscript. MH and BZK carried out breath tests. LK, GZ, ZS, ADT and DLT participated in the design of the study and contributed to recruiting and clinically characterizing patients. GL contributed to the design and coordination of the study. IH conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript. Acknowledgements Th d The study was supported by Hungarian Scientific Research Fund (OTKA 68808) and Hungarian Respiratory Society grant (to Andras Bikov). The study was supported by Hungarian Scientific Research Fund (OTKA 68808) and Hungarian Respiratory Society grant (to Andras Bikov). Author details 1 Important issues which are needed to be investigated in future studies include histological subtype of lung cancer, the role of chemotherapy and accompanying dis- orders. Studies examining exhaled breath showed dif- ferences in lung cancer compared to control subjects irrespectively from histology [5-7]. Some of our patients were on active oncologic treatment. Similarly to the previ- ous study [5], electronic nose could classify lung cancer patients correctly disregarding their medical treatment history. In addition, there were no differences between the treated and untreated groups. However, this study has not been powered to analyse the effect of chemotherapy. COPD is frequently accompanied with lung cancer and it 1Department of Pulmonology, Semmelweis University, 1/C Dios arok, Budapest 1125, Hungary. 2Department of Pediatrics, Heim Pal Children’s Hospital, 86 Ulloi street, Budapest 1082, Hungary. 3Department of Radiology and Oncotherapy, Semmelweis University, 78/a Ulloi street, Budapest 1082, Hungary. Received: 12 October 2013 Accepted: 11 December 2014 Published: 16 December 2014 Conclusions In summary, expiratory flow rate, breath hold and ana- tomic dead space may affect “breathprints” significantly. These effects may also influence the classification accur- acy of electronic noses to separate disorders from health; by altering collection-related factors the discrimination ability may even be improved. Therefore, our study points to the need of methodological recommendations to standardise sample collection for electronic nose measurements. Abbreviations ANOVA: Analysis of variances; COPD: Chronic obstructive pulmonary disease; FEV1: Forced expiratory volume in 1 second; FVC: Forced vital capacity; GC- MS: Gas-chromatography mass-spectrometry; PC: Principal component; PCA: Principal component analysis; VOC: Volatile organic compound. Discussion Of note, previous studies showed that age does not influence the discrimination ability between healthy controls and patients with obstructive airway diseases [13,14]. Although smoking may change exhaled volatile substances [49], it was also shown that it did not affect the discrimination potential of exhaled volatile compound profile to detect lung cancer [7,23]. Confirming the previ- ous results [49], we also found a significant difference be- tween active-smoker and never-smoker healthy subjects. Most notably, similarly to previous reports [7,23], the dif- ference between healthy volunteers and lung cancer pa- tients was still present irrespectively whether the control group consisted of smokers or non-smokers. However, due to the potential effect of smoking, a comparison enrolling four groups of subjects (i.e. smoking healthy, non-smoking healthy, smoking patient and non-smoking patient) would be more accurate to exclude the potential effect of cigarette smoking. We found no difference in “breathprints” between active and ex-smoker lung cancer patients which was similar to the data found in COPD [12,13]. The relationship between lung function and “breathprint” is not fully understood. We found a signifi- cant association between “breathprint” and lung function in lung cancer, however other authors showed no relation- ship between acute changes in airway calibre and exhaled volatile compound pattern [50]. 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https://openalex.org/W4251284726	https://nhess.copernicus.org/articles/18/397/2018/nhess-18-397-2018.pdf	English	null	Brief communication: Post-seismic landslides, the tough lesson of a catastrophe	null	2,017	cc-by	4,785	Brief communication: Post-seismic landslides, the tough lesson of a catastrophe Xuanmei Fan, Qiang Xu, and Gianvito Scaringi The State Key Laboratory of Geohazards Prevention and Geoenvironment Protection (SKLGP), Chengdu University of Technology, Chengdu, Sichuan, 610059, China Xuanmei Fan, Qiang Xu, and Gianvito Scaringi The State Key Laboratory of Geohazards Prevention and Geoenvironment Protection (SKLGP), Chengdu University of Technology, Chengdu, Sichuan, 610059, China orrespondence: Qiang Xu (xuqiang_68@126.com) and Gianvito Scaringi (g.scaringi@qq.com) Correspondence: Qiang Xu (xuqiang_68@126.com) and Gianvito Scaringi (g.scaringi@qq.co Received: 13 October 2017 – Discussion started: 18 October 2017 Accepted: 17 December 2017 – Published: 26 January 2018 Received: 13 October 2017 – Discussion started: 18 October 2017 Accepted: 17 December 2017 – Published: 26 January 2018 Abstract. The rock avalanche that destroyed the village of Xinmo in Sichuan, China, on 24 June 2017, brought the is- sue of landslide risk and disaster chain management in highly seismic regions back into the spotlight. The long-term post- seismic behaviour of mountain slopes is complex and hardly predictable. Nevertheless, the integrated use of ﬁeld mon- itoring, remote sensing and real-time predictive modelling can help to set up effective early warning systems, provide timely alarms, optimize rescue operations, and perform sec- ondary hazard assessments. We believe that a comprehensive discussion on post-seismic slope stability and on its implica- tions for policy makers can no longer be postponed. seismic landslides left large amounts of loose material along the slopes which, in turn, caused deadly debris ﬂows and avalanches during every rainy season for decades – just like what happened, and is still happening, after the Ms 8.0 2008 Wenchuan earthquake (Fan and Huang, 2013), not far away. However, what does not collapse during the quake is not ex- empt from damage. An earthquake can produce cracks and fractures in the rock which, paradoxically, can be noticed from space by satellites but yet hidden to the human eye if covered by dense vegetation on high-elevation ridges (e.g. Fan et al., 2017). The damaged rock can hold in place for decades or even centuries, but rainwater can inﬁltrate within the fractures, dissolve minerals, ﬁll the cracks, freeze, and pull the blocks apart with its pressure until, more or less sud- denly, the proverbial last straw – a rainfall or a minor shake (Qiu, 2016) – makes it collapse. 1 Introduction: the 2017 Xinmo landslide, the lasting legacy of earthquakes p The long-term effect of strong earthquakes on the geolog- ical hazards in mountainous areas seems to be an underesti- mated issue. While coseismic landslides are well described (e.g. Parker et al., 2011; Zhang et al., 2016), and large at- tention is being given to the short and mid-term effects of earthquakes on debris ﬂow occurrence and sediment yield (Hovius et al., 2011), the delayed effects on slope stability is often neglected. Rock weathering and crack propagation are complex time-dependent processes. Thus, the occurrence of post-seismic landslides does not follow a clear trend, and destructive events might happen “randomly”, decades after the quake (see Towhata, 2013). Rock slopes damaged by the Mw 7.7 Chi-Chi earthquake in 1999 in Taiwan (Lin et al., 2006) are still collapsing, year after year during the rainy sea- sons, causing hundreds of fatalities. Post-seismic landslides and long-term rock degradation have been reported in several On 24 June 2017, after days of not-so-heavy rain, a 13 million m3 rock and debris avalanche submerged the vil- lage of Xinmo (in the eastern margin of the Tibetan Plateau, Sichuan, China) with impressive energy, rushing towards the river and blocking its course for more than 1 km. The rescue operations were launched promptly, and all possible efforts were done by local heroes and professionals. Nonetheless, 10 people were found dead and a further 73 were reported missing in one of the deadliest landslides in recent history (Fan et al., 2017; Fig. 1). Almost a century earlier, in 1933, Xinmo was struck by a magnitude Ms 7.5 earthquake, during which large-scale landslides destroyed villages and choked rivers, producing dammed lakes that, collapsing, produced enormous ﬂoods, killing thousands of people (Cheng et al., 2008). Many co- Nat. Hazards Earth Syst. Sci., 18, 397–403, 2018 https://doi.org/10.5194/nhess-18-397-2018 © Author(s) 2018. This work is distributed under the Creative Commons Attribution 4.0 License. Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. X. Fan et al.: Brief communication: Post-seismic landslides, the tough lesson of a catastrophe X. Fan et al.: Brief communication: Post-seismic landslides, the tough lesson of a catastrophe 398 Figure 1. Panoramic view of the Xinmo rock avalanche as seen from the opposite slope. Notice the red trucks for the scale. Figure 1. Panoramic view of the Xinmo rock avalanche as seen from the opposite slope. Notice the red trucks for the scale. With the leitmotif “safety ﬁrst”, the government listed disas- ter prevention and reduction in its economic and social devel- opment plan as an important guarantee of sustainable devel- opment. One year after the 2008 Wenchuan earthquake, with the white paper “China’s Actions for Disaster Prevention and Reduction”, the State Council deﬁned the strategic goals and tasks of disaster reduction and built a legal framework, an institutional setup, and an operative mechanism for disas- ter reduction (Chinese Government, 2009). To strengthen the capacity of emergency rescue and relief work, the National Emergency Plan for Sudden Geological Disasters was also enforced, featuring centralized command, sound coordina- tion, clear division of tasks, and level-by-level control with local authorities. The geo-disasters have been classiﬁed into four size-dependent categories according to the estimated fa- talities and money loss (Fig. 2). Different levels of govern- ment have been given responsibility for handling disasters of different magnitude. areas of Japan (Okamoto et al., 2012), and a clear tendency for landslides to occur more frequently due to past earth- quakes has been found in New Zealand (Parker et al., 2015). Several recent landslides in Sichuan, China, have been corre- lated with the Ms 7.5 Xichang earthquake (Wei et al., 2014), which occurred more than 160 years ago. It is estimated that 5000 to 10 000 die because of a non- coseismic landslide each year in the world, with China be- ing the most exposed country (Petley, 2012) and with a wor- risome increasing trend over time. A signiﬁcant number of events can be regarded as post-seismic landslides, such as the recent one in Xinmo, for which the rock weakening ac- tion of past strong earthquakes likely acted as a predisposing factor. 2 An example of policy: disaster prevention and emergency response in China In the wake of a highly catastrophic event, the local-level divisions are required to report to the State Council directly within no more than 4 h, and the latter will take immediate action. The commanding and coordination headquarters, led by the State Council directly, will be set up with a cross- functional steering committee, consisting of experts from dif- ferent ﬁelds, to conduct rescue work, evacuation, temporary relocation, information and data gathering, geological sur- veying, weather forecasting, medical and epidemic preven- tion, lifeline engineering repair, and so on. The headquarters have also the power to command the People’s Armed Police (the Chinese army) directly. Conversely, in the case of small- and medium-scale disasters, the local government will trig- ger the emergency response immediately and autonomously China has been doing great efforts in funding research, al- locating special budget to professional teams and training the general public and government divisions at various lev- els with the aim of improving early reconnaissance, warning, and prevention of geological hazards. Nevertheless, tragedies still happen and, unfortunately, the Xinmo landslide was not an isolated event. In 2013, the Wulipo landslide – an event with similar characteristics – claimed 166 victims. It was considered as a lagged effect of the 2008 Wenchuan earth- quake (Yin et al., 2016). According to Huang (2009), most of the catastrophic landslides in China might be caused by the joint effect of earthquakes and rainfall (Huang, 2009). On the other hand, China has gained solid experience in the emergency response and rescue in catastrophic events. Fan et al.: Brief communication: Post-seismic landslides, the tough lesson of a catastrophe 399 X. Fan et al.: Brief communication: Post-seismic landslides, the tough lesson of a catastrophe X. Fan et al.: Brief communication: Post-seismic landslides, the tough lesson of a catastrophe Figure 2. The emergency response to disasters in the People’s Republic of China. Figure 2. The emergency response to disasters in the People’s Republic of China. Figure 2. The emergency response to disasters in the People’s Republic of China. some extent, in the landslide source area (Fan et al., 2017). InSAR images highlighted noticeable deformations (Fig. 3a, b) in the rock mass during the months preceding the landslide and, reasonably, the inﬁltration of recent rainfalls within the cracks speeded up the failure process (Fan et al., 2017). and will set up a local emergency command, with the local government’s heads serving as chief commanders, to jointly set up the emergency response and disaster relief, organize the ﬁeld work, and report on the disaster details and work progress to the higher governmental level. Tragedies such as the one occurred in Xinmo might be avoided if the same scrupulous and systematic early recon- naissance and monitoring activity is carried out in due time. Satellite imagery can help detect and prioritize potential haz- ardous areas. Then, through ﬁeld and aerial investigations, using UAV, lidar, and InSAR, detailed mapping can be done. Potentially critical situations can be recognized and then han- dled through continuous monitoring of deformations, for in- stance through ground-based SAR interferometry (Crosetto et al., 2014; Monserrat et al., 2014; Fan et al., 2017). Further- more, in situ recording and interpretation of the characteris- tics of ambient noise, i.e. the low energy and low-frequency vibrations of the ground due to natural or anthropic sources (e.g. Del Gaudio, 2017), can be used to characterise the me- chanical properties of the soil or rock mass (e.g. anisotropy, sets of directional discontinuities; Di Giulio et al., 2009) and their changes with time (e.g. shear wave velocity decrease due to crack opening or water level rise; Daskalakis et al., 2016; Behm, 2016), potentially providing an indirect assess- ment of strength degradation or stress changes. Furthermore, monitoring of acoustic emissions, i.e. the high-frequency transient elastic waves originating from the sudden release of energy at localized points within a loaded material (Nomikos et al., 2010), can provide information on the occurrence of microcracks within the rock, that may signal progression of 3 Open discussion: the lesson of a killer landslide The recent catastrophic event in Xinmo received consider- able attention by the scientiﬁc community and by the me- dia worldwide, bringing the issue of landslide risk and dis- aster chain management in highly seismic regions back into the spotlight. Through this brief, work-in-progress paper, we hope to trigger a comprehensive open discussion within the scientiﬁc community and gather and share ideas on the best handling of long-term post-seismic slope stability problems and on its implications for policy makers. Soon after the Xinmo landslide, the Sichuan Province Ad- ministration began a detailed investigation to identify poten- tial geohazards before the beginning of the rainy season. The satellite radar interferometry technique (InSAR) has been ap- plied to identify hot spots of deformation within the large search region. Laser scanning (lidar) and drone ﬂights (UAV) were then been used to further conﬁrm the potential haz- ardous sites. We think that this is perhaps the most effec- tive way to proceed in densely vegetated mountainous areas. Satellite images of the Xinmo village in the visible spec- trum taken subsequent to 2003 showed that there were in- deed several cracks, up to 150 m long and interconnected to www.nat-hazards-earth-syst-sci.net/18/397/2018/ Nat. Hazards Earth Syst. Sci., 18, 397–403, 2018 www.nat-hazards-earth-syst-sci.net/18/397/2018/ www.nat-hazards-earth-syst-sci.net/18/397/2018/ Nat. Hazards Earth Syst. Sci., 18, 397–403, 2018 www.nat-hazards-earth-syst-sci.net/18/397/2018/ www.nat-hazards-earth-syst-sci.net/18/397/2018/ X. Fan et al.: Brief communication: Post-seismic landslides, the tough lesson of a catastrophe 400 Figure 3. (a, b) Differential interferogram of the landslide obtained by processing Sentinel-1 satellite data. The arrow points to the deforma- tion occurred in the landslide source area. (c–g) Seismic signal of the Xinmo landslide event recorded by the Maoxian MXI station, about 43 km apart (modiﬁed from Fan et al., 2017): vertical component (c); frequency–time Hilbert spectrum (d); high-frequency time–magnitude spectrum, f = 2.73 Hz (e); low-frequency time–magnitude spectrum, f = 0.4 Hz (f); frequency–magnitude spectrum (g). Figure 3. (a, b) Differential interferogram of the landslide obtained by processing Sentinel-1 satellite data. The arrow points to the deforma- tion occurred in the landslide source area. (c–g) Seismic signal of the Xinmo landslide event recorded by the Maoxian MXI station, about 43 km apart (modiﬁed from Fan et al., 2017): vertical component (c); frequency–time Hilbert spectrum (d); high-frequency time–magnitude spectrum, f = 2.73 Hz (e); low-frequency time–magnitude spectrum, f = 0.4 Hz (f); frequency–magnitude spectrum (g). al., 2015), and thus the volume involved, V = 13 million m3 (quite close to the estimation based on ﬁeld observation and topographic difference), and the average mobilized friction coefﬁcient, µ = 0.29 (following Lin, 2015). For compari- son, similar evaluations resulted in E = 150 TJ, µ = 0.12, v = 298 km h−1, and V = 107 m3 for the 2009 Hsiaolin land- slide in Taiwan (Lin et al., 2010, 2015; Lin, 2015), and E = 55 TJ, v = 101 km h−1 and V = 2.1 × 107 m3 for the 2011 Atakani landslide in Japan (Yamada et al., 2013). Sys- tematic evaluations of landslide characteristics based on seis- mic recordings are also given by Chen et al. (2013). rock damaging, crack coalescence, deformations, and incipi- ent failure (e.g. Manthei and Eisenblätter, 2008; Agioutantis et al., 2016). Such techniques, combined with hydrological monitoring, would be extremely valuable for setting up early warning systems and help the authorities to take informed decisions on possible evacuation or relocation of the exposed people. p p In addition, if the event happens too suddenly to take coun- termeasures, something could still be done to optimize the alarm-and-rescue chain, for instance, by using the existing seismic networks. X. Fan et al.: Brief communication: Post-seismic landslides, the tough lesson of a catastrophe the interpretation of UAV images. The mass was likely dis- placed by the shearing and dragging action of the Xinmo landslide, but it stabilized after sliding for about 40 m after encountering a natural obstacle. During the emergency res- cue operations, the mass was believed to be in a state of in- cipient failure and received considerable attention. In order to provide a reliable evaluation of its stability condition, and to ensure the safety of people near the landslide deposition area and preventing further disasters, a ground-based SAR was installed. Subsequently, numerical modelling with vari- ous methods (ﬁnite elements, discrete elements) was carried out to evaluate the potentially affected areas in case of a new failure (Scaringi et al., 2017). The model results showed that the potential new landslide would likely affect several more buildings and a further portion of river and of road infrastruc- ture. Furthermore, the resulting river damming would pose a serious risk for the population living downstream in the case of dam breach and for the population living upstream with the possible water level rise. In the wake of these re- sults, different modelling approaches have been discussed comparatively, and the opportunity of an integrated real-time monitoring-and-modelling system arose. As pointed out by Molinari et al. (2014), a physically based numerical model capable of re-computing a new solution in a very short time (i.e. within seconds) based on spatially distributed real-time ﬁeld monitoring data can be extremely useful in dynamic risk assessment systems at a scale of detail to provide early warn- ing to the authorities and implement timely risk mitigation countermeasures. Competing interests. The authors declare that they have no conﬂict of interest. Acknowledgements. After the occurrence of the 24 June Xinmo landslide, the State Council, the Ministry of Land and Resources, and various governmental departments at all levels in Sichuan Province immediately devoted their efforts to emergency rescue operations, secondary hazard relief, and geological surveying and monitoring. To them go our most sincere appreciation and gratitude. g g pp g We thank the Sichuan Provincial Surveying and Mapping Geo- graphic Information Bureau, the High-resolution Earth Observation System Sichuan Data and Application Center, the Sichuan Shu Tong Geotechnical Engineering Company, and the Beijing Digital Green- ﬁeld Technology Co. Ltd. X. Fan et al.: Brief communication: Post-seismic landslides, the tough lesson of a catastrophe We express our gratitude to Yueping Yin from China Geological Environment Monitoring Institute, to Xiao Li from China Geological Survey; to Zhenhong Li from Newcastle University, to Lu Zhang and Mingsheng Liao from Wuhan Univer- sity; to Qin Zhang from Chang’an University; to Shizhong Hong from Chengdu Earthquake Prevention and Hazard Mitigation Bu- reau; to Xinghui Huang from China Earthquake Networks Center; to Yong Li from Chengdu University of Technology; and to Chong Xu from the Institute of Geology, China Earthquake Administra- tion, for providing valuable information at the earliest time. We express sincere thanks to Yanan Jiang, Jing Ran, Xianxuan Xiao, Weiwei Zhan, Jing Ren, Yuanzhen Ju, Chen Guo, and other post- graduate students from Chengdu University of Technology for their hard work on the UAV aerial photography, ground-based SAR mon- itoring, and other ﬁeld work. This research is ﬁnancially supported by National Science Fund for Outstanding Young Scholars of China (grant no. 41622206), the Funds for Creative Research Groups of China (grant no. 41521002), National Science Fund for Distinguished Young Scholars of China (grant no. 41225011), the Fok Ying-Tong Education Foundation for Young Teachers in the Higher Education Institutions of China (grant no. 151018), the AXA fund, and the Fund from Land and Resources Department of Sichuan Province (grant no. KJ-2015-01). 4 Concluding remarks The Xinmo tragedy taught us a tough lesson, but it also showed us how the use of new technologies and the collabo- rative work of experts and professionals can prove successful in identifying potential hazards and performing quick assess- ments in “inaccessible” areas. As the hazard chain of earth- quakes can last for centuries, we argue that a dedicated haz- ard prevention and mitigation department should be estab- lished in every region where strong earthquakes can strike. It would provide the necessary coordination and integration of resources, information, equipment, and labour. It would be able to set up big data centres and platforms, automatic reconnaissance, and warning and alarm algorithms. It would carry out comprehensive research on geological hazard pre- vention and promote the practical application of new tech- nologies. Finally, it would comprehensively enhance our ca- pacity to prevent and mitigate geological hazards, as well as avoid tragedies. The authors are grateful to the editor, Kang-tsun Chan, for handling the manuscript, and to Wei-An Chao and one anonymous referee for the useful comments and for encouraging the publication of this work. Edited by: Kang-tsung Chang Edited by: Kang-tsung Chang Reviewed by: Wei-An Chao and one anonymous referee www.nat-hazards-earth-syst-sci.net/18/397/2018/ In fact, it is known that landslides generate seismic signals, “landquakes”, which contain a speciﬁc sig- nature: low-frequency waves released by the bedrock when the mass detaches, and high-frequency waves produced by the landslide mass while it is sliding, peaking when it im- pacts the deposition area (e.g. Yamada et al., 2012; Chen et al., 2013). If analysed separately, they can give information on both landslide initiation and impact (Fig. 3c–g). In the- ory, two distinct epicentres can be identiﬁed automatically by the seismic networks, if they are sensitive enough and they are taught to do so. In the case of the Xinmo land- slide, the seismic recording showed that just 1 min elapsed from the initiation to the deposition, during which the mass slid along the slope for more than 2.5 km, with more than 1000 m of height relief, and hit the population at an im- pressive velocity v = 250 km h−1 (Fan et al., 2017, following Lin, 2015). The potential energy released by the event could be estimated, E = 290 TJ (Fan et al., 2017, following Lin et If all these calculations were done automatically, within minutes from the event, the authorities would have received a detailed alarm report containing the coordinates and mag- nitude of the landslide, the runout, the rock/soil involved, the volume and impact velocity, the number of people and infras- tructure potentially affected, and the estimated damage. Such quantitative information can be extremely useful for launch- ing the rescue operations in the most efﬁcient way. Some work has been done on this path already (Chao et al., 2017), and so far it seems very promising. Finally, after the landslide event, an accurate and contin- uous secondary hazard assessment is fundamental. Fan et al. (2017) reported on a preliminary evaluation of the sec- ondary hazard deriving from potential further failures in the source area and its surroundings. Various potentially unsta- ble masses have been identiﬁed. Among them, a large-scale deformation of a 4.5 million m3 mass was detected through www.nat-hazards-earth-syst-sci.net/18/397/2018/ Nat. Hazards Earth Syst. Sci., 18, 397–403, 2018 401 X. Fan et al.: Brief communication: Post-seismic landslides, the tough lesson of a catastrophe Data availability. No data sets were used in this article. 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https://openalex.org/W2783212251	https://zenodo.org/records/3991362/files/38%207625.pdf	English	null	Personal Security Tracking based on Android and Web Application	Telkomnika	2,018	cc-by-sa	2,639	TELKOMNIKA, Vol.16, No.2, April 2018, pp. 771~775 ISSN: 1693-6930, accredited A by DIKTI, Decree No: 58/DIKTI/Kep/2013 DOI: 10.12928/TELKOMNIKA.v16i2.7625 TELKOMNIKA, Vol.16, No.2, April 2018, pp. 771~775 ISSN: 1693-6930, accredited A by DIKTI, Decree No: 58/DIKTI/Kep/2013 DOI: 10.12928/TELKOMNIKA.v16i2.7625  771  771 Abstract Nowadays, the frequency of abduction is increasing significantly. Information technology provide many social media which is causing the leaked of personal information where abductor can find out the information. Using social media, people can share location, life event, and anything related about their life event, these become the useful information for the abductor. To anticipate this, a lot of personal tracking device is developed, but the response is too late. Because, the family or relative of the victims will notice that abduction had happen after the victim is lost. According to the problem, this research will focus on develop the personal tracking device which trigger by the victims who own the phone based on android and web application. Whenever the abduction happens, the victim just need to click the button and the system will send a message to the relatives and family in their contact also trigger the notification to the local security. Software development life cycle that will be used is scrum which is divided into several processes like backlog, sprints, scrum meetings, and demos. The result of this research is android application for user and web application for managing application. Keywords: decision support system; tracking application; android application; web application 1. Introduction Information technology provides many simplicities in human life. Social media is one of the most popular simplicity to share life event in public. Instead of many benefits comes from it, there are also become the way to collect information about the victim’s life event. Abduction is one of the crimes which is increase significantly. There are a lot of tools and applications developed to prevent this crime. But, most of them are trigger the victim’s family or relative. At that time, the abduction process is already in progress. So, the number of abduction victims is still high. Children’s safety is one of the issues that need attention. This is because the lack of supervision of parents against their children, especially when parents are working/traveling, not be able to supervise their children throughout the day. Mobile tracking application supposed to have several features such as tracking location, trigger, and notification alert [1]. Mobile tracker is focusing on tracking device which can locate the device, collect the SIM Card information, collect contact information, and call log information. These features are developing under the concept to track down lost phone [2]. Both research before are able to collect the location of the user in the realtime whether using Global Positioning System or Location Based System. These systems suggested on previous research are lack of several feature such as do not store the history location, not be able to take different kinds of evidence (such as photo and sound), and trigger by related of victims. ( p ) gg y In this research, a new product is developed. The user which is in victim position is having control of this application. This application will record location of the user in specific time range which defined by the user, collect sound record in secret, and taking photo from every camera of the user’s phone in secret. Whenever the user click the panic button, the application will send the notification to their relatives and family member which is already registered in that application by the user. Also, this will trigger a notification to the local security and send the evidences to the server. The evidences are location, sound, and photo recorded secretly on the user’s phone and will be taking repeatedly in specific time range. The application also stores the location of the user’s phone since the application is activated. Personal Security Tracking based on Android and Web Application Angry Ronald1, Violitta Yesmaya2, Muhammad Danaparamita3 Computer Science Department, School of Computer Science, Bina Nusantara University, Jakarta, Indonesia 11480 Corresponding author, e-mail: aadam@binus.edu1, vyesmaya@binus.edu2, mdanaparamit Angry Ronald1, Violitta Yesmaya2, Muhammad Danaparamita3 Computer Science Department, School of Computer Science, Bina Nusantara University, Jakarta, Indonesia 11480 ng author, e-mail: aadam@binus.edu1, vyesmaya@binus.edu2, mdanaparamita@binus.edu3 Angry Ronald1, Violitta Yesmaya2, Muhammad Danaparamita3 Computer Science Department, School of Computer Science, Bina Nusantara University, Jakarta, Indonesia 11480 *Corresponding author, e-mail: aadam@binus.edu1, vyesmaya@binus.edu2, mdanaparamita@binus.edu3 2.1. Location Based Services Location Based Services (LBS) is a service provider that provides location information. LBS is a value-added service from cellular services provider to collect location information of the user. To collect the location, this service will collect information from the device of the user that send back the signal from the tower and mapping it into the map to get the location. Even though this service is provided by the cellular services provider, but the accuracy is quite lower than GPS (Global Positioning System) which is depending on satellite to get the location [3]. 2.2. Global Positioning System Global Positioning System (GPS) is a feature that embedded to a tool that can send and receive the signal from the satellite to collect the exact location of the user. Nowadays, GPS is already attached to all smartphones that makes smartphone able to locate its own location. Because of this feature send signals from the device to the satellite then the location is more accurate rather than LBS [4]. Using GPS, position of user will be able to be tracked by the longitude and latitude of the user. Different transmitter having different accuracy. GPS on mobile device are having several kinds of GPS transmitter which affecting the accuracy of the user position. The transmitter been used on this research is A-GPS using Samsung Galaxy Tab 4. User’s device has a configuration to upload user location for every specific interval time which is configure by user. But, when incident is already triggered the device will change the status and send the location with photo of front and back camera together with sound recording on phone. In mobile phone, GPS works by transmit the signal to the satellites. The satellites receive the signal and monitor the position by control station which are sent back the signals through the satellites using ground antenna. The radio signals from satellites are pick up by GPS receiver on mobile phone [7]. In order to used google maps API, GSM is needed. GPS will focusing on collecting location while GSM will send the location which are longitude and latitude to Google Maps API to display the maps [8]. 1. Introduction So, the local security could be Received October 5, 2017; Revised January 28, 2018; Accepted February 12, 2018 ISSN: 1693-6930  772 able to respond quickly and accurately according to the data given. There are several applications which are available in google play store such as GPS Tracker, Track Any Phone, and Friend Locator: Phone Tracker. The comparison as shown in Table 1: able to respond quickly and accurately according to the data given. There are several applications which are available in google play store such as GPS Tracker, Track Any Phone, and Friend Locator: Phone Tracker. The comparison as shown in Table 1: Table 1. Comparison Table of Family Locator, iLocateMobile, and Friend Locator. Features Family Locator- GPS Tracker Track Any Phone (iLocateMobile) Friend Locator: Phone Tracker Route assistance Yes No No Tracking location Yes Yes Yes Emergency alert Yes No Yes Manage emergency contact Yes Yes Yes Messaging Yes No Yes History location No Yes No Sound recording No No No Photo taking No No No Manage crime status No No No Dual interface (for user and admin) No No No Table 1. Comparison Table of Family Locator, iLocateMobile, and Friend Locator. Features Family Locator- GPS Tracker Track Any Phone (iLocateMobile) Friend Locator: Phone Tracker 2.3. Questionnaire Questionnaire is one of the tool to acquire information by giving the constraint and problem of the research on questions and collecting the feedback from the respondents as the answer of that problem given [6]. To be able to collect the right information, the questionnaire must be having principal requirement which are sequenced in a logical order, allowing a smoth transition from on topic to the next. By applying these principal methods, participant should be able to understand the purpose of research and able to give useful information [6]. TELKOMNIKA Vol. 16, No. 2, April 2018 : 771 – 775 TELKOMNIKA 773 ISSN: 1693-6930  3. Results and Analysis y In this section, the picture of the web application is shows. The result of this research is android application and web application which are consists of many pages including main page in android where user can access the panic button and trigger to the incident page on admin in web application. On web application, admin can also track down the location which shown in maps with the time range also can collect any other evidence in the real time. User can set the configuration of range time to upload location in normally, but when incident happen the system will collect the location per minute. Figure 1 shows Incident List’s Page on web application where admin can see all the incidents happen. Figure 2 shows android main page where user can easily press the panic button which change the status on the server and collecting the evidence such as location periodically. Figure 3 shows the detail victims movement location in maps since that day. Figure 4 shows the evidence page which already collected also on this page admin can collect evidence such as photo from back and front camera and sound evidence. From 30 Respondents, below are the description of the feedback. From 30 Respondents, below are the description of the feedback. From 30 Respondents, below are the description p p 1. How fast you understand and remember how to use this application? 1. How fast you understand and remember how to use this application? a. 20 Respondents response “Very Fast”. b. 9 Respondents response “Fast”. c. 1 Respondents response “Slow”. 1. How fast you understand and remember ho a. 20 Respondents response “Very Fast”. b. 9 Respondents response “Fast”. c. 1 Respondents response “Slow”. 2. How good are you using this application? a. 20 Respondents response “Very Good”. b. 10 Respondents response “Good”. 2. How good are you using this application? a. 20 Respondents response “Very Good”. b. 10 Respondents response “Good”. 3. Is this application can fulfil your expectation about the problem? a. 5 Respondents Strongly Agree. 3. Is this application can fulfil your expectation about the problem? 5 R d t St l A 3. Is this application can fulfil your expectation about a. 5 Respondents Strongly Agree. b 16 R d A a. 5 Respondents Strongly Agree. b. 16 Respondents Agree. b. 16 Respondents Agree. c. 9 Respondents Neutral. 3. Results and Analysis On the appendix diagram of the process when the incident happen and collecting evidence will c. 9 Respondents Neutral. p On the appendix, diagram of the process when the incident happen and collecting evidence will be shown. Personal Security Tracking based on Android and Web Application (Angry Ronald) Figure 1. “List of incidents” page in web application Figure 2. Homescreen on android application Figure 2. Homescreen on android application Figure 2. Homescreen on android application Figure 1. “List of incidents” page in web application Personal Security Tracking based on Android and Web Application (Angry Ronald) Personal Security Tracking based on Android and Web Application (Angry Ronald)  774 774  774 ISSN: 1693-6930 Figure 3. Incident’s page on web application Figure 4. Evidence’s page on web application Figure 3. Incident’s page on web application Figure 3. Incident’s page on web application Figure 3. Incident’s page on web application Figure 4. Evidence’s page on web application Figure 4. Evidence’s page on web application 4. Conclusion According to the result application and questionnaire given, this product has an easy and simple user interface. Also, this product can fulfill and solved the problem where the trigger control is also located not only on admin control but also in user. This application can collect different kinds of evidences such as photo, sound, and location which is not provide by other applications. [6] Bird DK. The use of questionnaires for acquiring information on public perception of natural hazards and risk mitigation–a review of current knowledge and practice. Natural Hazards and Earth System Sciences. 2009; 9:1307-1325. References e e e ces [1] Kinage R, Kumari J, Zalke P, Kulkarni M. Mobile Tracking Application. International Journal of Innovative Research in Science, Engineering and Technology. 2013; 2(3): 617-623. [2] Shirin S, Dipina DB, Surekha MV. Mobile Tracker. International Journal on Cybernetics and Informatics. 2016; 5(2): 213–219. [3] Abhijeet T, Ahemad T, Ravindra S, Pranay D, Sumit H. Mobile Tracking Application for Locating Friends using LBS. International Journal of Innovative Research in Computer and Communication Engineering. 2013; 1(2): 303–308. [4] Abha D, Hardik S, Krishna S. Global Positioning System for Object Tracking. International Computer Application. 2015; 109(8): 40-45 p pp ( ) [5] Pressman RS, Maxim BR. Software Engineering: A Practitioner's Approach, Eighth Edition. Jakart McGraw-Hill Education. 2010. [6] Bird DK. The use of questionnaires for acquiring information on public perception of natural hazards and risk mitigation–a review of current knowledge and practice. Natural Hazards and Earth System Sciences. 2009; 9:1307-1325. TELKOMNIKA Vol. 16, No. 2, April 2018 : 771 – 775 TELKOMNIKA ISSN: 1693-6930  Personal Security Tracking based on Android and Web Application (Angry Ronald) 775 [7] Singal P, Chhillar RS. A Review on GPS and its Applications in Computer Science. International Journal of Computer Science and Mobile Computing. 2014; 3(5):1295-1302. [8] Verma P, Bhatia JS. Design and development of GPS-GSM based tracking system with Google Map based monitoring. International Journal of Computer Science, Engineering and Application. 2013; 3(3):33-40 Appendix Diagram 1. Process of when incident happen Diagram 2. Process of collecting evidence User Mobile Apps Web Apps Start Click “PANIC” Button Change status, trigger to send alert to related registered partner, and collecting location per minute Change status, send alert to related registered partner, and collecting location per minute End Admin Start Click “Collect Evidence” Button (Photo \| Sound) Collecting Evidence (Photo \| Sound) Trigger Mobile Device to Collect Evidence (Photo \| Sound) Silently End Mobile Apps Web Apps Upload Evidence (Photo \| Sound) Show Evidence list ISSN: 1693-6930 775 TELKOMNIKA  [7] Singal P, Chhillar RS. A Review on GPS and its Applications in Computer Science. International Journal of Computer Science and Mobile Computing. 2014; 3(5):1295-1302. Journal of Computer Science and Mobile Computing. 2014; 3(5):1295-1302. [8] Verma P, Bhatia JS. Design and development of GPS-GSM based tracking system with Google Map based monitoring. International Journal of Computer Science, Engineering and Application. 2013; 3(3):33-40 Appendix Diagram 1. References Process of when incident happen User Mobile Apps Web Apps Start Click “PANIC” Button Change status, trigger to send alert to related registered partner, and collecting location per minute Change status, send alert to related registered partner, and collecting location per minute End Click “PANIC” Button Change status, trigger to send alert to related registered partner, and collecting location per minute Change status, send alert to related registered partner, and collecting location per minute Diagram 1. Process of when incident happen Diagram 1. Process of when incident happen Diagram 2. Process of collecting evidence Admin Start Click “Collect Evidence” Button (Photo \| Sound) Collecting Evidence (Photo \| Sound) Trigger Mobile Device to Collect Evidence (Photo \| Sound) Silently End Mobile Apps Web Apps Upload Evidence (Photo \| Sound) Show Evidence list Click “Collect Evidence” Button (Photo \| Sound) Trigger Mobile Device to Collect Evidence (Photo \| Sound) Silently Collecting Evidence (Photo \| Sound) Upload Evidence (Photo \| Sound) Show Evidence list End Diagram 2. Process of collecting evidenc Diagram 2. Process of collecting evidence Personal Security Tracking based on Android and Web Application (Angry Ronald)
https://openalex.org/W2124461864	https://europepmc.org/articles/pmc4381774?pdf=render	English	null	Price promotions on healthier compared with less healthy foods: a hierarchical regression analysis of the impact on sales and social patterning of responses to promotions in Great Britain	The American journal of clinical nutrition	2,015	cc-by	8,379	1 From the Behaviour and Health Research Unit, University of Cambridge, Cambridge, United Kingdom (RN, MS, SAJ, RP, and TMM); the Health Economics Group, Norwich Medical School, University of East Anglia, Norwich, United Kingdom (RN and MS); the Centre for Health Economics, University of York, York, United Kingdom (RN and MS); the UKCRC Centre for Diet and Activity Research, Cambridge, United Kingdom (RN and MS); the Nufﬁeld Department of Primary Care Health Sciences, University of Oxford, Oxford, United Kingdom (SAJ); and the Medical Research Council Human Nutrition Research, Elsie Widdowson Laboratory, Cambridge, United Kingdom (EA-R). ABSTRACT possibly stimulating impulsive purchases by increasing the prominence of items in store (e.g., via tags and placement). There is a growing concern that such promotional activities by the food industry may contribute to poor dietary intake particularly in individuals who are more socially deprived (1–3). It was also suggested that price promotions on less-healthy products might lure consumers away from healthier, higher-priced options and that the industry has disproportionately promoted less-healthy but more-proﬁtable options (4). If so, there might be a case for public policy to regulate the promotional activities of industries to help achieve, or at least not hamper, public health nutrition goals. possibly stimulating impulsive purchases by increasing the prominence of items in store (e.g., via tags and placement). There is a growing concern that such promotional activities by the food industry may contribute to poor dietary intake particularly in individuals who are more socially deprived (1–3). It was also suggested that price promotions on less-healthy products might lure consumers away from healthier, higher-priced options and that the industry has disproportionately promoted less-healthy but more-proﬁtable options (4). If so, there might be a case for public policy to regulate the promotional activities of industries to help achieve, or at least not hamper, public health nutrition goals. Background: There is a growing concern, but limited evidence, that price promotions contribute to a poor diet and the social patterning of diet-related disease. Objective: We examined the following questions: 1) Are less- healthy foods more likely to be promoted than healthier foods? 2) Are consumers more responsive to promotions on less-healthy prod- ucts? 3) Are there socioeconomic differences in food purchases in response to price promotions? Design: With the use of hierarchical regression, we analyzed data on purchases of 11,323 products within 135 food and beverage categories from 26,986 households in Great Britain during 2010. Major supermarkets operated the same price promotions in all branches. The number of stores that offered price promotions on each product for each week was used to measure the frequency of price promotions. We assessed the healthiness of each product by using a nutrient proﬁling (NP) model. However, there is a paucity of empirical evidence available in the public domain, and the existing claims about a bias in the use of price promotions toward less-healthy items largely rest on anecdotal reports. ABSTRACT Although the general responsiveness of con- sumers to price promotions received substantial attention in the marketing literature (5, 6), and there is a fast-growing body of research on the effect of price per se on healthier compared with Results: A total of 6788 products (60%) were in healthier cate- gories and 4535 products (40%) were in less-healthy categories. There was no signiﬁcant gap in the frequency of promotion by the healthiness of products neither within nor between categories. However, after we controlled for the reference price, price discount rate, and brand-speciﬁc effects, the sales uplift arising from price promotions was larger in less-healthy than in healthier categories; a 1-SD point increase in the category mean NP score, implying the category becomes less healthy, was associated with an additional 7.7–percentage point increase in sales (from 27.3% to 35.0%; P , 0.01). The magnitude of the sales uplift from promotions was larger for higher–socioeconomic status (SES) groups than for lower ones (34.6% for the high-SES group, 28.1% for the middle-SES group, and 23.1% for the low-SES group). Finally, there was no signiﬁcant SES gap in the absolute volume of purchases of less-healthy foods made on promotion. 2 The Department of Health had no role in the study design, data collec- tion, analysis, or interpretation. The research was conducted independently of the funder, and the views expressed in this article are those of the authors and not necessarily those of the Department of Health in England. 3 The study was funded by the Department of Health Policy Research Programme (http://prp.dh.gov.uk/) [Policy Research Unit in Behaviour and Health (PRUN-0409-10109)]. This is an open access article distributed under the CC-BY license (http://creativecommons.org/licenses/by/3.0/). Conclusion: Attempts to limit promotions on less-healthy foods could improve the population diet but would be unlikely to reduce health inequalities arising from poorer diets in low-socioeconomic groups. Am J Clin Nutr 2015;101:808–16. 4 Supplemental Figure 1, Supplemental Tables 1–12, and additional tech- nical details of empirical methods (Supplemental Data sections 2, 3 and 6) are available from the “Supplemental data” link in the online posting of the article and from the same link in the online table of contents at http://ajcn. nutrition.org. Keywords: food purchasing, Great Britain, price promotion, pub- lic health, public policy 5 Address correspondence to TM Marteau, Behaviour and Health Research Unit, Institute of Public Health, Cambridge CB2 0SR, United Kingdom. E-mail: theresa.marteau@medschl.cam.ac.uk. Ryota Nakamura, Marc Suhrcke, Susan A Jebb, Rachel Pechey, Eva Almiron-Roig, and Theresa M Marteau Ryota Nakamura, Marc Suhrcke, Susan A Jebb, Rachel Pechey, Eva Almiron-Roig, and Theresa M Marteau Data We used a secondary data source, the Kantar WorldPanel survey (14, 16), which includes purchase records of 26,986 households in the United Kingdom throughout 2010. Households were recruited by a data company (Kantar WorldPanel), and the authors were not involved in the data collection. Data-collection procedure was as follows: the United Kingdom Ofﬁce of National Statistics census information and the United Kingdom Broad- casters’ Audience Research Board Establishment survey were used to determine quotas for recruitment. The data company purchased potential participant lists from another company and recruited participants by sending postal mails and e-mails. Re- cruited participants received vouchers for high-street retailers and/or vouchers for leisure activities [in total for an average monetary equivalent of £100 (;$160) per household per year]. Recruited households were nationally representative in terms of region, age group, and household size. The survey includes purchase records of all foods and beverages that were taken home from supermarkets and similar stores in 2010. Sampled households were asked by the data company to record all purchases using barcode scanners and to send digital images of cash- register receipts to the company. The data contain rich information on purchases, including the price at which they were bought, whether they were bought on promotion, the number of packets purchased, and the retail chain from which the product (Stock Keeping Unit) was purchased. The data also include detailed in- formation on product characteristics, including information on the brand, manufacturing company, and nutritional content. The frequency of promotions for each product was deﬁned by the number of branches (22) that ran a promotion on the product in a given week aggregated across the 11 supermarket chains and 52 wk. Each branch could run a promotion on a given product (Stock Keeping Unit) only once at a given point in time, and hence, the number of branches that ran a promotion on a product gave the number of promotions on the same product. See Supplemental Data section 2 for additional details. INTRODUCTION Price promotions are commonly used in store with the aims of boosting purchasing by reducing the price of products as well as ReceivedJune 30, 2014. Accepted for publication January 8, 2015. ReceivedJune 30, 2014. Accepted for publication January 8, 2015. , p p y , First published online February 11, 2015; doi: 10.3945/ajcn.114.094227. 808 Am J Clin Nutr 2015;101:808–16. Printed in USA. SUPERMARKET PRICE PROMOTIONS 809 chains [i.e., the “main parties” as deﬁned by the United King- dom Competition Commission (17)]. The food and drink cate- gories reﬂected those used in the retailing sector (Kantar WorldPanel; see Supplemental Table 1 for more information). With the use of transaction records in the data, we calculated the total number of units of each product sold to panel households across the country over 52 wk. In keeping with common practice in the related literature (13, 18–20), we restricted the set of products to the more-popular items; in particular, we included only products that were purchased at least once in each of the 52 wk by any of the panel households, irrespective of whether the products were on promotion or not. less-healthy purchasing or consumption (7–13), there has been relatively little research to consider whether consumer uptake of promoted products differs for healthier or less-healthy products. Moreover, it is unclear whether the impact of promotions varies by the socioeconomic characteristics of consumers. In this study, we sought to ﬁll this evidence gap. We examined whether pro- motions on less-healthy products increased sales more than pro- motions on healthier products by using data from supermarkets across the United Kingdom. We also sought to explore whether social disparities in the healthiness of food purchased were attributable to differences in responses to retail price promotions. In an earlier study, by using the same survey data, we showed signiﬁcant socioeconomic pat- terning in the healthiness of food purchases (14). However, the mechanisms that could account for such patterns remain under- explored. In the current study, we tested one potential mechanism, i.e., price promotions; we investigated the differential use of price promotions across socioeconomic groups to explore whether this may be one contributor to diet-related health inequalities (15). Frequency of promotions To assess consumer responses to price promotions, it is es- sential to measure the number of price promotions available for each product. However, in commonly available data sets (in- cluding ours), a price promotion is recorded for a given item in a given store only if that item has been purchased on promotion from that store by panel households. This purchase-based nature of existing data sets has thus far prevented researchers from measuring the frequency of price promotions at population level. We designed our study to address the following 3 questions: 1) Are less-healthy foods more likely to be promoted than healthier foods? 2) Are consumers more responsive to promotions on less- healthy products than promotions on healthier ones? 3) Are there socioeconomic differences in food purchases in response to price promotions? In the absence of a directly observable measure in the data, we estimated the frequency of promotions for each product by exploiting a particular feature of the retail policy in major United Kingdom grocery retailers. We focused on the following 11 “main parties” of United Kingdom multiple grocers: Tesco (sales market share in 2010 was 24%), Asda (sales market share in 2010 was 12.8%), Sainsbury (sales market share in 2010 was 12.5%), Morrisons (sales market share in 2010 was 9.8%), Waitrose (sales market share in 2010 was 3.1%), Iceland (sales market share in 2010 was 1.7%), Lidl (sales market share in 2010 was 1.6%), Aldi (sales market share in 2010 was 1.5%), M&S (sales market share in 2010 was 1.3%), Netto (sales market share in 2010 was ,1%), and Budgens (market share information not available), as deﬁned by the United Kingdom Competition Commission (17). The total market share of these grocers in 2010 was ;70% (21). The Commission conﬁrmed that the stores followed a national pricing policy according to which stores operated the same pricing (and, thus, the same price promotions) in all branches. This institutional feature provided us with the opportunity to estimate the number of pro- motions run in the country in a given time period; if we observed any transaction involving a product on promotion in a given store, we could assume that the product was also on price promotion in the other branches of the same supermarket chain. Product and category healthiness We constructed a cross-sectional data set of 11,323 individual products in 135 food and drink categories, which were purchased by panel households in leading United Kingdom supermarket The nutrient proﬁling (NP) model developed by the United Kingdom Food Standards Agency was used to capture the 810 NAKAMURA ET AL. healthiness of products (23). This method assigns a score for each food calculated from the energy density, saturated fat, sugar, sodium, ﬁber, and protein contents together with an estimate of the fruit, vegetable, and nut contents, thereby providing a uniﬁed measure of healthiness across all available food and drink products. The NP model applies to all food and drink products equally without exemptions or category-speciﬁc criteria (23). However, the deﬁnition of healthier and less-healthy products typically uses different cut points for foods and beverages, reﬂecting the very different energy densities of the 2 groups, and we adopted this convention. Note that, as the NP score increases, the healthiness of the product declines. Compared with other NP models, this NP score was shown to perform well when matched to a standard ranking of foods by .700 nutrition professionals (24). Intake of high-scoring foods was shown to act as a risk factor for obesity (25). Category-level healthiness was calculated by taking the mean NP score for products within the category. products. The analysis assessed whether price promotions in- creased sales of less-healthy compared with healthier foods (between-category effect). The analysis also addressed whether sales of less-healthy versions within a given food category in- creased more in response to promotions than did healthier ver- sions in the same food category (within-category effect). Again a similar hierarchical regression approach was used. The baseline product-level purchases equation is given by log Salesjc ¼ b0c þ b1c log FoPjc þ b2cNPjc þ b12c log FoPjc 3 NPjc þ Zjcd þ ejc ð3Þ ð3Þ The outcome variable was the log of total number of products j in category c that were purchased by the panel households over 52 wk. The interaction term [log(FoPjc)3NPjc] was used to mea- sure whether and, if so, to what extent the effect of promotions varied by the healthiness of the product. RESULTS Table 1 shows characteristics of participating households (main shoppers) by socioeconomic groups. The total number of households was 26,986 with 5667 households in the high-SES groups, 14,870 households in the middle-SES group, and 6449 households in the low-SES group. There were gradients in household income, education level of the main shopper, and BMI. Although all households were included to calculate Analytic framework Are less-healthy foods more likely to be promoted than healthier foods? Are less-healthy foods more likely to be promoted than healthier foods? Similar to the previous analysis, category-speciﬁc coefﬁcients were modeled as follows: To address this question, we set up a product-level regression model of sales and assessed the relation between the frequency of promotions and NP score. In supermarkets, each product was nested by product category (135 categories in our data), and hence, our product-level data set had a 2-level structure (i.e., between-category and within-category variations in healthiness). First, we estimated the association between the frequency of promotions and NP score of various food categories (i.e., be- tween-category differences). Next, we estimated the relation between promotions and the NP score at the product-level separately by the food category (i.e., within-category differ- ences). These 2 step estimations were conducted simultaneously via a hierarchical regression approach (26). For item j in category c, the following base model was speciﬁed: b0c ¼ c00 þ c01NPc þ u0c; b1c ¼ c10 þ c11NPc þ u1c; b2c ¼ c20 þ c21NPc þ u2c; b12c ¼ c30 þ c31NPc þ u3c ð4Þ ð4Þ The model nested the within-category and between-category sales effects of promotion by healthiness, which, again, were es- timated separately in the regression analysis. All models were estimated via a restricted maximum likelihood technique (27). See Supplemental Data section 3 for full technical details. The model nested the within-category and between-category sales effects of promotion by healthiness, which, again, were es- timated separately in the regression analysis. All models were estimated via a restricted maximum likelihood technique (27). See Supplemental Data section 3 for full technical details. Are there socioeconomic differences in food purchases in response to price promotions? To address this question, we constructed 3 subsamples that focused on purchases that were made by 1) high–socioeconomic status (SES) households, 2) middle-SES households, and 3) low- SES households and repeated the previous analysis (on the basis of NP scores) for each group. The SES of the household was deﬁned by the occupation of the household head using the United Kingdom Registrar General’s classiﬁcation [high: higher managerial and professional; middle: white collar and skilled manual; and low: semiskilled and unskilled manual (28, 29)]. Other socioeconomic indicators such as household income and education were not used because of a substantial number of nonresponses. Observations with missing information (such as the NP score) were excluded from the analysis (6 cases, and no imputation was made for missing variables). Stata MP Version 12 software (StataCorp) was used for all analyses. log FoPjc ¼ b0c þ b1cNPjc þ ejc ð1Þ ð1Þ FoPjc refers to the frequency of promotion of item j in category c and NP represents the nutrient proﬁle. The term ejc is the idio- syncratic error. This basic estimation was used to tell whether less-healthy items were more frequently promoted than healthier ones. We further speciﬁed that the baseline frequency of pro- motions (intercept: b0c) and the association with the NP score (slope: b1c) varied by dietary category, and this variation was a function of the genuine healthiness of each dietary category b0c ¼ c00 þ c01NPc þ u0c; b1c ¼ c10 þ c11NPc þ u1c ð2Þ ð2Þ NPc is the mean NP score of products in category c. Now, the model could distinguish between product-level (i.e., within- category) effects and category-level (i.e., between-category) effects of healthiness, which were estimated separately in the regression analysis. See Supplemental Data section 3 for full technical details. NPc is the mean NP score of products in category c. Now, the model could distinguish between product-level (i.e., within- category) effects and category-level (i.e., between-category) effects of healthiness, which were estimated separately in the regression analysis. See Supplemental Data section 3 for full technical details. Product and category healthiness The vector Zjc included a set of product-level covariates known to affect sales, including the reference price, average rate of price discount when promoted, and a set of indicators of brands (which captured the brand-speciﬁc features of each product). Frequency of promotions by NP score product sales, note that there were substantial item nonresponses in the information on the above characteristics (household in- come, education, and BMI). Figure 1 summarizes the estimation results of the hierarchical model (see Supplemental Table 3 for complete results and Supplemental Table 4 for sensitivity checks). The mean (6SD) NP score for food products at a product- category level was 4.54 6 6.96 and ranged from the healthiest at approximately 210 (fruit and vegetables) to the least healthy at ;22 (butter, margarine, and chocolate confectionery). At the individual product level, the mean was 3.72 6 9.17. The mean frequency of promotions (i.e., number of branches that ran a promotion on a product in a given week) for each product was 481.4 6 735.8 branches/wk. Figure 1A illustrates the estimated frequency of promotions by food category and shows that the frequency of promotions varied substantially across categories. The estimated mean of the log frequency was 7.25 (i.e., 1405.3 branches running promotions per product per week), with an SD across categories of 1.40 (Sup- plemental Table 3). The straight line in the graph shows the overall relation between promotions and the category-level NP score (i.e., the between-category relation). The slope coefﬁcient was 20.022 (P = 0.272), which was small and statistically in- distinguishable from zero, implying that promotions were equally likely in healthier and less-healthy food categories. Table 2 presents descriptive statistics for the number of packs of each product purchased per 1000 households in 2010 sepa- rately by NP score and sales made on and off promotion. In this table, food categories that scored $4 in the category mean NP score, and beverages that scored one or higher were classiﬁed as less-healthy categories and, otherwise, as healthier ones. Prod- ucts that scored above the median NP score within each category were classiﬁed as less-healthy versions and, otherwise, as healthier ones. In total, 6788 products were in healthier categories, and 4535 products were in less-healthy categories. Results were qual- itatively similar when a different cutoff of healthiness was used (see Supplemental Table 2 for sensitivity checks). Figure 1B shows the relation between NP score and pro- motions within each category (i.e., the within-category relation). Gradients representing the association between frequency of promotions and NP scores within each category were plotted against the mean NP score of the category. Are consumers more responsive to promotions on less-healthy products? To address this question, we investigated differential effects of the frequency of promotions on product sales by the NP score of SUPERMARKET PRICE PROMOTIONS 811 TABLE 1 TABLE 1 Household (main shoppers’) baseline characteristics by socioeconomic group1 All n High-SES2 group n Middle-SES group n Low-SES group n Age, y 48.60 6 15.843 26,986 47.59 6 15.20 5667 47.83 6 15.77 14,870 51.25 6 16.27 6449 Age groups (y), % 26,986 5667 14,870 6449 #29 11.9 10.3 12.7 11.1 30–44 36.0 41.4 37.3 28.4 45–59 26.5 25.9 26.0 28.1 $60 27.7 24.4 26.0 34.3 Sex (F), % 78.9 26,986 75.5 5667 79.7 14,870 80.3 6449 Ethnicity (whites), % 95.2 25,473 93.3 5429 95.2 14,008 96.8 6036 Household income,4 £ 172,621 6 11,002.5 20,474 25,123.1 6 12,317.1 4367 17,271.8 6 9844.2 11,299 10,099.2 6 6523.7 4808 Age ﬁnished education (y), % 25,369 5469 13,952 5948 0–15 18.6 8.6 17.3 30.8 16–18 42.4 30.6 45.6 45.8 $19 37.9 59.8 35.8 22.6 Currently in education, % 1.1 1.0 1.3 0.8 BMI, kg/m2 27.39 6 5.89 12,008 26.62 6 5.26 2898 27.27 6 5.81 6563 28.57 6 6.58 2547 Country of residence, % 26,986 5667 14,870 6449 England 86.3 87.3 86.5 85.1 Scotland 8.6 7.9 8.5 9.1 Wales 5.1 4.7 5.0 5.8 Total households, n 26,986 5667 14,870 6449 Household (main shoppers’) baseline characteristics by socioeconomic group1 4Household income was adjusted for household size and composition. Differential consumer responses to promotions by NP score Figure 2 summarizes key results of the regression analysis regarding the association between unit sales and the frequency of promotions by NP score (see Supplemental Table 9 and Supplemental Figure 1 for complete results and additional technical details). A 10% increase in the frequency of promotions was associated with an increase in sales of 27.3% (95% CI: 20.6%, 33.9%; P , 0.01) for the whole population (average effect). The sales uplift from price promotions was signiﬁcantly larger for less-healthy than for healthier food categories. An SD point increase (6.96 points) in the category mean NP score (implying that the food category became less healthy) was associated with, all else be- ing equal, an additional 7.7–percentage point increase in sales (P , 0.01; Supplemental Table 9) (i.e., the overall effect in- creased from 27.3% to 35.0%). The sales uplift was also shown within each SES group. However, the magnitude of sales uplift was greater in higher- than for lower-SES groups for both healthier and less-healthy food categories (Supplemental Table 9 and Supplemental Table 10). Moreover, SES differences in the sales uplift were more marked in healthier than in less-healthy food categories; for less-healthy food categories, the sales uplift for high-, middle-, and low-SES group was 39.5%, 35.1%, and 31.5%, respectively, whereas in healthier food categories, it was 29.7%, 21.1%, and 14.7%, respectively. By contrast, within a given category, the NP score of the product did not uniformly or signiﬁcantly moderate the effect of promotions, although for some categories, a moderation effect did exist (see Supplemental Table 9 and Supplemental Table 11 for separate regressions by product category). r WorldPanel Survey 2010. Households: n = 26,986; total products: n = 11,323. SES, socioeconomic status. red $4 nutrient proﬁling score and beverages that scored $1 were grouped in the less-healthy category and, otherwise, in the healthier category. lues). S differences in the purchasing outcome were tested. Mean numbers that do not share an asterisk () were signiﬁcantly different from each other at the 5% level on the basis of nferroni corrected The overall result was also replicated when applied to the fol- lowing 2 speciﬁc types of promotion separately: simple price reductions and multibuys (e.g., “buy-one-get-one-free” and “X for $Y” (Supplemental Tables 6 and 7). However, promotions on less-healthy versions of foods were characterized by a bigger discount rate than were those for healthier foods (gradient: 0.00163; P = 0.058; Supplemental Table 8). 1Data are from the Kantar WorldPanel Survey 2010. Households: n = 26,986; total products: n = 11,323. SES, socioeconomic status. 2Food categories that scored $4 nutrient proﬁling score and beverages that scored $1 were grouped in the less-healthy category and, otherwise, in the healthier category. 3Mean 6 SD (all such values). 4Within each column, SES differences in the purchasing outcome were tested. Mean numbers that do not share an asterisk () were signiﬁcantly different from each other at the 5% level on the basis of 2 sided t tests Tests were Bonferroni corrected TABLE 2 Number of packs purchased on and off promotion per product per 1000 households by nutrient proﬁling score1 Healthier category (n = 78 food/beverage categories)2 Less-healthy category (n = 57 food/beverage categories) Healthier version Less-healthy version Healthier version Less-healthy version On promotion Off promotion On promotion Off promotion On promotion Off promotion On promotion Off promoti Proﬁling score, n 23.2 6 3.43 1.6 6 5.5 7.3 6 7.7 16.9 6 6.3 Branches running price promotion per product per week, n 446.9 6 714.1 536.2 6 823.8 477.7 6 726.8 492.9 6 670.5 Packs purchased per product per 1000 households, n All households 30.8 6 111.9 46.1 6 107.2 34.3 6 104.6 67.1 6 262.5 18.9 6 47.0 29.0 6 38.0 21.8 6 57.3 30.0 6 38.7 High SES households (n = 5667)4 33.3 6 138.0* 47.9 6 110.8* 33.6 6 97.0* 65.5 6 247.6* 18.2 6 45.7* 27.7 6 34.9 20.7 6 58.4* 28.3 6 40.8 Middle SES households (n = 14,870)4 31.6 6 118.1* 46.6 6 116.2* 34.3 6 110.6* 68.0 6 274.6* 19.1 6 48.9* 28.8 6 39.7* 21.9 6 57.8* 29.8 6 37.0 Low SES households (n = 6449)4 26.1 6 92.2 42.7 6 102.4 34.0 6 120.1* 65.5 6 273.0* 18.1 6 57.6* 29.6 6 44.8* 21.7 6 63.3* 31.3 6 47.7 Products included in the analysis, n 4316 2472 2560 1975 1Data are from the Kantar WorldPanel Survey 2010. Households: n = 26,986; total products: n = 11,323. r WorldPanel Survey 2010. Households: n = 26,986; total products: n = 11,323. SES, socioeconomic status. red $4 nutrient proﬁling score and beverages that scored $1 were grouped in the less-healthy category and, otherwise, in the healthier category. lues). S differences in the purchasing outcome were tested. Mean numbers that do not share an asterisk () were signiﬁcantly different from each other at the 5% level on the basis of nferroni corrected SES, socioeconomic status. 2Food categories that scored $4 nutrient proﬁling score and beverages that scored $1 were grouped in the less-healthy category and, otherwise, in the healthier category. 3Mean 6 SD (all such values). 4Within each column, SES differences in the purchasing outcome were tested. Mean numbers that do not share an asterisk () were signiﬁcantly different from each other at the 5% level on the basis 2-sided t tests. Tests were Bonferroni corrected. Frequency of promotions by NP score A positive gradient implied that promotions were more frequent in less-healthy than in healthier versions within a given category. The horizontal line and associated dotted lines show the overall (average) gradient, which was 0.0165 (P = 0.462) and insigniﬁcant. Therefore, by looking at the within–food category variation, promotions were overall equally likely on healthier and less-healthy versions of the foods. At the individual category level, gradients were generally small and insigniﬁcant. However, there were a few cases in which price promotions were skewed toward less-healthy versions (e.g., cakes, cheese, and sauces; Supplemental Table 5). As for healthier food categories, Table 2 suggests that higher- SES groups bought more products from healthier versions of healthier food categories than did lower-SES groups for pur- chases made both off and on promotion. In terms of less-healthy food categories, socioeconomic differences were predominantly shown in off-promotion sales; the sales of less-healthy foods off promotion were signiﬁcantly greater for the lower-SES group than for the highest-SES group. 812 NAKAMURA ET AL. Price elasticity Effects of the reference price (or nonpromotional price) and price discount associated with a price promotion were also es- timated as control variables (Supplemental Table 9). The elas- ticity of the reference price within category was 20.64 (95% CI: 20.67, 20.61; P , 0.01), which implied that a 1% increase in the reference price led to a decrease in sales by 0.64% within a given category. The elasticity was larger for lower- than for higher- SES groups; the elasticity equaled 20.47 (95% CI: 20.51, 20.43; P , 0.01) for the high-SES group, 20.63 (95% CI: 20.66, 20.60; P , 0.01) for the middle-SES group, and 20.82 (95% CI: 20.86, 20.78; P , 0.01) for the low-SES group. The within-category elasticity of the price discount was 1.44 (95% CI: 1.32, 1.55, P , 0.01); a 1% increase in the depth of price discount led to a sales uplift by 1.44% within a given category. The effect was similar in size across SES groups, whereby it was 1.44 (95% CI: 1.31, 1.57; P , 0.01) for the high-SES group, 1.44 (95% CI: 1.32, 1.56; P , 0.01) for the middle-SES group, and 1.43 (95% CI: 1.29, 1.58; P , 0.01) for the low-SES group. Our results for the price elasticity between categories were nonsigniﬁcant for both the reference price and price discount. 813 SUPERMARKET PRICE PROMOTIONS FIGURE 1 Empirical Bayes predictions of the log frequency of price promotion for individual categories, i.e., the between-category effect (A), and empirical Bayes predictions of the association between promotions and NP score within each category, i.e., the within-category effect (B). Effects represented were derived from results of the hierarchical regression analysis (see Supplemental Data section 3 for technical details and Supplemental Table 3 for complete regression results). For both panels A and B, 95% CIs of predictions are presented. The coefﬁcient of the slope in panel A was 20.022 (P = 0.272; z test; n = 11,323; Supplemental Table 3). A positive gradient in panel B meant that promotions were more frequent in less-healthy than in healthier versions of foods within the category. The horizontal line and associated dashed lines show the overall size of effects with 95% CIs (0.0168; P = 0.462; z test; n = 11,323). NP, nutrient proﬁling. Price elasticity FIGURE 1 Empirical Bayes predictions of the log frequency of price promotion for individual categories, i.e., the between-category effect (A), and empirical Bayes predictions of the association between promotions and NP score within each category, i.e., the within-category effect (B). Effects represented were derived from results of the hierarchical regression analysis (see Supplemental Data section 3 for technical details and Supplemental Table 3 for complete regression results). For both panels A and B, 95% CIs of predictions are presented. The coefﬁcient of the slope in panel A was 20.022 (P = 0.272; z test; n = 11,323; Supplemental Table 3). A positive gradient in panel B meant that promotions were more frequent in less-healthy than in healthier versions of foods within the category. The horizontal line and associated dashed lines show the overall size of effects with 95% CIs (0.0168; P = 0.462; z test; n = 11,323). NP, nutrient proﬁling. DISCUSSION associated with price promotions was larger in less-healthy than in healthier food categories, which conﬁrmed our main hy- pothesis. Products from less-healthy food categories are often nonperishable, whereas those from healthier food categories (in particular fruit and vegetables) are perishable. Therefore, Despite earlier anecdotal evidence to the contrary, we showed that, overall, less-healthy items were no more frequently pro- moted than were healthier ones. However, after controlling for the price, price discount, and brand-speciﬁc effect, the sales uplift FIGURE 2 Effects of price promotions on sales by category-level NP score and socioeconomic group. Effects represented were predicted from the hierarchical regression analysis (see the regression model in the Analytic framework section and Supplemental Table 9). The gray bar shows the average percentage increase in sales when the frequency of promotions was raised by 10% [the bar corresponds to 10 times the coefﬁcient of log(FoP)] presented separately by socioeconomic groups. Black and white bars show effects on less-healthy and healthier food categories, respectively, in which the category-level NP score was greater or smaller, respectively, than the mean by 1 SD point, whereas other factors remained ﬁxed. The effect size corresponds to the coefﬁcient of log(FoP)3NP) multiplied by the SD. The ﬁgure shows the between-category effect only. Within-category effects were indistinguishable from zero for all groups (Supplemental Table 9) and, therefore, are not visualized. See Supplemental Data sections 3 and 6 for additional technical details. FoP, frequency of promotion; NP, nutrient proﬁling. FIGURE 2 Effects of price promotions on sales by category-level NP score and socioeconomic group. Effects represented were predicted from the hierarchical regression analysis (see the regression model in the Analytic framework section and Supplemental Table 9). The gray bar shows the average percentage increase in sales when the frequency of promotions was raised by 10% [the bar corresponds to 10 times the coefﬁcient of log(FoP)] presented separately by socioeconomic groups. Black and white bars show effects on less-healthy and healthier food categories, respectively, in which the category-level NP score was greater or smaller, respectively, than the mean by 1 SD point, whereas other factors remained ﬁxed. The effect size corresponds to the coefﬁcient of log(FoP)3NP) multiplied by the SD. The ﬁgure shows the between-category effect only. Within-category effects were indistinguishable from zero for all groups (Supplemental Table 9) and, therefore, are not visualized. See Supplemental Data sections 3 and 6 for additional technical details. DISCUSSION FoP, frequency of promotion; NP, nutrient proﬁling. 814 NAKAMURA ET AL. stockpiling during a promotion may be more likely to happen for less-healthy food categories, which could explain the ﬁnding. supermarkets. However, the policy is known to be imperfectly adhered to in places characterized by a highly competitive market, such as in central London (17). Moreover, because the original data were purchase based, we did not cover all products that were available in the market, which was a feature that could have biased our estimate of the distribution of the availability of price promotions. Higher-SES groups were more responsive than lower SES groups to promotions for both healthier and less-healthy foods. The reasons for this could not be determined from these data but may have been because the ability to respond to promotions is a function of shopping-related cognitive abilities, information, and skills [all of which have been shown to correlate with SES (30)] rather than the need to make monetary savings (31). In addition, making the most effective use of promotions may involve stockpiling items while they are on promotion, thereby requiring ﬁnancial and spatial resources, which may also have contributed to the observed social patterning in the use of promotions. Second, the current study highlighted differential responses to price promotions by social groups, with the assumption that different social groups were exposed to the same promotional environment at the national level. However, United Kingdom supermarket chains have different main target consumers and operate in different parts of the country, and hence, the pro- motional environment may be segmented by social groups. Our sensitivity analyses that looked at shoppers’ exposure to pro- motions (by taking into account the usual shopping environment for different socioeconomic groups) showed similar socioeco- nomic patterning in responses to promotions (Supplemental Table 4, Supplemental Table 12). Moreover, we did not address potential differences in purchasing across social groups within a given store. These SES differences in the responsiveness to promotions were more pronounced in healthier than in less-healthy categories (Figure 2). Table 2 also revealed that there was a signiﬁcant SES gap in the sales of healthier foods on promotion, whereas there was no such gap in the sales of less-healthy foods on promotion. These results suggested that the socioeconomic gap in the on- promotion sales was driven by differences in purchases of healthier rather than less-healthy foods. Strengths and limitations To our knowledge, the current study provides the ﬁrst pop- ulation-level quantitative assessment of the relation between the frequency of price promotions and healthiness of food purchases in the main supermarket chains in the United Kingdom. The analysis involved a considerably larger sample size than in existing studies on price promotions. Our study focused on temporary price changes that often augment the prominence of items in the store through tags and placement. Hence, the re- search usefully complemented existing studies on the role of price in healthy food purchasing more generally, which have had implications mainly for taxation or subsidization (i.e., permanent price changes). In the current study, we examined overall differences in re- sponses to price promotions by SES groups at the population level. Future research could investigate the issue at the store level so that consumers’ responses to price promotions are analyzed within the same marketing strategy and variety of food products. Detailed analysis of various types of price promotion (e.g., simple price reductions and multibuys) could be valuable. Recent evidence showed that restricting multibuys has failed to change the overall volume of alcohol purchased (42). However, it would be worthwhile to investigate if this ﬁnding applies to a broader set of (healthier and less-healthy) dietary categories. In the current study, we examined overall differences in re- sponses to price promotions by SES groups at the population level. Future research could investigate the issue at the store level so that consumers’ responses to price promotions are analyzed within the same marketing strategy and variety of food products. To our knowledge, we also provided the ﬁrst systematic as- sessment of a channel through which social disparity of food purchases and intake may occur. Although we and others pre- viously showed social patterning of diet quality (proxied, for instance, by the proportion of less-healthy foods in total intake of energy) (14, 32–34), the underlying mechanisms as well as potential policy implications have rarely been tested, except for studies on food price (35–40). Although we had hypothesized that price promotions on less-healthy foods may be a plausible mechanism, our ﬁndings led us to reject this hypothesis. Detailed analysis of various types of price promotion (e.g., simple price reductions and multibuys) could be valuable. Recent evidence showed that restricting multibuys has failed to change the overall volume of alcohol purchased (42). Implications for future research Our measure of the frequency of price promotions provided an indication of variability of price promotions for individual products at the national level, but more-reﬁned ways to measure the frequency of price promotions (e.g., via direct routine ob- servations) should be developed in future research (41). More- over, retailers and manufacturers have their own target customers. Hence, their strategies of operationalizing price promotions differ according to the social characteristics (including income and food goals) of their target consumers (15). Future studies should fully take into account the potentially different food environments provided by different retailers. DISCUSSION Third, we restricted our sample to sales data from the 11 main parties of the United Kingdom grocery retail market (which account for ;70% of the total grocery market share), thereby excluding relatively smaller grocery chains and privately owned stores. Purchasing patterns of consumers as well as marketing strategies in those stores may have been different from that of the main parties. Therefore, our ﬁndings may not be entirely generalizable. There was also an SES gap in the sales of both healthier and less-healthy foods that were made off promotion (Table 2). This result was broadly in line with the SES patterning in terms of overall purchasing shown in the previous literature (14, 32, 33). Hence, SES differences in the larger proportion of off-promotion sales laid the foundation of the SES gap in food purchasing, which was exacerbated by promotional activities. 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https://openalex.org/W4313423925	https://bmcnutr.biomedcentral.com/counter/pdf/10.1186/s40795-022-00653-1	English	null	Stunting and thinness in school-attending adolescents in Addis Ababa	BMC nutrition	2,022	cc-by	7,450	© The Author(s) 2022, corrected publication 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. Abstract Background Adolescent population Global Diet Quality Score (GDQS) is uncommon in low-income settings. Though Global Diet Quality Score is a good measure of dietary diversity, it has not been used in assessing nutritional out- comes among adolescents. Therefore, the aim of this study is to assess school-attending adolescents stunting and thinness status and associations with global diet quality scores in Addis Ababa. Methods A school-based cross-sectional study was conducted among school-attending adolescents in the age group of 10–14 years in urban settings in Ethiopia. A multi-stage stratified random sampling procedure was used to select schools and students. A total of 1200 adolescents were included in the study. Binary and Multinomial logistic regression analyses were used to determine the predictors of stunting and thinness respectively. Results The proportion of school-attending adolescents stunting was 8.17% (95% CI: 6.74%,9.85%) and their thinness status 12.66% (95% CI: 10.89%, 14.67%). The overall mean GDQS food groups for Seven days period consumed were 19.99 + 2.81 SD. Male adolescents were 1.95 times more likely to be stunted compared to female adolescents (1.95; 95%CI: 1.11,3.39). Frequent consumption of low-fat dairy increased the risk of thinness, while frequent consumption of citrus fruits and white root tubers decreased the risk of school-attending adolescents’ thinness. Conclusion The proportion of thin or stunted adolescents attending school was high still, about 1 in 10. Stunting and thinness have no association with the overall GDQS. Nutritional interventions need to consider frequent con- sumption of citrus fruits, low-fat dairy, and white roots and tubers in school adolescents’ nutritional programs. Further studies should validate the GDQS for stunting and thinness of school adolescents. Keywords School adolescents, Thinness, Stunting, Global diet quality score, Ethiopia of school adolescents are affected by malnutrition [4]. Researches in Addis Ababa showed that 15. 15.9% of school age children are underweight [5]. In Ethiopia, stunting prevalence among school adolescents range from 5–29% [4, 6–8]. Risk factors for stunting include mothers’ occupation [7, 8], mothers’ education [7], ado- lescents age [6, 7] and grade [9], dietary diversity score [6], meal frequency [9], and food insecurity status [9]. In addition, in Ethiopia, most adolescents are dependent on their parents. Therefore, parents’ education and family income have an influence on adolescent nutritional status [10, 11]. Culturally boys had more free time to participate BMC Nutrition BMC Nutrition Yallew et al. BMC Nutrition (2022) 8:159 https://doi.org/10.1186/s40795-022-00653-1 Open Access Background Adolescents are vulnerable to malnutrition because of rapid growth and development and their high macro and micronutrient demand [1]. Adolescence is also a window of opportunity for establishing lifelong dietary habits that support nutritional well-being of generations [2]. The burden of malnutrition in developing countries among adolescents is high [3]. In Ethiopia, a significant number Correspondence: Walelegn Worku Yallew walelegnwaciph@gmail.com Full list of author information is available at the end of the article © The Author(s) 2022, corrected publication 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. Yallew et al. BMC Nutrition (2022) 8:159 Page 2 of 9 in outdoor physical activities than adolescent girls; which influence their body weight [12]. school exists in the city. The survey was conducted from May to June 2019 in 20 primary schools. Schools in sub- cities such as Addis Ketema, Akaky Kaliti, Arada, Bole, Gullele, Kirkos, Kolfe Keraino, Lideta, Nifas Silk-Lafto, and Yeka were included. l Studies indicate that thinness prevalence among school adolescent ranges from 4.5–29% [6–8, 13, 14]. In low- income countries, adolescents thinness is influenced by their parents education level [7], age of the adolescent [8, 14], family income [14], dietary diversity score [6], fre- quency of meal [6] and residency [8]. In addition, expo- sure of school adolescents to unhealthy food consumption [15] around school environment may have effect on their nutritional status. Adolescents’ food choices is based on peer pressure other than nutritional composition and adolescents may perceive either excess weight or thinness as a sign of well-being and attractiveness [16, 17]. The effect is eating disorders during adolescence [18]. Participants Sample size was calculated assuming stunting prevalence of 7.2% [32], level of significance 2%, design effect of 1.5, 20 school clusters and 10% non-response rate. A total of 1078 sample size was calculated to get adequate power to answer our objective. While we have taken all 1200 students collected information to the project to acquire adequate precise information from the available data. A multi-stage stratified random sampling procedure was used to select schools and students. First, randomly two government schools were selected from each sub-city. A total of 20 schools were included in the study. From each selected school, randomly 15 students enrolled whose age were 10–14 years old from each of the grades 5–8. A total of 60 students per school were enrolled to the study. An updated student roster was used as a sampling frame to select students. A total of 1,200 school-attending ado- lescents were included in the study. f Research recommend a need for consistently measured and standardized indicators for dietary diversity [19]. Dietary diversity is one dimension of diet quality. There are different type of dietary quality assessment methods [20, 21]. Global Diet Quality Score (GDQS) is one of the recent methods to capture both under- and overnutri- tion [22]. The GDQS was designed for non-pregnant, non-lactating women of reproductive age in low- and middle-income countries (LMICs) [23–25]. The method was tested for mothers but has not been tested for ado- lescents [20, 22, 26, 27]. Focusing on adolescents’ popula- tions will benefit present and future generations in terms of health and development. Variables measurement Outcome’s definitions i Stunting and thinness were assessed according to WHO definition for adolescent. Height was measured in meters to the nearest centimetre. Weight was measured using portable standing digital scale. Measurements were transformed into height-for-age z-scores based on sex and age in year using WHO Anthro-Plus software [33]. Height-for-age z-scores were categorized according to WHO stunting cut-offs (Stunted: <− 2SD; Not stunted: ≥ −2 SD) [34]. Thinness was defined as adolescents with BMI-for-age with Z-score < − 2 SD from the median value of WHO’s 2006 reference data [34]. Dietary diversity practice has an association with underweight for school adolescents [28]. Poor dietary diversity and food variety are directly associated with inadequate micronutrient intake [29]. A review in low- and middle-income countries show that daily consump- tion of nutritious foods is low for school adolescents. On average, 46% adolescents consume daily meat, 44% fruits, and 37% vegetables [19]. Although there are improvements in nutritional problem in Ethiopia [30], the government is still off-track to achieve nutritional targets at 2025 [31]. This may help to develop nutrition plans for health and development program for this age group among national priorities. Therefore, school adolescent nutritional status assessment is very important. The aim of this study is to assess stunting and thinness in school-attending adolescents in Addis Ababa. Predictors definitions In the study the primary exposures of interest were school adolescents Global Diet Quality Score (GDQS). Adoles- cents were asked food groups consumed in the reference for the past one-week period. Points were assigned for consumption of food groups based three classifications: never or 1/wk, 2–4/wk and 5–7/wk or > 1/day. Points for each food group were then summed to give an over- all score. Global Diet Quality Score were recorded based on the past 7 days for 25 GDQS food groups. GDQS was classified in to two GDQS sub-metrics, such as GDQS positive (GDQS+) sub-metric and the GDQS negative (GDQS–) sub-metric diet quality outcomes [22, 26]. Design and setting A cross-sectional study was conducted among school- attending adolescents aged 10–14 years. The study was conducted in Addis Ababa, the capital city of Ethiopia. Administratively the city is divided in to 10 sub-cities. Addis Ababa is the largest and one of the fastest growing cities in the continent. A total of 223 primary government Yallew et al. BMC Nutrition (2022) 8:159 Yallew et al. BMC Nutrition (2022) 8:159 Page 3 of 9 Page 3 of 9 Healthy food group The GDQS + is the total score across the 16 healthy GDQS food groups, with a possi- ble range of 0 to 32. Healthy food groups are; Dark green leafy vegetables, Cruciferous vegetables, Deep orange tubers, Deep orange vegetables, Deep orange fruits, other vegetables, Citrus fruits, other fruits, Legumes, Nuts and seeds, Poultry and game meat, Fish and shellfish, Whole grains, Liquid oils, Low-fat dairy, Eggs. The mean GDQS + expressed the adolescent population healthy diet quality [22]. 0.1 kg using a calibrated SECA weighing scale. Each sub- ject weighed with light clothing and no footwear. All the measurements were taken twice; when necessary, any discrepancies resolved by a third measurement. The mean values were used for data analysis. Multinomial logistic regression was conducted to investigate factors associated with adolescent nutritional status. Adolescents’ nutritional status outcome was clas- sified as thinness, normal and overweight. Normal weight as the reference category compared to thinness and over- weight. Binary logistic regression analysis was conducted to see the association between predictor variable and stunting status of school adolescents. For stunted adoles- cent were considered as height-for-age Z-score less than − 2 standard deviation of the new WHO 2007 reference population. Unhealthy food group The GDQS − is the total score across the 7 unhealthy GDQS food groups and the 2 GDQS food groups that are unhealthy when consumed in excessive amounts, with a possible range of 0 to 17. Unhealthy food groups are; White roots and tubers, red meat, refined grains and baked goods, Processed meat, Sugar-sweetened beverages, Sweets and ice cream, High-fat dairy, Juice, and Purchased deep fried foods. The mean GDQS- expressed the adolescent population unhealthy diet quality [22]. First, univariate analysis was conducted to identify associations between individual variables and school- attending adolescent nutritional status (Stunting and thinness). Variables in the univariate analysis with P-Value less than 0.2 were included in the final model to. control confounders. Design and setting In the final model for both stunt- ing and thinness sex, age, mother occupation, father’s education, Global diet Quality Score, Dietary Diversity score were included in the model. Clustering effect was observed using school as a cluster effect. P-value < 0.05 was considered as a cut-off point for statistical signifi- cance. Statistical analyses were conducted using STATA 14 software. Overall GDQS The overall GDQS is a sum of the points across all 25 GDQS food groups. The GDQS has a pos- sible range of 0 to 49. A GDQS ≥ 23 is associated with a low risk of nutrient inadequacy, scores ≥ 15 and < 23 indi- cate moderate risk, and scores < 15 indicate high risk [22]. Dietary Diversity Score (DDS) was calculated based on 10-item food indicators. These food items were; starchy staples, pulses, nuts and seeds, dairy, meat, poultry and fish, eggs, dark green leafy vegetables, other vitamin-A rich fruits and vegetables, other vegetables, and other fruits. For each food item a score of “1” was given for groups consumed and “0” for not consumed over the last one week consumed. The total DDS range from 0 to 10. Those who scores ≥ 5 were good Minimum Dietary Diversity for Women (MDD-W) and < 5 were recorded insufficient MDD-W [35]. Socio‑demographic characteristics A total of 1200 school-attending adolescents included in the study with a median age of 13 years with interquar- tile range of 12 and 14 years. The mean age of participant was 12 years ± 1.16 years standard deviations. A quar- ter of students 316 (26.38%) had only one sibling. For the majority of school adolescent, the education level of fathers and mothers was at the primary level (Table 1). Data management and statistical analysis The overall stunting prevalence was 8.17% with 95% CI: 6.74–9.85%. From the total stunted cases 61(62.24%) are males and 37(37.76%) female school adolescents. mean score for unhealthy (GDQS-) food groups con- sumed within one week was 11.17 with ± 1.59 SD. The overall stunting prevalence was 8.17% with 95% CI: 6.74–9.85%. From the total stunted cases 61(62.24%) are males and 37(37.76%) female school adolescents. Factors associated with stunting among school adolescents Sex of school-attending adolescents were significantly associated with stunting status of school adolescents. Males were 1.95 times more likely to be stunted com- pared to female adolescents with 95% CI: 1.11,3.39 in the final model controlling other factors (Table 3). Factors associated to thinness among school adolescents Multivariable multinomial logistic regression indicates that gender and grade were statistically significant with thinness status of school adolescents. The associations between consumption of citrus fruits, low fat dairy and white roots and tubers school adolescents’ thinness sta- tus were statistically significant. Except white roots and tubers all food groups (Citrus fruits, Low fat dairy) were under healthy diet food group category. Thinness com- pared with consumption of Low-fat dairy from 2 to 4 times per week compared to non-consumption, the rela- tive risk for thinness to normal would be expected to increase by a factor of 1.66. While consumption of cit- rus fruits and white roots and tubers 2–4 times per week compared to non-consumption or once per week the risk for thinness decreased by a factor of 0.52 and 0.28 respec- tively compared to normal school adolescents’ nutritional status (Table 4). Data management and statistical analysis y School-attending adolescents frequently (5–7 times a week) consumed wholegrains (95.25%) and liquid oils (86.58%). Poultry, fish, dark green leafy vegetable, cruci- ferous vegetables, deep orange fruits, deep orange tubers were rarely consumed food groups. Legumes food group were consumed 2–4 times per week by half of the stu- dents. Juice was one of the frequently consumed food groups and 94.17% of school-attending adolescents took 5–7 times per week. The second most frequently con- sumed unhealthy food group was sugar-sweetened bever- ages, 82.08% of students consumed 5–7 times per week. Except for legumes, other vegetables, low fat dairy, and Data were collected using a pretested standard ques- tionnaire by trained data collectors. WHO Anthro-plus was used to convert height and weight measurements to Z-scores for 5–19 years [36]. Questionnaires were programmed using ODK for electronic data collection and translated into local languages (Amharic in Ethio- pia). Data collectors and supervisors were trained on research ethics, how to take consents and anthropomet- ric measurements. Regular supervision was conducted during data collection in the field. Height and Weight were measured for each school adolescent. Height was measured to the nearest 0.1 cm and weight to the nearest Page 4 of 9 Yallew et al. BMC Nutrition (2022) 8:159 Yallew et al. BMC Nutrition Table 1 Socio demographic characteristics of urban school- attending adolescents in Addis Ababa ,2019 Ethiopia, Variables Frequency Percent (%) Gender Male 543 45.2 Female 657 54.7 Age in Years 10 years 54 4.5 11 years 188 15.6 12 years 294 24.5 13 years 344 28.6 14 years 320 26.6 Fathers Education No schooling 225 23.1 Primary 430 44.2 Secondary 251 25.8 Technical and University 67 6.9 Mother Education No schooling 62 9.4 Primary 264 39.9 Secondary 237 35.8 Technical and University 99 14.9 Father/male guardian occupation Private 487 63.3 Government workers 228 29.6 Unemployment 46 5.9 Others (Paster, religious leader) 8 1.0 Mother/female guardian occupation Private 407 37.62 Government 238 22.00 Unemployment 46 4.25 Homemaker 391 36.14 Number of siblings (brother and Sister in the Household) No sibling 145 12.1 One sibling 308 25.7 Two siblings 316 26.4 Three siblings 225 18.8 Four and Above sibling 204 17.0 Table 1 Socio demographic characteristics of urban school- attending adolescents in Addis Ababa ,2019 Ethiopia, mean score for unhealthy (GDQS-) food groups con- sumed within one week was 11.17 with ± 1.59 SD. Discussionh The prevalence of stunting and thinness were 8.17% and 12.67% respectively among school-attending adolescents in Addis Ababa. In this finding there was no association between adolescents stunting and thinness with the over- all GDQS. Males and older school-attending adolescents were significantly associated with stunting status. Con- sumption of citrus fruits and white roots tubers were positively associated to thinness. While consumption of low-fat dairy for 2–4 times per week was positively asso- ciated with thinness status of school adolescents.h liquid oils, most of the students consumed healthy food groups one time per week. Only other vegetables, whole grains, and liquid oils were consumed at least 5 days in a week. The students rarely ate red meat and high fat dairy food groups. But most frequently ate sugar sweetened beverages, sweets and ice cream, and juice (Table 2).h The prevalence of stunting in this study was low com- pared to previous studies among in school adolescent [6–8]. This low stunting prevalence in our study may be due to this study conducted in urban settings [8] .In urban setting families have an opportunity to get more media exposure compared to rural setting [37]. In addi- tion, reginal variation due to sociodemographic and cli- matic variation between regions in Ethiopia may be the The overall mean score GDQS food groups consumed by school-attending adolescents one week period was 19.99 ± 2.81 Standard Deviation (SD). The one-week period mean healthy (GDQS+) sub metrics score across 16 healthy food groups was 8.81 with ± 2.86 SD. The Yallew et al. BMC Nutrition (2022) 8:159 Page 5 of 9 Yallew et al. Discussionh BMC Nutrition Table 2 GDQS food groups consumed by school-attending adolescents and their Nutritional status in Addis Ababa Healthy Food groups Never or 1/wk# of students (%) 2–4/wk# of students (%) 5 d Table 2 GDQS food groups consumed by school-attending adolescents and their Nutritional status in Addis Ababa Healthy Food groups Never or 1/wk# of students (%) 2–4/wk# of students (%) 5–7/wk or ≥ 1/ day# of students (%) Dark green leafy vegetables 940 (78.3) 253 (21.1) 7 (0.5) Cruciferous vegetables 1011 (84.5) 184 (15.3) 5 (0.4) Deep orange vegetables 838 (69.3) 345 (28.7) 17 (1.4) Deep orange fruits 1060 (88.3) 134 (11.4) 6 (0.5) Deep orange tubers 1134 (94.5) 57 (4.7) 9 (0.7) Other vegetables 61 (5.08) 143 (11.9) 996 (83.0) Citrus fruits 952 (79.3) 223 (18.5) 25 (2.1) Other fruits 910 (75.8) 271 (22.5) 19 (1.5) Legumes 181 (15.1) 425 (35.4) 594 (49.5) Nuts and seeds 1051 (87.6) 121 (10.1) 28 (2.3) Poultry 1179 (98.2) 21 (1.7) 0 (0) Fish 1194 (99.5) 6 (0.5) 0 (0) Whole grains 13 (1.1) 44 (3.6) 1143 (95.2) Liquid oils 130 (10.8) 31 (2.6) 1039 (86.5) Low fat dairy 939 (78.2) 210 (17.5) 51 (4.2) Eggs 951 (79.2) 236 (19.6) 13 (1.1) Unhealthy Food groups Never or 1/wk 2–4/wk 5–7/wk or≥ 1/day White roots and tubers 35 (2.92) 461 (38.4) 704 (58.6) Red meat 1111 (92.6) 89 (7.4) 0 (0) Processed meat 9 (0.7) 219 (18.2) 972 (81) Refined grains and baked goods 727 (60.6) 361 (30.1) 112 (9.3) Sugar-sweetened beverages 24 (2) 191 (15.9) 985 (82.1) Sweets and ice cream 81 (6.7) 348 (29) 771 (64.2) High fat dairy 927 (77.2) 230 (19.1) 43 (3.6) Juice 5 (0.4) 65 (5.4) 1130 (94.1) Fried foods eaten away from home 42 (3.5) 301 (25.1) 857 (71.4) Table 2 GDQS food groups consumed by school-attending adolescents and their Nutritional status in Addis Ababa Table 2 GDQS food groups consumed by school-attending adolescents and their Nutritional status in Addis Ababa cause of the difference compared to previous studies among school adolescents [11]. This implies the need of different setting contextual action for the implemen- tation of nutritional intervention program for adoles- cent population. Boys are more stunted than girls in our finding. Our finding is supported by similar study conducted in Ethiopia [38]. Discussionh In Ethiopia culturally boys had more free time to participate in outdoor activities compared to adolescent girls; adolescent boys physical activity may influence their body weight [39]. The male school-attending adolescents were more likely to be stunted than female adolescents; which is consistent to previous studies [13, 42]. In Ethiopia, boys have more free time to participate in outdoor physical activities compared to adolescent girls; which influence their body weight [12]. School adolescents age increases the probability of stunted increased [11], while in this study there is no statistical significance between adoles- cents age to stunting. This may be due to available differ- ence between boys and girls towards their growth spurt age [43]. y yl y g The prevalence of thinness in Our finding is lower than studies conducted in the northern 29% [8] and 14.9% [14] and 15% [7] southern part of Ethiopia. This may be due to different sociodemographic character- istics and adolescents from rural settings which are more likely to be involved in activities which need more energy expenditure [40] and different cultural charac- teristics in the rural setting [41]. While our finding was higher than compared to previous studies conducted in Ethiopia 8.8% [6], 4.9% [13], and 5.2% [4]. The high prev- alence in the study setting needs further investigation. The overall GDQS was not statically significant with school adolescents stunting and thinness. In this study the mean overall GDQS food group score was 19.99 while in non-lactating and non-pregnant women in India overall GDQS food group score was 24 [25]. The overall mean difference GDQS food group score value between in this school adolescent group and non-lactating and non-pregnant women may be due to age of the two popu- lations. This indicate that the need for further food qual- ity measurement for adolescents. Yallew et al. BMC Nutrition (2022) 8:159 Page 6 of 9 Yallew et al. Discussionh BMC Nutrition Table 3 Factors to stunting, Urban school-at NB: = p < .001, = p < .01, = p < .05 Characteristic Stunti Not stu Gender Female 620 Male 482 Grade Garde 5 280 Grade 6 273 Garde 7 276 Grade 8 273 Age of Adolescents 10 years 50 11 Years 150 12 Years 223 13 Years 282 14 Years 234 Mother/female guardian occupation Private 373 Government worker 208 Unemployment 45 Homemaker 368 Fathers’/male guardian educational status No schooling 203 Primary 395 Secondary 234 Technical and University 63 Cruciferous vegetables Never or 1/wk 923 2–4/wk 175 5–7/wk or > 1/day 4 Other fruits Never or 1/wk 828 2–4/wk 257 5–7/wk or > 1/day 17 Legumes Never or 1/wk 171 2–4/wk 392 5–7/wk or > 1/day 539 Liquid oils Never or 1/wk 125 2–4/wk 30 5–7/wk or > 1/day 947 Sweets and ice cream Never or 1/wk 79 2–4/wk 321 5–7/wk or > 1/day 702 Table 3 Factors to stunting, Urban school-attending adolescents in Addis Ababa, 2019 Ethiopia NB: = p < .001, = p < .01, = p < .05 Characteristic Stunting Status Crude OR (95% CI) Adjusted OR (95% CI) Not stunted Stunted Gender Female 620 37 1 1 Male 482 61 1.99 (1.437,2.768) 1.95 (1.11,3.39) Grade Garde 5 280 20 1 1 Grade 6 273 27 1.38 (0.817,2.231) 0.72 (0.30,1.71) Garde 7 276 24 1.2 (0.70,2.057) 0.49 (0.18,1.32) Grade 8 273 27 1.38 (0.814,2.239) 0.33 (0.11,0.98) Age of Adolescents 10 years 50 1 1 1 11 Years 150 10 3.187 (0.507,20.039) 2.76 (0.32,23.82) 12 Years 223 17 3.612 (0.516,25.287) 4.74 (0.56,40.22) 13 Years 282 13 2.247 (0.283,17.879) 3.58 (0.39,33.03) 14 Years 234 36 6.799 (1.094,42.259) 16.19 (1.74,150.82) Mother/female guardian occupation Private 373 34 1 1 Government worker 208 30 1.509 (0.824,2.762) 1.45 (0.73,2.87) Unemployment 45 1 0.260 (0.038,1.764) 0.39 (0.05,3.18) Homemaker 368 23 0.704 (0.448,1.106) 0.64 (0.33,1.25) Fathers’/male guardian educational status No schooling 203 22 1 1 Primary 395 35 0.832 (0.571,1.212) 1.32 (0.63,2.77) Secondary 234 17 0.693 (0.368,1.304) 0.93 (0.40,2.15) Technical and University 63 4 0.611 (0.267,1.394) 1.16 (0.32,4.17) Cruciferous vegetables Never or 1/wk 923 88 1 1 2–4/wk 175 9 0.562 (0.304,1.037) 0.62 (0.27,1.44) 5–7/wk or > 1/day 4 1 2.297 (0.0558,9.459) 2.09 (0.34,13.07) Other fruits Never or 1/wk 828 82 1 1 2–4/wk 257 14 0.573 (0.331,0.991) 0.58 (0.27,1.24) 5–7/wk or > 1/day 17 2 1.168 (0.288,4.738) 1.19 (0.26,5.51) Legumes Never or 1/wk 171 10 1 1 2–4/wk 392 33 1.405 (0.777,2.543) 1.34 (0.56,3.23) 5–7/wk or > 1/day 539 55 1.676 (0.880,3.191) 1.72 (0.74,3.99) Liquid oils Never or 1/wk 125 5 1 1 2–4/wk 30 1 0.839 (0.122,5.748) 0.98 (0.09,9.82) 5–7/wk or > 1/day 947 92 2.302 (0.9147,5.795) 2.21 (0.74,6.59) Sweets and ice cream Never or 1/wk 79 2 1 1 2–4/wk 321 27 3.142 (0.700,14.096) 1.19 (0.25,5.64) 5–7/wk or > 1/day 702 69 3.624 (0.898,14.615) 1.83 (0.41,8.11) Table 3 Factors to stunting, Urban school-attending adolescents in Addis Ababa, 2019 Ethiopia scents in Addis Ababa, 2019 Ethiopia Crude OR (95% CI) Adjusted OR (95% CI) Stunted 37 1 1 61 1.99 (1.437,2.768) * 1.95 (1.11,3.39) 20 1 1 27 1.38 (0.817,2.231) 0.72 (0.30,1.71) 24 1.2 (0.70,2.057) 0.49 (0.18,1.32) 27 1.38 (0.814,2.239) 0.33 (0.11,0.98) 1 1 1 10 3.187 (0.507,20.039) 2.76 (0.32,23.82) 17 3.612 (0.516,25.287) 4.74 (0.56,40.22) 13 2.247 (0.283,17.879) 3.58 (0.39,33.03) 36 6.799 (1.094,42.259) 16.19 (1.74,150.82) 34 1 1 30 1.509 (0.824,2.762) 1.45 (0.73,2.87) 1 0.260 (0.038,1.764) 0.39 (0.05,3.18) 23 0.704 (0.448,1.106) 0.64 (0.33,1.25) 22 1 1 35 0.832 (0.571,1.212) 1.32 (0.63,2.77) 17 0.693 (0.368,1.304) 0.93 (0.40,2.15) 4 0.611 (0.267,1.394) 1.16 (0.32,4.17) 88 1 1 9 0.562 (0.304,1.037) 0.62 (0.27,1.44) 1 2.297 (0.0558,9.459) 2.09 (0.34,13.07) 82 1 1 14 0.573 (0.331,0.991) 0.58 (0.27,1.24) 2 1.168 (0.288,4.738) 1.19 (0.26,5.51) 10 1 1 33 1.405 (0.777,2.543) 1.34 (0.56,3.23) 55 1.676 (0.880,3.191) 1.72 (0.74,3.99) 5 1 1 1 0.839 (0.122,5.748) 0.98 (0.09,9.82) 92 2.302 (0.9147,5.795) 2.21 (0.74,6.59) 2 1 1 27 3.142 (0.700,14.096) 1.19 (0.25,5.64) 69 3.624 (0.898,14.615) 1.83 (0.41,8.11) NB: *= p < .001, = p < .01, = p < .05 NB: = p < .001, = p < .01, = p < .05 Page 7 of 9 Yallew et al. Discussionh BMC Nutrition (2022) 8:159 Yallew et al. Funding Funding The research was funded by United Nations Children’s Fund ‘UNICEF’. g The research was funded by United Nations Children’s Fund ‘UNICEF’. Discussionh BMC Nutrition Table 4 Specific food item factors to thinness among Urban school-attending adolescents in Addis Ababa ,2019 Ethiopia, (n = 1200) Specific food item frequency of consumption by adolescents Thinness Crude IRR (95% CI) Adjusted IRR (95% CI) Normal Thin Gender Female 501 68 1 1 Male 446 84 1.387 (0.98, 1.957) 1.45 (1.06,1.99) * Grade Garde 5 250 32 1 1 Grade 6 228 47 1.61 (0.993,2.612) 1.80 (1.03, 3.15) Garde 7 243 30 0.965 (0.569,1.636) 1.09 (0.70,1.69) Grade 8 226 43 1.486 (0.909,2.43) 1.56 (0.94,2.61) Fathers’/male guardian educational status No schooling 178 27 1 1 Primary 336 63 1.236 (0.76,2.01) 1.19 (0.73,1.97) Secondary 206 26 0.832 (0.468,1.478) 0.76 (0.42,1.38) Technical and University 48 11 1.511 (0.6993.263) 1.49 (0.738,3.03) Deep orange vegetables Never or 1/wk 675 102 1 1 2–4/wk 261 46 1.16 (0.91,1.49) 1.05 (0.72,1.52) 5–7/wk or > 1/day 11 4 2.41 (0.79,7.36) 1.81 (0.63,5.18) Deep orange tubers Never or 1/wk 899 139 1 1 2–4/wk 44 9 1.32 (0.53,3.31) 1.14 (0.38,3.43) 5–7/wk or > 1/day 4 4 6.46 (1.57,26.55) 6.05 (0.52,70.78) Citrus fruits Never or 1/wk 740 131 1 1 2–4/wk 189 17 0.51 (0.29,0.87) 0.52 (0.26,0.99) 5–7/wk or > 1/day 18 4 1.25 (0.60,2.61) 1.63 (0.64,4.16) Low fat dairy Never or 1/wk 752 107 1 1 2–4/wk 158 35 1.56 (1.08,2.24) 1.66 (1.07,2.58) 5–7/wk or > 1/day 37 10 1.89 (0.91,3.97) 1.85 (0.76,4.49) Egg Never or 1/wk 757 120 1 1 2–4/wk 181 28 0.97 (0.67,1.42) 0.84 (0.48,1.48) 5–7/wk or > 1/day 9 4 2.80 (1.14,6.91) 2.25 (0.65,7.71) White roots and tubers Never or 1/wk 22 10 1 1 2–4/wk 347 68 0.43 (0.20,0.91) 0.44 (0.15,1.30) 5–7/wk or > 1/day 578 74 0.28 (0.13,0.59) 0.28 (0.09,0.83) Refined grains and baked goods Never or 1/wk 563 103 1 1 2–4/wk 294 34 0.63 (0.41,0.97) 0.69 (0.45,1.05) 5–7/wk or > 1/day 90 15 0.91 (0.44,1.86) 0.98 (0.44,2.18) High fat dairy Never or 1/wk 743 107 1 1 2–4/wk 172 37 1.49 (1.03,2.16) 0.99 (0.61,1.63) 5–7/wk or > 1/day 32 8 1.74 (0.65,4.62) 1.09 (0.34,3.55) Fried foods eaten away from home Never or 1/wk 35 3 1 1 2–4/wk 242 29 1.39 (0.41,4.73) 0.95 (0.27,3.33) 5 7/wk or >1/day 670 120 2 09 (0 68 6 43) 1 79 (0 56 5 72) Urban school-attending adolescents in Addis Ababa ,2019 Ethiopia, (n = 1200) Crude IRR (95% CI) Adjusted IRR (95% CI) Thin 68 1 1 84 1.387 (0.98, 1.957) 1.45 (1.06,1.99) 32 1 1 47 1.61 (0.993,2.612) 1.80 (1.03, 3.15) 30 0.965 (0.569,1.636) 1.09 (0.70,1.69) 43 1.486 (0.909,2.43) 1.56 (0.94,2.61) 27 1 1 63 1.236 (0.76,2.01) 1.19 (0.73,1.97) 26 0.832 (0.468,1.478) 0.76 (0.42,1.38) 11 1.511 (0.6993.263) 1.49 (0.738,3.03) 102 1 1 46 1.16 (0.91,1.49) 1.05 (0.72,1.52) 4 2.41 (0.79,7.36) 1.81 (0.63,5.18) 139 1 1 9 1.32 (0.53,3.31) 1.14 (0.38,3.43) 4 6.46 (1.57,26.55) 6.05 (0.52,70.78) 131 1 1 17 0.51 (0.29,0.87) 0.52 (0.26,0.99) 4 1.25 (0.60,2.61) 1.63 (0.64,4.16) 107 1 1 35 1.56 (1.08,2.24) 1.66 (1.07,2.58) 10 1.89 (0.91,3.97) 1.85 (0.76,4.49) 120 1 1 28 0.97 (0.67,1.42) 0.84 (0.48,1.48) 4 2.80 (1.14,6.91) 2.25 (0.65,7.71) 10 1 1 68 0.43 (0.20,0.91) 0.44 (0.15,1.30) 74 0.28 (0.13,0.59) 0.28 (0.09,0.83) 103 1 1 34 0.63 (0.41,0.97) 0.69 (0.45,1.05) 15 0.91 (0.44,1.86) 0.98 (0.44,2.18) 107 1 1 37 1.49 (1.03,2.16) 0.99 (0.61,1.63) 8 1.74 (0.65,4.62) 1.09 (0.34,3.55) 3 1 1 29 1.39 (0.41,4.73) 0.95 (0.27,3.33) 120 2.09 (0.68,6.43) 1.79 (0.56,5.72) Table 4 Specific food item factors to thinness among Urban school-attending adolescents in Addis Ababa ,2019 Ethiopia, (n = 1200) Specific food item frequency of consumption by Thinness Crude IRR Adjusted IRR Table 4 Specific food item factors to thinness among Urban school-attending adolescents in Addis Ababa ,2019 Ethiopia, (n = 1200) NB: = p < .001, = p < .01, =p < .05 Yallew et al. Availability of data and materials The data that support the findings of this study are available from Addis Continental Institute of Public Health, but restrictions apply to the availability of these data, which were used under license for the current study, and so are not publicly available. Data are however available from the authors upon reasonable request and with permission of Addis Continental Institute of Public Health. References 1. Das JK, Salam RA, Thornburg KL, Prentice AM, Campisi S, Lassi ZS, et al. Nutrition in adolescents: physiology, metabolism, and nutritional needs. Ann N Y Acad Sci. 2017;1393:21–33. 2. Ivers LC, Cullen KA. Food insecurity: special considerations for women1234. Am J Clin Nutr. 2011;94:1740S–1744S. 2. Ivers LC, Cullen KA. Food insecurity: special considerations for women1234. Am J Clin Nutr. 2011;94:1740S–1744S. 3. Manyanga T, El-Sayed H, Doku DT, Randall JR. The prevalence of underweight, overweight, obesity and associated risk factors among school-going adolescents in seven african countries. BMC Public Health. 2014;14:887. Consent for publication Not applicable. Consent for publication Not applicable. Consent for publication Not applicable. Not applicable. Discussionh BMC Nutrition (2022) 8:159 Page 8 of 9 Healthy food groups like citrus fruits, deep orange tubers, low fat dairy are an important diet components for humans [44]. In this study indicated that school adolescents’ thinness has association with consump- tion of citrus fruits, Low fat dairy and white roots and tubers. Almost 80% adolescents did not eat for the past seven days either citrus fruits or low-fat dairy prod- ucts. In the other way 58% and 94% of adolescents take unhealth food groups like white root tubers and Juice respectively. This is an indication of malprac- tice for the frequency of consumption of health food group. Low-fat dairy products are reduced or natu- rally low-fat dairy products (≤ 2% milk fat) [45]. The frequency and consumption of healthy diets like citrus fruits and low-fat dairy products increased the over- all GDQS score. While Consumption of unhealth food group like White roots and tubers has decreased the overall GDQS score. This inverse finding may be due to adolescent population difference compared to adult population. In addition, it indicated that the need for well-designed strong design to implement for adoles- cent population. Acknowledgements h k d k l 8. Arage G, Assefa M, Worku T. Socio-demographic and economic factors are associated with nutritional status of adolescent school girls in Lay Guyint Woreda, Northwest Ethiopia. SAGE Open Med. 2019;7:2050312119844679. Acknowledgements We thank and acknowledge team members involved in adolescent school health nutrition survey. 9. Tamrat A, Yeshaw Y, Dadi AF. Stunting and Its Associated Factors among Early Adolescent School Girls of Gondar Town, Northwest Ethiopia: A School-Based Cross-Sectional Study. BioMed Res Int. 2020;8850074. Competing interests The authors declare that they have no competing interests. Conclusionh 4. Teferi DY, Atomssa GE, Mekonnen TC. Overweight and Undernutrition in the Cases of School-Going Adolescents in Wolaita Sodo Town, Southern Ethiopia: Cross-Sectional Study. J Nutr Metab. 2018;2018:e8678561. 4. Teferi DY, Atomssa GE, Mekonnen TC. Overweight and Undernutrition in the Cases of School-Going Adolescents in Wolaita Sodo Town, Southern Ethiopia: Cross-Sectional Study. J Nutr Metab. 2018;2018:e8678561. This study revealed that the prevalence of thinness and stunting was high among urban school attending adoles- cents. The overall GDQS food score has no association with stunting and thinness status of school adolescents’ Nutritional interventions need to consider health food consumption. Consider the need for dietary quality, gen- der focused promotion interventions through effective behaviour change communications. 5. Degarege D, Degarege A, Animut A. Undernutrition and associated risk factors among school age children in Addis Ababa, Ethiopia. BMC Public Health. 2015;15:375. 6. Kahssay M, Mohamed L, Gebre A. Nutritional Status of School Going Adolescent Girls in Awash Town, Afar Region, Ethiopia. J Environ Public Health. 2020;2020:e7367139. 7. Gagebo DD, Kerbo AA, Thangavel T. Undernutrition and Associated Fac- tors among Adolescent Girls in Damot Sore District, Southern Ethiopia. J Nutr Metab. 2020;2020:5083140. Author details 1 1 Addis Continental Institute of Public Health, Po. Box 196, Addis Ababa, Ethiopia. 2 Department of Infectious Diseases Epidemiology, London School of Hygiene and Tropical Medicine, London, UK. 3 United Nations Children’s Fund, Addis Ababa, Ethiopia. 4 Department of Nutrition, Harvard TH Chan School of Public Health, Boston, MA, USA. 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W2938619174.txt	https://www.econjournals.com/index.php/ijeep/article/download/7319/4321	en		ASYMMETRIC EFFECT OF OIL SHOCKS ON FOOD PRICES IN NIGERIA: A NON LINEAR AUTOREGRESSIVE DISTRIBUTED LAGS ANALYSIS	International journal of energy economics and policy	2,019	cc-by	4,905	International Journal of Energy Economics and Policy ISSN: 2146-4553 available at http: www.econjournals.com International Journal of Energy Economics and Policy, 2019, 9(3), 128-134. Asymmetric Effect of Oil Shocks on Food Prices in Nigeria: A Non Linear Autoregressive Distributed Lags Analysis Abdulaziz Shehu1, Abdullahi Sayuti Shafii1, Nasiru Alhaji Yau2 Department of Economics, Federal University Birnin Kebbi, Nigeria, 2Department of Banking and Finance, Federal Polytechnic, Kauran Namoda, Nigeria. Email: abazizu@gmail.com 1 Received: 29 October 2018 Accepted: 13 February 2019 DOI: https://doi.org/10.32479/ijeep.7319 ABSTRACT This paper examines the asymmetric effect of oil shocks on food prices in Nigeria, using quarterly time series data for the period of January 2010 to December, 2017. Non Linear Autoregressive distributive lag framework was used to analyse both the asymmetric short run and long run effect of oil shocks on food prices. The results indicate that positive oil price shocks have a positive and significant effect on food price while negative changes have no significant effect on food prices. Thus, the paper suggests the need for the government to strengthen its effort in the development of agricultural sector as well as diversifying its sources of income. There is also need for an institution that is mandated to save and invest the excess liquidity that arises from positive oil shocks in the development of agricultural sector and other sectors of the economy. Keywords: Asymmetric, Oil Price Shocks, Food Prices, Non Linear Auto regressive Distributive Lag, Nigeria JEL Classifications: B4, E3 I. INTRODUCTION Oil sector has been one of the vital sectors in the global economy. The sector served as a source of input and energy to almost all other sectors of the economy. It also accounts for about 2.5% of the world gross domestic product (Irakli, 2015). However, in recent years, the sector suffered from serious persistent shocks in the energy market which consequently lead to obstinate fluctuations in oil price. The shocks outrageously increased oil price from US$55 in 2005 to US$147 in mid-2008. By January 2016, the oil shocks declined oil price sharply to US$30 per barrel and remained less than US$50 to the end of third quarter of 2016. Afterwards, it steadily rose oil price to around US$50 per barrel up to end of second quarter of 2017 and sluggishly stabilised around US$65 to US$70 per barrel from third quarter of 2017 to end of the second quarter of 2018 (CBN, 2018). The persistent fluctuations in crude oil price witnessed over the years have stimulated the interest of researchers in identifying its relationship with recent hike in food prices (Baffes and Dennis, 2013; Ibrahim, 2015). Meanwhile, some argued that the oil shocksfood price relations is symmetric (Baffes and Dennis, 2013; Teera, 2014; Nwako et al., 2016), meaning that that the effect of increase and decrease in oil prices on food prices are equal in opposite directions. While, others (Ibrahim, 2015; Abdulaziz et al., 2016; Olasunkanmi and Oladele, 2018) found that the relationship is asymmetric, implying that the effect of oil price decrease on food prices significantly differs from that of oil price increase. However, in both perspectives the oil shocks transmits into food prices through cost channel in which an increase in oil price upsurges cost of agricultural inputs and consequently creates an inflationary pressure on food prices. The oil shocks also passes on exchange rate of oil exporting countries by reducing their foreign earnings which abruptly increase their food import bills. Oil price shocks is reckoned to have a direct effect on the Nigerian economy as the country heavenly depends on oil export for its foreign earning and food import for the feeding of its general This Journal is licensed under a Creative Commons Attribution 4.0 International License 128 International Journal of Energy Economics and Policy \| Vol 9 • Issue 3 • 2019 Shehu, et al.: Asymmetric Effect of Oil Shocks on Food Prices in Nigeria: A Non Linear ARDL Analysis populace. In the years of global oil shocks, Nigerian economy has recorded persistent fluctuations in prices of food products. The average yearly food inflation rate in the country rose from 7.88 in 2011 to 13.05 in 2012. It then reduced to 9.27 in 2013 and despicably increased to about 17.82 in 2017 (CBN, 2017). The food price instability experienced in the country is inclined to have a significant adverse effect on the overall wellbeing of households, especially the poor ones. In an effort to prescribe policies that would reduce the adverse effect of oil shocks on food prices, several scholars have assessed oil-food price relations (Yu et al., 2006; Baffes, 2007; Zhang and Reed, 2008; Nazlioglu and Soytas, 2011; Gilbert, 2010; Nazlioglu, 2011; Baffes and Dennis, 2013; Ibrahim, 2015; Nwako et al., 2016). However, most of the studies have only given attention to their symmetric relationship despite positive and negative oil shocks may have an independent diverse effect on food prices (Nazlioglu, 2011; Ibrahim, 2015). Moreover, the outcome of their studies were mixed with different findings. Some argued that oil price has a significant relationship with food prices (Baffes, 2007; Baffes and Dennis, 2013). While others found that oil price shocks did not cause any reaction on food prices (Yu et al., 2006; Zhang and Reed, 2008; Nwako et al., 2016). The few studies on the effect of oil price shocks on food prices in Nigeria (Nwako et al, 2016; Olasunkanmi and Oladele, 2018) however focused on prices of only 5 agricultural products. It is against this background that this study examined the long and short run asymmetric effect of oil price shocks on food prices in Nigeria, using quarterly data from January 2010 to December, 2017. The period was chosen in order to capture the influence of the recent energy market shocks on food prices as well as the current recession that gripped the country between 2016 and 2017. The paper improves on existing studies by estimating the independent asymmetric short and long run effect of positive and negative changes in oil price on food prices using nonlinear autoregressive distributed lags (NARDL) model. It also extends it sample period to 2017 which made it possible to capture the recent global energy crisis on food prices in the country. The relevance of this paper to policy formulation particularly in an oil-producing economy like Nigeria cannot be over emphasised as it would help the policy makers and monetary authorities to anticipate the likely effects of oil price fluctuations on food prices in the country. It would also provide them with proper policy prescriptions to control any emerging food price crisis in the country. The paper will also be beneficial the academia seeking to understand the oil price-food asymmetric relations. To achieve the aforementioned objective, the paper is structured into five sections. Section one presents the introduction. Next section reviews related literature on the impact of oil price shocks on food prices. Section three presents the methodology. Section four discusses the empirical results, while section five gives the conclusion and policy implications. Nazlioglu and Soytas, 2011; Gilbert, 2010; Nazlioglu, 2011; Baffes and Dennis, 2013; Ibrahim, 2015; Nwako et al., 2016; Abdulaziz et al., 2016; Olasunkanmi and Oladele, 2018). In this perspective, oil price shocks refers to the rate of variation in oil price over a successive period of time due to fluctuations in either the demand or supply of oil in the international energy market. At a regional level, Jongwanich and Park (2009; 2011) examined the effect of oil shocks on food price shocks in nine Asian countries using a vector autoregressive framework. They revealed that oil price shocks exerts a minimal influences on the food inflation performance in Asia. However, food price inflation are domestically driven by domestic factors. In the same vein, Nazlioglu and Soytas (2012) examined the relationship between oil price shocks and agricultural commodity prices of 24 Asian countries. But, on the contrary they found strong evidence of transmission from world oil price to agricultural commodity prices as well a positive impact of the weak dollar on food prices. In the same vein, Teera (2014) used generalized autoregressive conditional heteroskedasticity (ARCH) (1-1) and vine copula estimation techniques to found a slight relationship between crude oil price and palm oil and soyabean oil prices in Asian countries. At individual country level, Baffes (2007) found evidence of a strong impact of oil price fluctuations on food prices. Further evidence was provided by Zhang and Reed (2008) who found that oil price changes have a significant effect on the prices of corn, soya meal and pork uptrend in China. Baffes and Dennis (2013) reaffirmed the significant relationship of oil price shocks to food prices. On the contrary, Mutuc et al. (2010) provides a strong evidence of a weak effect of petroleum prices using data on US cotton prices. Nazlioglu and Soytas (2011) reaffirms this finding by revealing neutrality of the prices wheat, maize, cotton, soybeans and sunflower to oil price changes in Turkey. A more recent study by Ibrahim (2015) revealed that there exist a long-run relation between oil price increase and food price in Malaysia while the long term oil price reduction and food price is non-existent. However, Jiranyakul (2015) failed to detect the long-run impact of oil price shocks on consumer prices in the case of Thailand. More recently, Abdulaziz et al. (2016) found a strong evidence of long- and short-run co-integration between positive oil shocks oil and food price in Indonesia. But, they revealed that negative oil price shocks has no significant impact on food prices. 2. LITERATURE REVIEW Nwako et al., 2016) provides an evidence of the neutrality of symmetric effect of oil price shocks on prices of wheat, maize, cotton, soybeans and sunflower in Nigeria. More recently, Olasunkanmi and Oladele (2018) examined the asymmetric impact of oil price shocks on prices on agricultural commodities in Nigeria using monthly data on oil prices, maize, wheat and soybean. The study found that positive oil price changes has a significant and positive impact on prices of agricultural commodities, implying that increases in oil price lead to increases in agricultural commodities. A considerable amount of literature have explored the effect of oil price shocks on food prices in developed and developing countries ((Yu et al., 2006; Baffes, 2007; Zhang and Reed, 2008; In sum, the literature suggest that there are few studies that examined the effect of the oil price shocks on food prices in Nigeria and other developing countries. The review also indicate International Journal of Energy Economics and Policy \| Vol 9 • Issue 3 • 2019 129 Shehu, et al.: Asymmetric Effect of Oil Shocks on Food Prices in Nigeria: A Non Linear ARDL Analysis that most of the existing studies have focused on the aggregate price inflation and predominantly given attention to the symmetric effect of oil price shocks on food prices. It also portrayed that there are relatively a small number of studies that examined the relationship between oil price shocks and food prices in Nigeria. Meanwhile, none of the studies captured effect of oil shocks on aggregate price of food products within the context of recession that country experienced between 2016 and 2017. This paved way for further research on the asymmetric impact of oil price shocks on food prices in Nigeria and other developing countries. 3. DATA AND METHODOLOGY 3.1. Source of Data The paper used quarterly secondary data of crude oil price, food price index and monetary policy rate of Nigeria for the period of 2010 to 2017 to examine the asymmetric effect of oil shocks on food prices. The data for all the variables were obtained online from the website of Central Bank of Nigeria (CBN), www.cenbank. org. The sample period was chosen as to capture the recent oil shocks and food price hike in the country. 3.2. Model Specification The study adopt NARDL model advanced by Shin et al. (2011) for its empirical analysis. The framework deemed most appropriate than existing methods as it allows the estimation of potential long-run and short-run asymmetries as well as dynamic relationship between oil shocks and food prices. Thus, the model for the study is specified as: FP=  0 + 1OPt + +  2 OPt − + +  3 MPRt + et t (1) Where FP =Food price index, OP+_ =Positive changes in oil price, OP =Negative changes in oil price, MPR =Monetary Policy Rate to capture monetary policy effect, β0,β1,β2,β3=long run parameters of the independent variables. Equation 1 is framed in an ARDL setting so as to capture both short run and long run impact of oil price shocks on food prices as: a ∆FPt =  0 + 1 FPt −1 +  2 OPt +−1 +  3OPt −−1 +  4 MPRt −1 + ∑1∆FPt − i i =1 b c d + ∑ 2 ∆ OPt +−1 + ∑ 3 ∆OPt −− i ∑ 4 ∆MPRt − i + et =i 1 =i 1 =i 1 (2) _ Where FP, OP+, OP and MPR are as defined in Equation 1 while a, b, c and d are lag orders of the variables. B0 is the intercept, β1 denotes coefficient of the lag of the dependent variable, β2 and β3 denotes the long run impacts of both the increase and decrease in the oil price shocks on the food price inflation respectively. Β4 represents the coefficient of monetary policy rate, which control monetary policy effect. a b i =1 i =1 ∑ϕ1 , ∑ϕ 2 , c d i =1 i =1 ∑ϕ 3 and ∑ϕ 4 measures the short-run impact of the lag of dependent variable, positive oil shocks, negative oil shocks and monetary policy rate on food 130 prices. Thus, Equation (2) reflects short run and long run asymmetric relationship of oil price shocks with food prices. 3.3. Technique of Data Analysis The paper subject each of the time series variables to the modified and robust unit root tests developed by Ng-Perron (1995) and Elliott, Rothenberg and Stock (1996). The tests give the best overall performance than the commonly used ADF and PP unit root tests in terms of small-sample size and power. The essence of the tests is to avoid spurious regression and satisfy the requirement of ARDL bound cointegration test which suggest that I (2) variable should not be involved in the estimation as it renders the computed F-statistics invalid. The study also performs a test for the presence of cointegration among the variables using ARDL bounds test approach. With the presence of cointegration in the results, the study examine the long and short-run asymmetries effect of oil price shocks on food price. The paper also conducts various diagnostic statistics tests to checked adequacy of the dynamic specification. These tests include the Jarque-Bera statistics for error normality (J-B), the LM statistics for autocorrelation up to order 2, and the ARCH statistics for ARCH up to order 2. It is worthy to note that the paper used dollar and naira equivalent price of crude oil in all its analysis in order to capture the possible oil shocks - exchange rate pass through. The naira equivalent price of crude oil was obtained by multiplying crude oil price in dollar by the prevailing naira dollar exchange rate. Hence, converting the oil price into the Nigerian Currency (Naira) capture the effect of exchange rate fluctuations on food price inflation in Nigeria. 4. RESULTS AND DISCUSSION 4.1. Trend of Oil and Food Prices in Nigeria The trend of monthly time series data of oil and food price in Nigeria presented in Figure 1a to 2b indicates that both oil and food price index have been unstable over the sample period of January to 2010 to December 2017. Figure 1 depicted that oil price outrageously increased from US$65 per barrel in 2010 to around US$120 per barrel in 2012. It then sharply decrease to about US$30 per in 2016 and remained less than US$50 to the end of third quarter of 2016. Afterwards, it steadily rise oil price to US$55 per barrel and sluggishly stabilise around US$65 per barrel to the end of 2017 (CBN, 2017). Similarly, food inflation data depicted in Figure 2 indicate that the average yearly food inflation rate in the country rose from 7.88 in 2011 to 13.05 in 2012. It then reduced to 9.27 in 2013 and despicably increased to about 17.82 in 2017. As may be observed in Figures 1 and 2 food price has exhibited higher volatility than oil price over the years with its upswing to be relatively steeper despite various policy mechanisms such as price controls and subsidies. 4.2. Unit Root and ARDL Bound Cointegration Tests The results of both Dickey Fuller -GLS and Ng-Perron unit root tests presented in Table 1 are in agreement indicating that positive and negative oil price shocks in dollar and naira models are stationary at level. Meanwhile, food price inflation and monetary International Journal of Energy Economics and Policy \| Vol 9 • Issue 3 • 2019 Shehu, et al.: Asymmetric Effect of Oil Shocks on Food Prices in Nigeria: A Non Linear ARDL Analysis Figure 1: (a) Trend of oil price in US$, (b) Trend of differenced in oil a b Figure 2: (a) Trend of Food Price, (b) Trend of differenced in food price a b Figure 3: CUSUMSQ graph for testing robustness of the stability of the mode policy rate are stationary at first difference, meaning that they are integrated of order 1. The results implies that none of the variables is I (2), which satisfies the requirement of ARDL bounds cointegration test presented in Table 2. Accordingly, the results of ARDL bounds cointegration test presented in Table 2 indicates that the respective values of F-statistics, −11.90 and 12.48 for US dollar and Nigerian currency oil price models exceed the values of critical upper bound. This International Journal of Energy Economics and Policy \| Vol 9 • Issue 3 • 2019 131 Shehu, et al.: Asymmetric Effect of Oil Shocks on Food Prices in Nigeria: A Non Linear ARDL Analysis Table 1: Unit root tests Variables FP OP+$ OP‑$ MPR FP OP+₦ OP‑₦ MPR Dickey‑fuller GLS Level First difference 1.509 −2.806* −6.705* ‑‑‑‑‑‑‑‑ −6.496* ‑‑‑‑‑‑‑‑ −1.375 −9.277* 1.509 −2.806* −7.597* ‑‑‑‑‑‑‑‑ −9.533* ‑‑‑‑‑‑‑‑ −1.375 −9.277* Level 2.837* −4.556* −4.625* −1.320 2.837* −4.721* −4.857* −1.320 NG‑Perron First difference −2.483 ‑‑‑‑‑‑‑‑ ‑‑‑‑‑‑‑‑ −4.843 −2.483 ‑‑‑‑‑‑‑‑ ‑‑‑‑‑‑‑‑ −4.843* Integration order of variable I (1) I (0) I (0) I (1) I (1) I (0) I (0) I (1) The constant and trend terms are included in the test equations and the SIC is used to select the optimal lag order in the ADF test equation. , * and * indicates 1%, 5% and 10% levels of significant respectively, Source: Authors’ Computation from Eviews 9 Output. Table 2: NARDL bounds test for cointegration Model specification U.S. Dollar ($) oil price model Domestic currency (Naira‑₦)) oil price model F‑Statistics 5.104 4.526 95% Lower boundary 2.79 2.79 95% Upper boundary 3.67 3.67 Conclusion Cointegration Cointegration The critical values are obtained from Narayan (2005), Source: Authors’ Computation from Eviews 9 Output Table 3: Short run effect of oil shocks on food price (U.S. Dollar ($) oil price model) Dependent variable: Food price Variable Coefficient P‑value −0.142 0.21 Δ (FD(−1)) −0.073 0.45 Δ (FD(−2)) 0.036 0.05 Δ(OP+$) −0.023 0.18 Δ(OP+$(−1)) −0.012 0.29 Δ(OP−$)(−1) −0.165 0.06 Δ (MPR) −0.150 0.08 Δ (MPR(−1)) 0.583 0.02 Δ (MPR(−2)) ECM (−1) −0.522 0.01 Model summary and diagnostic tests R2=0.44, F‑statistics=4.516 (0.0000), D.W=1.89 LM−5 test=0.216 (0.90) J‑B=1.98 (0.36) ARCH−1 = 2.59 (0.11) (Domestic Currency [Naira‑₦] oil price model) Dependent variable: Food price Variable Coefficient P‑value −0.091 0.46 Δ (FD(−1)) −0.073 0.42 Δ (FD(−2)) 0.001 0.03 Δ(OP+$) 0.001 0.53 Δ(OP+$(−1)) −0.001 0.17 Δ(OP−$)(−1) −0.175 0.20 Δ (MPR) −0.464 0.12 Δ (MPR(−1)) 0.589 0.06 Δ (MPR(−2)) ECM (−1) −0.611 0.01 R2=0.44, F‑statistics=3.120 (0.0000), D.W=1.85, LM−5 test=0.4075 (0.6664) JB=1.85 (0.32) ARCH=0.75 (0.38) Values in parenthesis in the lower part represents the P value, Source: Authors’ Computation from Eviews 9 Output implies that the variables are integrated in the long run and satisfies the requirement of assessing the dynamic effect of oil price changes and monetary policy rate on food prices in Nigeria. 4.3. Model Stability Test The results of pre and post estimation tests for adequacy and specification of the model on the effect of oil price shocks on food prices presented at the lower panel of Table 3 and Figure 3 indicates that both dollar and naira oil price models passes all diagnostic tests suggesting fitness of the model, error normality, model stability, absence of autocorrelation and heterokeasticity. However, the CUSUMSQ graph for testing robustness of the stability of the model presented in Figure 3 indicates an existence of structural break in 2015. Meanwhile, the break was associated with the crash in the price of crude oil experienced in the last quarter of 2015. The results of diagnostic tests validates the short and long run effect of oil shocks on food price in Nigeria. 4.4. Effect of oil price shocks on food price The short run results presented in Table 3 reveals an existence of asymmetric relation between oil price shocks and food price 132 in both dollar and naira denominated models with only positive changes having contemporaneous and significant effect on food price. An increase in oil price by 10% leads to an immediate increase in food price by 36% in dollar model and 10% in Naira model respectively. The possible reason why the coefficient of dollar model is higher than that of naira denominated oil price model is that dollar currency is what is absolutely being used for the importation of food items into the country. The results also indicate that the lag 1 and 2 of monetary policy rate has a negative and significant effect on food prices in dollar denominated oil price model. However, the sign of the coefficient turns out to be positive at lag 3 in both models. This implies that at the initial stage, monetary policy rate reduces food prices however after the first two month it turns out to have positive and significant effect on food prices due to direct relation of amount of money in circulation and general price level. As expected, the coefficient of error correction term of the two models are significant at 5%, indicating that all the models reverts back to equilibrium roughly after two months. International Journal of Energy Economics and Policy \| Vol 9 • Issue 3 • 2019 Shehu, et al.: Asymmetric Effect of Oil Shocks on Food Prices in Nigeria: A Non Linear ARDL Analysis Table 4: Long run effect of oil shocks on food price (U.S. Dollar ($) oil price model) Dependent variable: Food price price Variable Coefficient P‑value OP+$ 0.167 0.04 OP−_$ −0.009 0.75 MPR 0.610 0.05 (Domestic currency [Naira‑₦] oil price model) Dependent variable: Food price Variable Coefficient P‑value OP+$ 0.003 0.03 OP−_$ −0.001 0.53 MPR 0.81 0.02 Source: Authors’ computation from Eviews 9 Output The long run coefficients of ARDL models presented in Table 4 suggest an asymmetric long run relation between oil price shocks and food price. The asymmetries reveals that positive oil price shocks have a positive and significant effect on food price while negative changes have no significant effect on food price. From the estimates, an increase in the price of oil by one dollar is associated with increase in food price by 16% while an increase in oil price by one naira is related to increase in food price by 0.3%. Hence, coefficient of dollar price model is higher than that of naira price model because of high rate of naira exchange rate to dollar in the country to have more impact on domestic currency than foreign currency. Our results of asymmetric relations corroborates with the finding of Ibrahim (2015) and Abdulaziz et al. (2016) who revealed that positive oil price shocks has a positive and significant effect on food inflation in Malaysia and Indonesia respectively. It also corroborates with the findings of Olasunkanmi and Oladele (2018) who found that positive oil price shocks has a positive and significant effect on prices of 3 agricultural commodities in Nigeria. Accordingly, the results revealed that monetary policy rate has a positive and significant effect on food prices. An increase in the rate by 1% will lead to increase in food prices by 61% and 81% in dollar and naira denominations oil price models respectively. One possible explanation for the slight difference in the coefficients of the two models is that increase in money supply tend have more impact on the economy at the local level than international level. 5. CONCLUSION AND RECCOMENDATIONS The paper empirically examined the asymmetric effect of oil price shocks on food prices in Nigeria from the first quota of 2010 to the last quota of 2017 using non Linear Auto regressive distributive lag model. The study reveals an existence of asymmetric relation between oil price shocks and food price in both the short run and long run. The results indicate that positive oil price shocks have a positive and significant effect on food price while negative oil price shocks have no significant and dynamic effect on food price. Accordingly, the results revealed that monetary policy rate has a positive and significant effect on food prices. The findings suggest that the government reforms that has been adopted over the years to cushion the adverse effect of oil price shocks on the economy have not made significant impact in reducing its adverse effect on food prices. It is obvious from the aforementioned findings that positive oil shocks has a significant adverse effect on food prices in Nigeria. Thus, there is the need for the government to strengthen its effort in the development of agricultural sector as well as diversifying its sources of income. There is also need for the concerned authorities to establish an institution that is mandated to save and invest the excess liquidity that arises from positive oil shocks in the development of agricultural sector and other sectors of the economy. The government also need to reduce interest rate on agricultural loans and strengthen its effort towards enlightened famers on modalities of assessing agricultural loans to the extent that they see themselves active players in the policy making process. There is also need to improve the agricultural information system as this would reduce market speculations, price hikes, and uncertainty in the economy. Lastly, the monetary authorities in the country need to rigorously pursue expansionary monetary policy that would minimise the adverse effect of oil price fluctuations on agricultural output and geared towards development of agricultural sector in the country. REFERENCES Abdulaziz, R.A., Rahim, K.A., Adamu, P. (2016), Oil and food prices co-integration nexus for Indonesia: A non-linear autoregressive distributed lag analysis. International Journal of Energy Economics and Policy, 6(1), 82-87. Baffes, J. (2007), Oil spills on other commodities. Resources Policy, 32(3), 126-134. 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(2018), Oil price shock and agricultural commodity prices in Nigeria: A non-linear autoregressive distributed lag International Journal of Energy Economics and Policy \| Vol 9 • Issue 3 • 2019 133 Shehu, et al.: Asymmetric Effect of Oil Shocks on Food Prices in Nigeria: A Non Linear ARDL Analysis (NARDL) approach. African Journal of Economic Review, 6(2), 74-91. Rothenberg, T.J., Stock, J.H. (1996), Efficient tests for an autoregressive unit root. Econometrica, 64(4), 813-836. Teera, K.S.S. (2014), Relationship between exchange rates, palm oil prices, and crude oil prices: A vine copula based GARCH approach. Advances in Intelligent Systems and Computing, 251, 399-413. Yu, T.H.E., Bessler, D.A., Fuller, S. (2006), Cointegration and Causality 134 Analysis of World Vegetable Oil and Crude Oil Prices. Long Beach: Selected Paper Prepared for Presentation at the American Agricultural Economics Association Annual Meeting. Zhang, Q., Reed, M. 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https://openalex.org/W2120706885	https://europepmc.org/articles/pmc4371823?pdf=render	English	null	Applying Value Sensitive Design (VSD) to Wind Turbines and Wind Parks: An Exploration	Science and engineering ethics	2,014	cc-by	11,930	I. Oosterlaken (&) Section Economics of Technology and Innovation; Faculty of Technology, Policy and Management, Delft University of Technology, P.O. Box 5015, 2600 GA Delft, The Netherlands e-mail: e.t.oosterlaken@tudelft.nl Sci Eng Ethics (2015) 21:359–379 DOI 10.1007/s11948-014-9536-x Sci Eng Ethics (2015) 21:359–379 DOI 10.1007/s11948-014-9536-x ORIGINAL PAPER Introduction The large-scale introduction of onshore and offshore wind turbines remains a societal challenge, despite its potential for providing sustainable energy. In the Netherlands and elsewhere issues of social acceptance have meant that offshore wind energy is increasingly being considered as an option, despite greater technical and economic difﬁculties as compared to onshore wind. However, so Hagget (2011, p. 503) points out, ‘‘the ﬁrst offshore wind farms in the UK and elsewhere around the world have not The large-scale introduction of onshore and offshore wind turbines remains a societal challenge, despite its potential for providing sustainable energy. In the Netherlands and elsewhere issues of social acceptance have meant that offshore wind energy is increasingly being considered as an option, despite greater technical and economic difﬁculties as compared to onshore wind. However, so Hagget (2011, p. 503) points out, ‘‘the ﬁrst offshore wind farms—in the UK and elsewhere around the world—have not been free from opposition.’’ Many social acceptance issues for onshore wind also apply to offshore wind (Wolsink 2010), although ‘‘maybe with slightly different character- istics than for onshore’’ (Huber and Horbaty 2010, p. 29). Considering political goals to increase sustainable energy production, it is not surprising that this issue of social acceptance of wind energy has received a lot of attention, both in practice and from researchers. In a seminal article on renewable energy innovation Wu¨stenhagen et al. (2007)identiﬁedthreeinterrelatedtypesofsocialacceptance:socio-politicalacceptance (of wind energy in general by politicians, policy makers and citizens), market acceptance (by e.g. electricity ﬁrms and investors), and community acceptance (by stakeholders, of concrete wind energy projects). This paper will focus on the latter. This paper proposes that the adoption of Value Sensitive Design or VSD may be helpful to achieve a responsible, socially acceptable implementation of wind energy. VSD is an approach that originates from the ﬁeld of ICT, but that is increasingly being applied to other technologies (Van den Hoven et al. forthcoming). VSD is based on an assumption that the conﬁguration of technology is not value-neutral, and that generally different alternatives exist, which can and should be compared and assessed against relevant values (Van de Poel 2009). It therefore aims to pro-actively take values into account throughout the design process. Any VSD project will of course have some speciﬁc object of design. As ‘wind energy’ is rather broad, a range of design projects could be distinguished within this domain. 1 This is a claim made by the NWO-funded project ‘‘Platform wars for socially responsible smart grids: the inﬂuence of stakeholder networks and platform ﬂexibility’’ (http://www.nwo.nl/onderzoek-en- resultaten/onderzoeksprojecten/26/2300178426.html, accessed June 18th 2013). Keywords Aesthetics Ecology Justice Community acceptance Social acceptance Keywords Aesthetics Ecology Justice Community acceptance Social acceptance Keywords Aesthetics Ecology Justice Community acceptance Social acceptance Keywords Aesthetics Ecology Justice Community acceptance Social acceptance Applying Value Sensitive Design (VSD) to Wind Turbines and Wind Parks: An Exploration Ilse Oosterlaken Received: 27 January 2014 / Accepted: 15 March 2014 / Published online: 18 April 2014 The Author(s) 2014. This article is published with open access at Springerlink.com Abstract Community acceptance still remains a challenge for wind energy pro- jects. The most popular explanation for local opposition, the Not in My Backyard effect, has received ﬁerce criticism in the past decade. Critics argue that opposition is not merely a matter of selﬁshness or ignorance, but that moral, ecological and aesthetic values play an important role. In order to better take such values into account, a more bottom-up, participatory decision process is usually proposed. Research on this topic focusses on either stakeholder motivations/attitudes, or their behavior during project implementation. This paper proposes a third research focus, namely the ‘objects’ which elicit certain behavioral responses and attitudes—the wind turbine and parks. More concretely, this paper explores Value Sensitive Design (VSD) as way to arrive at wind turbines and parks that better embed or reﬂect key values. After a critical discussion of the notion of acceptance versus acceptability and support, the paper discusses existing literature on ecology and aesthetics in relation to wind turbine/park design, which could serve as ‘building blocks’ of a more integral VSD approach of the topic. It also discusses the challenge of demarcating wind park projects as VSD projects. A further challenge is that VSD has been applied mainly at the level of technical artifacts, whereas wind parks can best be conceptualized as socio-technical system. This new application would therefore expand the current practice of VSD, and may as a consequence also lead to interesting new insights for the VSD community. The paper concludes that such an outcome-oriented approach of wind turbines and park is worth exploring further, as a supplement to rather than a replacement of the process-oriented approach that is promoted by the current literature on community acceptance of wind parks. 12 123 123 360 I. Oosterlaken Introduction One could think of wind turbine components, wind turbines, complete wind parks, storage facilities, HVDC converter stations, transmission networks, the integration of large-scale offshore wind parks into the electricity net, or even the electricity system as a whole—for example, a European super grid or smart grids enabling the large-scale integration of intermittent, renewable energy sources like wind energy. All of them may in principle raise certain value issues, possibly making VSD a sensible approach. However, at the moment both value and social acceptance issues are especially salient in smart grids and wind parks. For example, smart grids raise issues of privacy, security and reliability, for which VSD may offer a partial solution.1 This paper focusses on wind parks, and as a 12 3 361 Applying Value Sensitive Design derivative also on wind turbines—as these are obviously the deﬁning element of wind parks. The application of Value Sensitive Design to wind parks and turbines could arguably contribute to solutions that are more acceptable from the perspective of relevant values such as justice, sustainability, and well-being. That implementing this idea in practice could contribute to a larger degree of actual social acceptance is merely a hypothesis at this moment, the proof of which is beyond the scope of this paper. The paper will also not engage in evaluating concrete turbine or wind park designs from a value perspective, or investigate any actual design processes in this domain. Rather, the paper takes a step back and provides an in-depth exploration of the idea of applying VSD to wind parks and turbines, informed by different bodies of literature. One key issue is that wind parks may be best understood as socio- technical systems, whereas VSD has traditionally focused on technical artifacts. Another, somewhat related, key issue is the demarcation wind park projects as feasible VSD projects. This new application would therefore expand the current practice of VSD, and may as a consequence also lead to interesting new insights for the VSD community. The structure of the paper is as follows. It will ﬁrst discuss the importance of values in community acceptance of wind parks. After a brief critical reﬂection on acceptance—contrasting it with both acceptability and support—the VSD approach will be introduced. Next, I will discuss what leads and building blocks for a VSD approach are being offered in the current literature on wind energy. Introduction This sets the stage then for a discussion of the two key issues or challenges mentioned above. The paper will end with some conclusions. The Importance of Values in Wind Park Community Acceptance According to the well-known NIMBY (‘‘Not in My Backyard’’) explanation, local residents reject a wind project in their geographical vicinity because they are trying to maximize individual utility, despite them having—in recognition of the common good—a positive attitude towards wind energy in general. It is thus, just like the prisoner’s dilemma and the tragedy of the commons, a speciﬁc form of a social dilemma. Invoking the NIMBY explanation is often accompanied by authorities and experts judging local people to be ignorant, irrational or selﬁsh. The solution is generally sought in providing people with more knowledge or information about why the project would be beneﬁcial to society or why the risks would be acceptable, or in introducing more strict top-down planning procedures—ignoring the real arguments at stake (Wolsink 2006). In the past decade or so, however, a range of studies has quite forcefully criticized the NIMBY explanation (see e.g. Wolsink 2006; Haggett 2011), by showing that people often (also) have non-selﬁsh and more complex reasons for their opposition. This opposition is often closely tied up with moral or public values. As Kempton et al. (2005 p. 124) put it: We have three reasons for our not using this term [NIMBY]. First, it is generally used as a pejorative implying selﬁshness as an underlying cause; 12 3 362 I. Oosterlaken second, it appears to incorrectly describe much local opposition to wind projects; and third, the actual causes of opposition are obscured, not explained, by the label. In this section I will discuss some of the many research ﬁndings that point towards the importance of various kinds of values for the explanation of either opposition to or acceptance of wind energy projects.2 Wolsink (2000), for example, concludes from a survey in the US and the Netherlands that ‘‘most people with [alleged] NIMBY-feelings are not so much in favor of wind power at all’’ (p. 54), and that ‘‘the strongest impact on the [general] attitude [towards wind power] concerned the aesthetic value of wind turbines (p. 51).’’3 The importance of aesthetics is conﬁrmed by the results of a study of Ladenburg and Dubgaard (2007, p. 3 Note though that according to Wolsink (2007, p. 1193) ‘‘the variance of the landscape and scenic aspects of wind power is greater than for the other categories [of possible consequences of the application of wind energy], indicating that this is the aspect about which there is most disagreement.’’ 2 It is worth noting though hat according to Bidwell (2013 p. 190) ‘‘there is [still] a lack of empirical evidence for how values inﬂuence attitudes towards wind energy development.’’ 2 It is worth noting though hat according to Bidwell (2013 p. 190) ‘‘there is [still] a lack of empirical evidence for how values inﬂuence attitudes towards wind energy development.’’ 3 Note though that according to Wolsink (2007, p. 1193) ‘‘the variance of the landscape and scenic aspects of wind power is greater than for the other categories [of possible consequences of the application of wind energy], indicating that this is the aspect about which there is most disagreement.’’ The Importance of Values in Wind Park Community Acceptance 4068/69) in Denmark, which ‘‘strongly indicate that even if a large proportion of respondents (and people in general) are unable to see offshore wind farms on a daily basis [because there are none within sight from their residence or summer house], the visual disamenities are still perceived as being important.’’ This is indicated by their ‘‘willingness to pay’’ for ‘‘siting wind farms further offshore to reduce the visual disamenities.’’ And for those who do have a positive general attitude towards wind power, says Wolsink (2000) ‘‘the decision to support or oppose such a [concrete] project will depend primarily on the visual quality of the [selected] site’’ (p. 51). Either way, contra NIMBY, ‘‘the personal assessment of the beneﬁts of wind power hardly enters the argument’’ (p. 56). It is rather aesthetic values held by people that seem to be crucial. y p p Furthermore, the NIMBY explanation—ascribing selﬁsh motives to people— seems to completely ignore the possibility that local opposition may actually be based on a plausible claim of injustice taking place. Overall societal cost-beneﬁt analyses tend to ignore the question whether the beneﬁts and the costs or risks of an initiative are fairly distributed over different groups in society (distributive justice), whereas this is actually an important ethical issue for many new technologies or technological projects (Asveld and Roeser 2009). Indeed, so Wolsink (2007, p. 1188) concludes for wind power implementation more speciﬁcally, ‘‘feelings about equity and fairness appear the determinants of ‘backyard’ motives, instead of selﬁshness.’’ Perceptions of fairness, says Wolsink (2007, p. 1203), are amongst others ‘‘strongly connected with […] core values about how society should take such decisions, not only within the public, but among all stakeholders involved in such processes’’ (procedural justice). Based on seven Australian wind farm cases and using grounded theory, Hall et al. (2013) recently concluded that the stakeholder concerns with the most impact on social acceptance were related to four value themes: trust, distributive justice, procedural justice, and place attachment. Distributive justice and procedural justice were already mentioned. Trust is closely connected to procedural justice, and to 12 23 363 Applying Value Sensitive Design values such as honesty and transparency. The last one, place attachment, is relevant for people’s assessment of visual changes to a place or landscape—so for people’s aesthetic evaluation. 4 Other conceivable public responses mentioned by them are e.g. uncertainty, apathy, agreement, and resistance. The Importance of Values in Wind Park Community Acceptance In a UK offshore case study Devine-Wright and Howes (2010) found that the degree to which people’s ‘attachment to place’ leads to opposition depends on whether the project is situated in an area that is considered to be of great natural beauty, or in an area that is already industrialized or in decline. This is in line with the before mentioned ﬁnding of Wolsink (2000) that the quality of the project site is an important factor. Again, opposition is thus not a matter of people simply trying to maximize individual utility—although it may of course be that some people are actually ‘NIMBY’s’ (Bell et al. 2013). 5 It should be noted though that power relations between stakeholders may prevent a participatory design or decision-making process leading to a fair/just outcome (a point that was already raised by the discussion on acceptance versus ‘acceptability’ in the previous section). See also e.g. the case discussed by Anderson (2013), in which a minority group with strong social capital was able to prevail in a local wind farm conﬂict. Another group, larger but with low social capital, was unable to bring their view on the development across and inﬂuence the process. Anderson suggests that different participatory processes are needed for both types of groups. See also Gray et al. (2005) on the difference in bargaining power between ﬁshers and wind park developers in the case of offshore wind farms in the UK. ‘Support’ and ‘Acceptability’? A Critique of the Notion of ‘Acceptance’ The literature, so it was shown in the previous section, highlights a range of values as particularly important for the social acceptance of wind energy projects. Before discussing VSD as one way to pro-actively address value issues in wind energy, I will reﬂect brieﬂy on the notion of ‘acceptance’ itself—a reﬂection intended to lend further support to the exploration of alternative approaches to implementing wind energy. Firstly, I would like to draw attention to an article by Batel et al. (2013). They point out that the focus on social acceptance, and a certain interpretation of this concept, has been taken for granted in the renewable energy literature. They present some empirical evidence that a distinction between acceptance and support exists.4 Acceptance, so they explain, is a passive reaction to something which is proposed externally, and the absence of active opposition against something is generally taken as a sign of acceptance. Support, on the other hand, is a more action-oriented response, where people actually approve of something and are willing to defend or promote something. It implies ‘‘agency for and engagement with something’’ (p. 2). They speculate that a narrow focus on merely acceptance ‘‘might prevent the sustainability of these technologies in the long term’’ (p. 4), because it could—just like the NIMBY concept—contribute to maintaining and legitimizing a top-down planning approach. To this I would like to add that a narrow focus on acceptance could also encourage a narrow view on what values need to be taken into account, and in which way this needs to be done—namely focused on what seems most instrumentally efﬁcient in creating such acceptance in the short term. Long term support, however, might require taking a very broad range of values pro-actively and more seriously into account. Secondly, whereas Batel et al. contrast acceptance with support, Cowell et al. (2011) contrast acceptance with acceptability. Their focus is on the value of distributive justice, and the practice of developers to offer community ﬁnancial or economic beneﬁts in order to create local acceptance. ‘‘Care must be taken’’, they say, ‘‘not to elide ex ante acceptability with ex post acceptance’’ (p. 553/4)— noticing that ‘‘once a wind farm has been completed, people ﬁnd ways to accept it, 12 3 364 I. Oosterlaken as indeed people accept all sorts of unwanted outcomes […] and no longer actively resist the state of affairs’’ (p. 553). ‘Support’ and ‘Acceptability’? A Critique of the Notion of ‘Acceptance’ Furthermore, they say, apparent acceptance—in terms of lacking effective resistance—may also be a consequence of people anyways feeling/being powerless in the face of politics and societal change. There are indeed indications that the siting of renewable energy facilities tends to concentrate in areas that have the most vulnerable and marginalized communities. Blowers (2010, p. 169), looking at nuclear plant siting in the UK, concludes that the process ‘‘imposes a burden of risk on peripheral communities, least able to resist, offered neither compensation nor effective participation in decision making.’’ Similarly, Van der Horst and Toke (2010, p. 214), looking at wind farm siting in rural England, draw attention to ‘‘the strong signiﬁcance of local democratic deﬁcit (i.e. low voter turn-out) as a predictor of a ‘positive’ planning outcome.’’ In short, what is at stake is not mere acceptance, but the ethical question of acceptability. These two different ways of critiquing the focus on acceptance seem to supplement each other, and an interesting hypothesis for further research would be that a way to create long-term support for wind farms is not merely a more participatory process, but ensuring acceptability of the outcome in terms of key values. Process Versus Outcome Orientation If one accepts that values are salient for wind energy implementation, the question is then how to take them into account better. In principle it seems that there are two different approaches to doing so. The ﬁrst is improving the process of decision making at different levels, so that it reﬂects or incorporates relevant values better. The value of procedural justice is of course closely connected to this process- oriented approach, and also the value of trust comes into play here. Acknowledging the importance of values, Wolsink (2000, 2007) for example argues for more bottom-up, participatory, collaborative planning and decision-making arrangements with respect to wind energy. Indeed ‘‘frequently, participation is promoted within the [wind energy] literature as a tool with which to ensure greater public acceptance’’ (Aitken 2010 p. 1839).5 According to Jobert et al. (2007, p. 2752) ‘‘two general [research] approaches to the issue of social acceptance can be identiﬁed’’ in the literature on wind energy. The ﬁrst is ‘‘orientated towards public opinion (global and local), working with opinion polls or discussion groups to identify the motivations and attitudes of the public’’ (emphasis is mine). And the second ‘‘analyses how a project or a program is constructed to understand why it is accepted 12 123 Applying Value Sensitive Design 365 or rejected, focusing either on public policy or on actors’ behavior during the implementation’’ (emphasis is again mine). Both these types of research can provide valuable knowledge in support of this process-oriented approach to taking values seriously. or rejected, focusing either on public policy or on actors’ behavior during the implementation’’ (emphasis is again mine). Both these types of research can provide valuable knowledge in support of this process-oriented approach to taking values seriously. It seems then that the scholarly literature on the social acceptance of wind energy tends to ‘black box’ the materiality of wind parks and turbines, as design considerations and design alternatives/solutions have not received any systematic attention. This realization opens up avenues for a third research approach, one which is focused on the ‘objects’ towards which public motivation/attitudes are directed, the ‘objects’ which elicit certain behavioral responses in the process of their development: the wind parks and turbines themselves. Such research could be supportive of a second way to taking values more seriously into account in practice, namely making sure that outcomes—wind parks and turbines—better reﬂect these values. 6 Gross (2007) extensively discusses the complexity of the relationship between ‘process fairness/justice’ and ‘outcome fairness/justice’ in relation to a wind energy project. She shows amongst others that in her case study ‘‘attitudes regarding the legitimacy of the outcome were inﬂuenced by perceptions of fairness [of the process].’’ Value Sensitive Design VSD takes, according to Friedman and Kahn (2003, p. 1178), an ‘‘interactional’’ position on the question of ‘‘how exactly […] values become implicated in technological design.’’ This position means taking a middle road between technological and social determinism, acknowledging that both design features and usage or application in a certain context matter. A distinguishing feature of VSD is that it does not focus on a single value—such as e.g. ‘design for sustainability’ or ‘privacy by design’ does—but rather provides a general overarching framework to address a range of values throughout the design process. Its basic approach is that an iterative, tripartite process is needed in which conceptual, technical and empirical investigations are being integrated (Friedman et al. 2001; 2006). The conceptual phase concerns ‘‘philosophically informed analyses of the central constructs and issues under investigation’’ (Friedman et al. 2001, p. 2). Key questions include which values are relevant, how they should be understood (what do we mean with e.g. well-being or distributive justice?), and which trade-offs between conﬂicting values are acceptable (is lowering safety levels acceptable for achieving sustainability?). The technical investigation looks into the question ‘‘how existing technological properties and underlying mechanisms support or hinder human values’’ and involves ‘‘the proactive design of systems to support values identiﬁed in the conceptual investigation’’ (Friedman et al. 2001, p. 3). Empirical investigations, ﬁnally, complement conceptual and technical investigations. Examples are research into aspect of the context of implementation that co-determine to what degree values will in the end be realized, and stakeholder research into people’s perception of relevant values and their proper conceptual- ization. It has also been proposed that VSD always includes the activities of the discovery, translation and veriﬁcation of values (Flanagan et al. 2008). Van de Poel (2013) discusses the activity of translation in more detail. He proposes using a ‘‘value hierarchy’’ of three levels: values need to be translated into norms, which in turn have to be translated into design requirements.7 In practice a design may or may not realize the intended values, hence veriﬁcation is needed. The literature on VSD includes both cases studies (e.g. Cummings 2006; van Wynsberghe 2013) and more general reﬂections and discussions. One thing that has been discussed is its relationship with participatory design (PD). Borning and Muller (2012, p. 7 It may be helpful though to turn this into a hierarchy of four levels by splitting the level of value into two sub-levels, using a distinction that has amongst others been made by Rawls (1999p. 5): the distinction between the concept of a value, and the conception of a value. The concept is the general idea of a value, such as justice or sustainability. The conception is a speciﬁc interpretation or understanding of the meaning of that value. Process Versus Outcome Orientation This paper proposes Value Sensitive Design (VSD), being a pro-active approach to taking values systematically into account during the design phase, as a way to make this outcome-oriented approach more tangible and concrete. The next section will discuss VSD in more detail, but the general idea is that relevant values—such as distributive justice or well-being—should become embedded in new designs through systematic reﬂection on them during the full process of design. g g y g p g The proposal made here is not that such an outcome-oriented approach should replace a process-oriented approach. Both approaches are not mutually exclusive, but should rather be seen as interconnected6 and complementary. One can for example argue that the process matters both as an end in itself (i.e. as an expression of values like democracy, respecting people’s agency, procedural justice), and as a means towards a more high-quality, value-sensitive outcome—as stakeholders may e.g. be able to shed new light on relevant values and their meaning in a certain context. Furthermore, so Aitken (2010 p. 1839) claims, ‘‘meaningful participation must empower participants and facilitate relevant and sustainable outcomes’’ (emphasis is mine). Indeed, the design outcome is where relevant values become embedded and materialized in more or less comprehensive and suitable ways. It is the existence of realistic design alternatives that are substantially different from a value perspective that makes a deliberative, participatory decision process truly meaningful. If no such alternatives would exist and be feasible, such a process would be vacuous and redundant in an important sense. In addition one could argue that even if the conditions for making a process more fair or just are met, there is still no guarantee that the outcome of such a local process is per deﬁnition just. The outcome arguably also needs to be assessed against some wider moral standards. This is probably most obvious for moral standards or principles related to intergenerational justice; Future generations are per deﬁnition not participating in the process, even though their interests matter from a moral perspective. 12 123 I. Oosterlaken 366 Value Sensitive Design 1130) ‘‘suggest that the traditional PD commitments to co-design and power sharing be carefully considered in VSD projects as well,’’ and that VSD may beneﬁts from developments which have taken place in the PD literature. Manders-Huits (2011, p. 271), for example, voices the critique that ‘‘VSD does not have a clear methodology for identifying stakeholders’’, although Borning and Muller (2012, p. 1130) would emphasize the importance of ‘‘giving voice to the participants in the VSD study rather than prescribing particular methods.’’ 12 123 367 Applying Value Sensitive Design According to Friedman and Kahn (2003, p. 1183) VSD takes ‘‘a middle ground’’ in the debate around the universality or cultural relativity of values, ‘‘one that allows for an analysis of universal moral values, as well as allowing for these values to play out differently in a particular culture at a particular moment in time.’’ This is criticized by Manders-Huits (2011, p. 271), who ﬁnds it problematic that ‘‘the concept of values, as well as their realization, is left undetermined’’ and that ‘‘VSD lacks a complimentary or explicit ethical theory for dealing with value trade-offs’’. Contrary to that, Borning and Muller (2012, p. 1127) feel that these are the sort of issues that ‘‘VSD as such simply doesn’t need to take a position on’’, as ‘‘this doesn’t help advance the development and application of VSD’’, and may even impede it as people may dislike the speciﬁc answer given. In short, the idea of VSD still gives room to different concrete methods or normative background positions. The VSD approach has so far not been explicitly applied to wind energy, but two examples that do not concern wind energy, yet are in some respect relevant, can illustrate the idea of VSD and why it may be fruitful to explore a wind park application of VSD. The ﬁrst example concerns the Eastern Scheldt Storm Surge Barrier, which like offshore wind parks concerns the introduction of a major engineering work in a coastal area. This storm surge barrier was built in the 1970s in the Dutch province of Zeeland, as a response to a massive ﬂood disaster which took place in 1953. After the ﬂooding took place, a Delta plan was initially made that included closing off the Eastern Scheldt estuary. Value Sensitive Design This led, however, to a lot of opposition from environmental organizations, who pointed out that a valuable and unique ecological area would be lost if salty sea water and tides would no longer be part of the ecosystem. This conﬂict only became resolved when a proposal was developed to build a storm surge barrier instead, which is a barrier that is normally open, but can be closed when conditions are such that there is a risk of dangerous ﬂooding. This design solution was ‘‘a creative compromise to balance the two moral values, safety and ecological care, that were at stake’’ (Van de Poel and Royakkers 2011, p. 169). Even though this was at the time not conceptualized as VSD, the example illustrates how clever design may at least sometimes be able to solve a value conﬂict that was previously dividing people on the best way to deal with a societal challenge. The second example concerns the design of nuclear energy plants. A major decision for any plant design is whether the reactors should make use of a closed or an open fuel cycle. From an engineering perspective this choice depends on one’s assessment of the alternatives on criteria like cost, reliability, and efﬁciency. Research by Taebi and Kloosterman (2008, forthcoming) has shown, however, that these two fuel cycles are also very different from the perspective of the values of intergenerational justice, public health and safety, security and sustainability. Unfortunately it is not the case that one of the alternatives scores better on all these values, so that the choice means in effect prioritizing certain values over others. The idea of VSD is that such moral deliberation should be made explicit throughout the design process. Although this example can be taken to illustrate the idea behind VSD, it can also be taken to show that VSD is no panacea for all social acceptance issues. It is, after all, reasonable to reject both design alternatives for nuclear reactors by arguing that society should opt for other energy options instead—be it 12 3 3 368 I. Oosterlaken wind energy or something else. In fact, as every energy option will have some negative consequences, it does not sufﬁce to weigh risks and beneﬁts of a single energy technology, or try and optimize it; rather what is needed is evaluating complete energy scenarios (Dumke and Hillerbrand forthcoming). Aesthetics and Ecology in Wind Turbine and Park Design The previous section claimed that VSD has so far not been applied to wind energy. This seems to be true if one considers VSD as an approach advocated by a speciﬁc body of literature and with certain speciﬁc characteristics, such as always taking a wide range of values into account, and making use of the tripartite approach described before. Yet it is certainly not true that speciﬁc values, such as for example ecological or aesthetical values, have never before been explicitly considered in relation to wind turbine and wind park design. Concerning ecology, some examples have been included in a recent report on best practices in wind energy by the International Energy Agency (Huber and Horbaty 2013). It refers, for example, to a ‘‘nature inclusive design’’ process that has been developed in the Netherlands. Part of this approach is that ‘‘nature-development is planned in the same area [as wind turbines] and operated as one project. In consequence, the total effect of the project might be positive for nature.’’ The authors illustrate this approach with two Dutch projects, one near shore project in which ‘‘a ramp to safeguard the farm from collisions with ships is built in such a way that it will serve as a refuge for birds’’, and a project in which ‘‘turbines are built onshore on a dam. Within the wind farm project, an extra dam on the seashore was built as a nesting and refuge place for seagulls’’ (p. 18). In this way both projects achieved a positive effect on wildlife, which apparently contributed to respectively planning permission and extra support for the project in question. Another example can be found in a book by Beurskens (2011), reporting on the Dutch We@Sea research program (2004–2010) on offshore wind energy. It mentions an alternative, environmentally friendly monopole foundation which was developed for offshore wind turbines. This included research into the installation method, as monopole ramming normally causes a very sharp and intensive underwater sound, which is carried over long distances and which may harm the hearing ability of sea mammals and ﬁsh larvae. Still, gravity foundations, which do not require piling operations, might be even better if one would like to minimize wildlife disturbance (Kondili and Kaldellis 2012). And in France attempts have been made to develop and design wind parks in such a way that they lead to a bird-friendly landscape (Nadaı¨ and Labussie`re 2010). Value Sensitive Design Yet even when acknowledging this, it makes sense to also investigate the possibilities for value- sensitive design of speciﬁc energy technologies, so that the best possible design alternative can be taken into account in our scenarios and subsequent deliberations. 123 Aesthetics and Ecology in Wind Turbine and Park Design Aesthetics is amongst others discussed by Gipe (1993, 2002), who presents some concrete guidelines for how to design wind parks in such a way as to ‘‘minimize visual impact.’’ According to him the ‘‘single, most important consideration’’ for designing wind parks is ‘‘providing visual ‘unity’ in type of turbine, tower and spacing’’ (Gipe 1993, p. 245). Sharpe (2011) discusses the importance of aesthetics 123 123 369 Applying Value Sensitive Design and visual impact for a speciﬁc type of wind power application, namely single wind turbines applied in urban environments, integrated in or attached to buildings. The same type of wind power application is also being investigated by a group at Penn State University, which combines ‘‘technical, environmental and aesthetic research and design studies.’’8 An extensive treatment of the landscape aspects and aesthetics of wind turbine/park design can be found in a Dutch report by Scho¨ne (2007). According to Scho¨ne the visual effects of the youngest generation of wind parks is substantially different from the older wind parks with smaller turbines. He discusses many different aspects, such as the type of landscape, the micro-siting of the turbines and the design of the turbines themselves. Five ideas for a new way of looking at large-scale wind park design in relation to the surrounding landscape are discussed in an earlier publication by the same author (Scho¨ne 2004). This existing literature on ecology and aesthetics in relation to wind turbine and wind park design could be seen as providing building blocks for a more systematic VSD approach of wind energy in the future. A key reason to advocate such an approach is that this would enable identifying and investigating value conﬂicts and trade-offs, so that design decisions can be made in a transparent way. The examples of the design of nuclear reactors and of the Eastern Scheldt Storm Surge Barrier already illustrated why this is considered important in VSD. That this integral consideration of a range of values may also be desirable for wind turbine/parks design can be shown by the following example of aesthetics and ecology pointing in a different direction: The increased height of a wind turbine [leading to higher nominal power per turbine] means that the possible impacts of the turbine become more intensive, such as the visibility of the turbine from places of special interest, like archeological sites, tourist destinations and so on. 9 The reason why higher turbines reduce the probability of bird collision is mentioned by Mathew and Philip (2012, p. 96): as higher towers catch more wind, they have larger rotors. And ‘‘due to the larger rotor size, bigger turbines are designed to run slower to keep the optimal tip speed ratio. […] Lower rotational speed minimizes the risk of avian mortality’’. 8 See the project website at http://www.wind.psu.edu/BIWE/, accessed 29 July 2013. Aesthetics and Ecology in Wind Turbine and Park Design […] Generally, in sites with natural beauty and special esthetic, the installation of smaller wind turbines can be characterized as a secure selection, capable to protect the wind park project’s implementation from several [social acceptance] problems. On the other hand, the installation of a large number of wind turbines of lower nominal power [so low turbines] instead of few wind turbines of higher nominal power [so high turbines] increases the probability of birds’ collisions with the wind turbines’ spinning blades. […] Ornithologists [therefore] suggest the installation of [a] few [high] wind turbines of higher nominal power in large distances between them, in order to approach the total wind park’s nominal power (Al Katsaprakakis and Christakis 2012, p. 189).9 For reasons of efﬁciency larger distances often accompany the choice for higher wind turbines, as higher nominal power increases the distance needed between wind turbines to prevent ‘‘wake’’ effects between different turbines. Ornithologists, 12 3 370 I. Oosterlaken according to Al Katsaprakakis and Christakis (2012), apparently also recommend larger distances between wind mills. However large distances may—just like high wind turbines—be undesirable from an aesthetic perspective, as concentration (Scho¨ne 2007) and creating visual unity (Gipe 2002) are important for diminishing visual impact. Wind turbine height seems furthermore relevant in relation to another value, namely human well-being: ‘‘aerodynamic noise can […] be minimized by reducing the operating speed’’, and ‘‘due to the larger rotor size, bigger turbines are designed to run slower to keep the optimal tip speed ratio’’ (Mathew and Philip 2012). If such noise indeed negatively affects human well-being, this provides a reason to prefer high turbines. This short discussion is not meant to defend a particular design, but rather to illustrate that in designing a wind park explicit ethical deliberation needs to take place on how to balance values like human well- being, aesthetic pleasantness, ecological integrity, and distributive justice.10 g p g g y j An objection that one may have to the feasibility of applying VSD to wind turbines is the problem of cost-effectiveness. Cost-effectiveness is actually the main reason why wind turbines have tended to get bigger and bigger—it is still an important challenge for wind energy, although nowadays more for far-offshore than for near-shore or onshore wind parks. One might reply that cost-effectiveness seems like a very mundane consideration that should not override important value considerations. 10 These values may have further aspects or alternative interpretations not discussed so far. There may also be other consequences of this basic design choice (height/size of turbine) that have not been discussed here, but which may be relevant from a value perspective. And of course there will be other aspects of the full design that are also relevant from a value perspective. Demarcating Wind Parks as VSD Projects Whereas the emphasis in the previous section was on wind turbine design, this section will focus on the possibility of a VSD approach to wind park design. This possibility raises questions about the boundaries and scope of such design projects. I will subsequently discuss location choice, multi-space usage, and overall project set-up. Location choice is, as we also saw before, a major factor in community acceptance (Wolsink 2010)—and value-sensitivity may therefore be crucial in location choice. Whether location choice is also part of the design challenge depends. From a value perspective arriving at the best solution for an energy need may very well not be about a choice between one design or another, but about a choice between one design at one location, and another design at another location. However as planning procedures for wind farms are in general quite lengthy and costly, it may in practice not be realistic to expect a project developer to simultaneously look into several different combinations of design and location. Of course the design for a certain location will always partly depend on the features of that location. Another demarcation issue in wind park design is whether the project should concern merely the design of a wind farm, or whether the design should facilitate a multi-sector usage of the space involved. This question is speciﬁcally relevant for offshore wind farms, where competing usages include recreation, ﬁshery, (naval) transport and gas- and oil exploration. There have, for example, been proposals for technically integrating wind power production with offshore gas exploitation—the so-called ‘super wind concept’ (Hemmes et al. 2008). Another idea is that solar PV and/or wave energy converters are integrated with the supporting construction of the wind turbines (Marquis et al. 2012). The integration of marine aquaculture or ﬁsh farms within wind parks is another possibility—one which may provide a solution for conﬂicts of interest between the ﬁshery industry and wind farm developers (see Gray et al. 2005). Despite the existence of such ideas, multifunctional concepts for the design of offshore wind parks are currently, however, hardly systematically studied or even implemented.11 One reason may be that such multi-sector usage of wind parks still faces many practical obstacles, such as a lacking legal framework (Michler-Cieluch et al. 2009). 11 A positive exception is the European project Mermaid, whose systematic and structured approach to exploring ‘‘innovative multi-purpose offshore platforms’’ includes the integration of wind energy. See http://www.mermaidproject.eu/ for more information on the project. Aesthetics and Ecology in Wind Turbine and Park Design However, it might also be argued that cost-effectiveness—despite perhaps being (somewhat) at the expense of bird and landscape protection—is a requirement for achieving socio-political acceptance of a renewable energy source like wind energy, which is in turn needed to realize the value of inter-generational justice within our energy-intensive society. Whether any of these arguments make sense is partly an empirical question; For example, how important are cost considerations for creating socio-political acceptance in either the short and the long term? How big is the actual impact of certain turbines on wildlife? In that sense moral deliberation, and therefore also VSD as the context in which such deliberation takes place, could beneﬁt from ‘‘empirical investigations’’—although a moral conclusion cannot straightforwardly be drawn from any such facts. g y y With respect to the economic feasibility of the VSD of wind turbines it may also be objected that ‘‘to lower the cost of wind power still further takes mass production of turbines’’ (Wizelius 2007, p. 4). Considering economic realities it thus does not seem feasible to have a bespoke turbine design for each wind park. Yet ‘‘most manufacturers offer several models, with different hub heights and/or rotor diameters, so the turbines can be tailor-made for speciﬁc sites’’ (Wizelius 2007, p .75)—or at least to some degree. And it is not inconceivable that the problem of social acceptance in combination with the idea of VSD encourages the further development of alternative designs based on value considerations. Or at the least encourages a better articulation of the value-laden choices made in current turbine designs—which in turn would facilitate a more value sensitive turbine choice when 12 12 123 371 Applying Value Sensitive Design developing a wind park. In short, from the perspective of wind park design, the issue seems to be more a matter of value-sensitive turbine choice, than of value sensitive turbine design. developing a wind park. In short, from the perspective of wind park design, the issue seems to be more a matter of value-sensitive turbine choice, than of value sensitive turbine design. 14 Concerns have for example been expressed—based on recent psychological research ﬁndings - that such community beneﬁts may erode moral motivation (Walter 2012). A recent case study indicates that people might actually be more supportive of a local wind farm initiative when it is part of a larger vision of realizing the common good (Firestone et al. 2009). Furthermore, one may question the rationale of or justiﬁcation for community beneﬁts (compensation or fairness?), and their legitimacy (Walter 2012). 13 The expectation that it will lead to larger community acceptance is in practice a motivation for developers to provide community beneﬁts or creating community ownership (Hall et al. 2013), although there exists the risk that introducing community beneﬁts will backlash when perceived as ‘‘buying consent’’ by the local population (Huber and Horbaty 2010). Demarcating Wind Parks as VSD Projects A major reason for wanting to expand the scope of the design of offshore wind parks in such ways is that this enables cost sharing—as mentioned, making large and far offshore wind farms ﬁnancially viable is still a challenge. Nevertheless multifunctional wind parks may also contribute to the social acceptance of offshore wind, as it allows taking into consideration competing claims for the usage of space. Enlarging the scope of design in this way means allowing for 12 123 123 372 I. Oosterlaken non-conventional, innovative solutions, thereby increasing the solution space.12 This may in turn result in being able to accommodate or respect relevant values to a larger degree—just as in the case of the innovative solution for the Eastern Scheldt Storm Surge Barrier. A further question that one may ask about the scope of wind park design is whether it should, in addition to its physical and technical design, also include the overall project set-up—such as its form of ownership, or monetary schemes connected to the wind park. Widening the scope of wind park design to include such aspects would— just as with multi-space usage—increase the solution space and therefore possibly the range of values that can simultaneously be accommodated—especially distributive justice. Providing community beneﬁts is, for example, one possible way to increase the acceptability of a project.13 It does however raise questions with a moral component,14 and explicit moral deliberation seems to be desirable as certain ways of providing community beneﬁts may be more problematic than others. Cowell et al. (2011), for example, mention a case where local people preferred getting free electricity during the period that the wind turbines were in use. ‘‘Given that making electricity free might do little to reduce consumption’’, they notice (p. 552), ‘‘one can see here the tension between a rationale for community beneﬁts that prioritizes satisfying local communities, and a rationale that favors long-term environmental sustainability.’’ Thus explicit moral deliberation is required when choosing a solution. Interestingly, the speciﬁc options that are feasible and the solution ﬁnally chosen will partly depend on or be inﬂuenced by the broader institutional environment. Regarding the provision of community beneﬁts Cowell et al. 12 The author got this idea from a personal conversation with Kas Hemmes, which took place in the course of 2013. Demarcating Wind Parks as VSD Projects (2011) argue that the nature of such beneﬁt streams reﬂect wider institutional characteristics of renewable energy provision in those countries: thus in Denmark and parts of Germany, ‘community beneﬁts’ arise mainly from cooperative and farmer ownership of turbines; in France, from increased local tax revenues attendant on designating wind energy development zones; and in Spain from company agreements to invest in the regional economy. In the UK, the typical form of community beneﬁt arises where a major, commercial energy developer offers a fund, per annum, per megawatt of installed capacity, to community organisations, for spending on local projects (p. 540). VSD processes may thus be shaped by the wider institutional arrangements, and they should also be studied from this perspective. This may lead to the conclusion 12 123 373 Applying Value Sensitive Design that a re-design of such institutions, so in a sense VSD at a meta-level, might be desirable to expand the set of alternatives available in the process of the VSD of wind farms, or to increase the chances of the adoption of alternatives that seem more desirable from a value perspective. that a re-design of such institutions, so in a sense VSD at a meta-level, might be desirable to expand the set of alternatives available in the process of the VSD of wind farms, or to increase the chances of the adoption of alternatives that seem more desirable from a value perspective. One thing that would be interesting for future research is integral, in-depth case studies of different concrete design alternatives which were considered during wind park development processes. Such case studies could contribute to making this idea of VSD of wind parks more concrete and tangible. These case studies could look at e.g. the turbines involved and the micro-siting, but also at how the institutional setting constrained or facilitated the design process and/or feasible design alternatives. Such cases should address how different design alternatives were/ could/should be judged from the perspective of different values—so the reasoning involved in linking values to design proposals. Such case studies could provide content for some sort of ‘design library’, which would collect and make accessible a wide range of different design alternatives, and their evaluation from different value perspectives. This design library may become a helpful source of inspiration and information in developing new parks and choosing turbines for it. 15 This example is derived from a presentation by Douwe Monsma (inhabitant of the area) and Margrit Delmel (energy company Nuon), who have both worked on this project, at the Nationaal Windenergie Congres (May 16th 2013, the Netherlands). A further clariﬁcation of the example was acquired through a short e-mail exchange with Deimel, which took place in December 2013. Unfortunately, there are no articles or public reports that document or discuss the micro-siting choice or turbine choice made in this project. A Challenge: Wind Parks as Socio-Technical Systems The previous sections featured several ways in which institutional elements could come into play in wind park design, as ‘objects’ of design, as providing both possibilities for and constraints on design outcomes, and as either facilitating or constraining design processes. Taking a step further, it could be argued that wind parks can best not be understood as collections of technical artifacts at certain geographical locations, but rather as being integrated socio-technical systems, ‘‘engineering systems that need actors and some social/institutional infrastructure to be in place in order to perform their function’’ (Ottens et al. 2006, p. 135). Depending on where one draws the system’s boundaries, one may also say that wind parks are socio-technical systems which are embedded in an even larger socio- technical system—the national or even international energy system as a whole. Safeguarding critical functions within such large and complex systems is only possible if technology and institutions are well-aligned. Non-moral values such as reliability of these energy system may pose additional demands and conditions on the design of wind parks (Kunneke 2008; Ku¨nneke et al. 2010). The challenge is that ‘‘the design of social elements […] lies largely beyond the scope of current engineering practice’’ (Ottens et al. 2006, p. 141). In fact, the idea of designing socio-technical systems has been challenged in the literature. Not only is it the case that ‘‘appropriate comprehensive design processes and methods are still lacking’’, there is also ‘‘not even a consensus as to the prospects and limits of all- inclusive design in socio-technical systems’’ (Bauer and Herder 2009, p. 602). Although ‘‘design choices’’ in principle exist at all levels at which a socio-technical system could be deﬁned (from small and limited to large and comprehensive), at higher levels ‘‘deliberate design decisions become less prevalent and emergent characteristics become more important’’ (Bauer and Herder 2009, p. 605). Furthermore, ‘‘as all purposive decisions are made in social settings’’ what matters is not only functional and normative design criteria, but ‘‘the process of decision making and the participating stakeholders will also inﬂuence the outcomes’’ (Bauer and Herder 2009, p. 606). ‘‘Many actors within the socio-technical system are continuously changing (redesigning) the system’’, Kroes et al. (2006, p. 16 Fortunately the recent introduction of a new piece of legislation in the Netherlands, the so-called ‘‘rijkscoo¨rdinatieregeling’’, has now simpliﬁed this process by making one ministry responsible for coordinating all relevant permits and aligning the appeal procedures. Demarcating Wind Parks as VSD Projects A wind park project that could, for example, make an interesting case is Zuidlob,15 one of the biggest onshore wind parks in the Netherlands. It was developed by energy company Nuon, together with 63 agrarian companies in the area. The last of 36 turbines was installed in March 2013. The project paid a lot of attention to the distribution of costs (e.g. shadows, effects on real estate prices) and beneﬁts over the participating farmers—so to distributive justice. As the micro- siting of the wind turbines was considered important for this, ten different spatial designs were seriously taken into consideration. Crucial in the process was that that before the micro-siting design was ﬁnalized, an agreement was reached amongst participants on ‘rules’ that would lead to a fair distribution of beneﬁts. For example, the compensation for somebody who would get a windmill on his land, the compensation per kilometer of road over somebody’s land, etc. Such rules can subsequently become input for an evaluation of different design options. The project developers also wanted to keep their options with respect to turbine choice open until quite late in the process. This gave them more lead way in negotiating with turbine manufacturers—but as argued before, this could also create room for a more deliberate value sensitive choice of turbines (it is not known though if any value considerations played a role in this case). Yet postponing the turbine choice meant that the project had to apply for building permits for each of the different turbine designs considered—which was a demanding administrative undertaking. One thing that the Zuidlob case could be taken to illustrate, then, is that the feasibility of a 12 123 374 I. Oosterlaken VSD approach to wind energy projects will partly depend on the broader institutional environment enabling and constraining certain processes.16 A Challenge: Wind Parks as Socio-Technical Systems 814) notice, ‘‘which makes the idea of ‘total design control’ problematic.’’ This is reinforced, they say, by the fact that the function of socio-technical systems is often contested— whereas the intended artifact function is central to engineering design. Somewhat similarly, Bauer and Herder (2009, p. 608) claim that ‘‘socio-technical design issues often pose ‘‘wicked’’, poorly deﬁned and evolving problems. It is thus not surprising that the previous section raised questions about the boundaries and scope of wind 12 123 Applying Value Sensitive Design 375 parks as potential VSD projects. The literature on Value Sensitive Design (VSD) is not going to be helpful here either, as it has been focused mainly on software and artifact design. Within VSD there has been a lack of attention for the long-term impacts of design choices and for wider socio-technical systems (Nathan et al. 2008). In that sense, the application of VSD to wind energy may also expand our understanding of the possibilities and limitations of VSD, and provide a stimulus for expanding existing methods and tools that can be applied in VSD. p g g pp Although ‘‘the possibility of a comprehensive, outcome-oriented planning [and design] process’’ can be questioned for socio-technical systems, so Bauer and Herder (2009, p. 625) conclude, ‘‘practical experience has generated ample evidence that design choices and planning can make a signiﬁcant difference.’’ Wind parks are socio- technical systems at a level that is still concrete enough to expect that making this difference is indeed possible, and to pose that adopting ideas from the VSD literature may indeed be fruitful. Yet although VSD may lead to more acceptable solutions, it is unlikely that all parameters relevant for social acceptance will be within the scope of the design project in each case. Even with the most extensive way of framing a feasible VSD project within the wider socio-technical system, some speciﬁc design alternatives will not be feasible or within reach for all sorts of reasons—including institutional limitations. The scope and boundaries of a VSD project within this domain of wind energy may there itself become a topic for controversy. A Challenge: Wind Parks as Socio-Technical Systems One question that one may ask at this point is whether wind park development should be conceptualized and studied as a design process (adopting methods and approaches from design studies), or whether perhaps some ‘social shaping of technology’ perspective is more useful (adopting methods and approaches from the ﬁeld of science and technology studies, or STS). Insights from STS that may prove useful for the case of wind park design are for example that of the ‘‘interpretative ﬂexibility’’ and the ‘‘agency’’ of technical artifacts. However, these ways of looking at wind park development could also be considered as complementary, as they both share the assumption that the exact materiality and shape of wind parks and turbines matters from a moral and political perspective, and is not ﬁxed by what is scientiﬁcally ‘best’. Conclusion Inspired by research showing the importance of a range of moral values—such as distributive justice and sustainability—in creating social acceptance of wind parks, this article has explored the possibility of adopting a value sensitive design (VSD) approach towards wind turbines and wind parks. Research on the social acceptance of wind energy currently treats wind parks and turbines, the central ‘objects’ towards which motivation/attitudes are directed, and which elicit certain behavioral responses of stakeholders, as ‘black boxes’. As a result, this literature provides too little guidance on what concrete design alternatives are available when one wishes to develop a wind park that strikes an acceptable balance in respecting or accommodating the key values at stake. At the same time, the literature that is available on wind park and turbine design is often too much geared towards engineers and technical aspects, providing little in-depth discussion of value issues. 12 3 123 376 I. Oosterlaken Or it merely discusses a single value—such as aesthetics or sustainability—rather than taking an integral approach towards values. A VSD approach of wind parks and turbines is meant to supplement rather than replace the bottom-up, participatory process-oriented approach promoted by the current social acceptance literature. Arguably adopting an outcome-oriented, value sensitive design approach would lead to wind parks that are more acceptable from a value perspective, which in turn may lead to a greater actual community acceptance by stakeholders. Whether the latter is indeed the case remains to be seen, and will of course also depend on the process involved; Improving the acceptability of the outcome through VSD seems, at least in the short term, neither necessary nor sufﬁcient for creating community acceptance for a project. Yet such a VSD approach may be nevertheless helpful. It goes perhaps without saying that future research on this topic would have to be interdisciplinary (Taebi et al. 2014), drawing amongst others on the VSD literature and the STS literature. It was mentioned before that it would be useful to have more case studies looking—from a value perspective—into the design alternatives considered during a wind park development. These cases could provide input for some sort of ‘wind park VSD library’ which could become a resource for future wind park development projects. Conclusion Further research should—amongst others—also look into two somewhat related challenges which were discussed in the paper, namely that of establishing the boundaries of the wind energy project to which VSD will be applied, and that of applying VSD to a socio-technical system such as a wind park. Such research would not only lead to helpful insights for the application of VSD in this domain, but it could also further our understanding of VSD itself. Although this paper has identiﬁed gaps in the existing academic literature, this is of course done with the aim of stimulating research that will ultimately facilitate the transition to renewable energy—including wind energy. Considering the importance of this societal goal and the difﬁculties in achieving it, alternative approaches like the one discussed in this paper are worth considering. Acknowledgments This research has been made possible by a grant from NWO, the Netherlands Organisation for Scientiﬁc Research (grant MVI-12-E01). The basic idea of this paper—applying VSD to wind energy—was formulated in the grant proposal, which also emphasized the importance of looking into institutional design. I would like to thank the other members of the project team—Rolf Ku¨nneke (project leader), Kas Hemmes, Rafaela Hillebrand and Donna Mehos—for their input and discussions on the topic. 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https://openalex.org/W3107104675	https://www.nature.com/articles/s41438-020-00418-6.pdf	English	null	A CCAAT-binding factor, SlNFYA10, negatively regulates ascorbate accumulation by modulating the d-mannose/l-galactose pathway in tomato	Horticulture research	2,020	cc-by	10,493	A R T I C L E A R T I C L E O p e n A c c e s s © The Author(s) 2020 OpenAccessThisarticleislicensedunderaCreativeCommonsAttribution4.0InternationalLicense,whichpermitsuse,sharing,adaptation,distributionandreproduction 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 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 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 license, visit http://creativecommons.org/licenses/by/4.0/. Abstract Ascorbic acid (AsA), an important antioxidant and growth regulator, and it is essential for plant development and human health. Speciﬁcally, humans have to acquire AsA from dietary sources due to their inability to synthesize it. The AsA biosynthesis pathway in plants has been elucidated, but its regulatory mechanism remains largely unknown. In this report, we biochemically identiﬁed a CCAAT-box transcription factor (SlNFYA10) that can bind to the promoter of SlGME1, which encodes GDP-Man-3’,5’-epimerase, a pivotal enzyme in the D-mannose/L-galactose pathway. Importantly, SlNFYA10 simultaneously binds to the promoter of SlGGP1, a downstream gene of SlGME1 in the D- mannose/L-galactose pathway. Binding assays in yeast and functional analyses in plants have conﬁrmed that SlNFYA10 exerts a negative effect on the expression of both SlGME1 and SlGGP1. Transgenic tomato lines overexpressing SlNFYA10 show decreased levels of SlGME1 and SlGGP1 abundance and AsA concentration in their leaves and fruits, accompanied by enhanced sensitivity to oxidative stress. Overall, SlNFYA10 is the ﬁrst CCAAT-binding factor identiﬁed to date to negatively regulate the AsA biosynthetic pathway at multiple sites and modulate plant responses to oxidative stress. A CCAAT-binding factor, SlNFYA10, negatively regulates ascorbate accumulation by modulating the D-mannose/L-galactose pathway in tomato Weifang Chen1, Tixu Hu1, Jie Ye1, Bing Wang1, Genzhong Liu1, Ying Wang1, Lei Yuan1, Jiaming Li1, Fangman Li1, Zhibiao Ye1,2 and Yuyang Zhang 1,2 xu Hu1, Jie Ye1, Bing Wang1, Genzhong Liu1, Ying Wang1, Lei Yuan1, Jiaming Li1, Fangman Li1, Yuyang Zhang 1,2 Chen et al. Horticulture Research (2020) 7:200 https://doi.org/10.1038/s41438-020-00418-6 Chen et al. Horticulture Research (2020) 7:200 https://doi.org/10.1038/s41438-020-00418-6 Horticulture Research www.nature.com/hortres Introduction and abiotic stress so that plants can carry out normal physiological processes. For AsA biosynthesis, the major biosynthesis pathway is the D-mannose/L-galactose path- way (Wheeler & Smirnoff pathway), although an alter- native pathway in plants involving galacturonate has been proposed4,5. However, controversy exists between the D- glucose6 and myo-inositol pathways7. Between them, the pivotal biosynthetic pathway of D-mannose/L-galactose involves ten catalytic steps through which D-glucose is converted to AsA. The ﬁrst six steps involved the synth- esis of active riboside sugars, which are utilized as sub- strates for AsA synthesis, as well as precursors of cell wall polysaccharides and glycoproteins. The last four steps, starting from GDP-L-galactose, constitute the exclusive steps for AsA synthesis. The genes in the D-Man/L-Gal pathway have been identiﬁed; they encode GDP-D-Man pyrophosphorylase (GMP)8, GDP-Man-3’,5’-epimerase (GME)9, GDP-L-Gal phosphorylase (GGP)10, L-Gal-1-P Ascorbic acid (AsA, also referred to as vitamin C) contributes to nutritional quality and stress responses in plants and thus has attracted increased amounts of attention. Since humans and higher primates have lost the capability to synthesize AsA, this compound must be obtained from dietary sources rich in AsA, e.g., fresh vegetables and fruits1. AsA plays an important role in protecting human health and in disease prevention. For example, AsA can effectively control tumor progression2, and intracellular AsA helps maintain the integrity and function of several processes in the central nervous sys- tem3. In plants, AsA can remove reactive oxygen species produced by photosynthesis, respiration, biological stress, Correspondence: Yuyang Zhang (yyzhang@mail.hzau.edu.cn) 1Key Laboratory of Horticultural Plant Biology, Ministry of Education, Huazhong Agricultural University, 430070 Wuhan, China 2HZAU Chuwei Institute of Advanced Seeds, 430070 Wuhan, China Correspondence: Yuyang Zhang (yyzhang@mail.hzau.edu.cn) 1Key Laboratory of Horticultural Plant Biology, Ministry of Education, Huazhong Agricultural University, 430070 Wuhan, China 2HZAU Chuwei Institute of Advanced Seeds, 430070 Wuhan, China © The Author(s) 2020 OpenAccessThisarticleislicensedunderaCreativeCommonsAttribution4.0InternationalLicense,whichpermitsuse,sharing,adaptation,distributionandreproduction 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 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 permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. Introduction To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Page 2 of 12 Chen et al. Horticulture Research (2020) 7:200 Page 2 of 12 the CCAAT-box to regulate the expression of down- stream genes34. Sequences associated with the NFY het- erotrimer complex have been found in all the sequenced genomes of eukaryotes35, and there are one or two genes encoding NFYs in mammals and yeast36. However, in plants, the NFY family has undergone expansion34. The NFY complex was reported to be involved in the regula- tion of plant growth and development and the stress response. NFYA in wheat positively responds to low nitrogen and phosphorus: overexpression of TaNFYA-B1 stimulated root development by upregulating nitrate and phosphate transporters and, subsequently, nitrogen and phosphorus absorption in roots37. Overexpression of the transcription factor NFYC9 mediates ABA signaling by targeting ABA-responsive transcription factors such as ABI538. The expression of NFYA5 was strongly induced by drought in an ABA-dependent manner, and NFYA5 overexpression could reduce moisture loss and drought sensitivity39. However, overexpression of the wheat NFYA10 gene increased plant sensitivity to salinity, as judged on the basis of seed germination and root growth40. phosphatase (GPP)11, L-Gal dehydrogenase (GalDH)12, and L-galactono-1,4-lactone dehydrogenase (GLDH)13. GME converts GDP-D-mannose into GDP-L-galactose, yielding an alternative product of GDP-L-glucose. In most plants, there is a single copy of the GME gene14, whereas in tomato (Solanum lycopersicum), two GME genes named GME1 and GME2 have been found15. Over- expression of GME1 and GME2 could increase the AsA content, while knockdown of either GME gene decreased the AsA content16. Simultaneous suppression of the GME1 and GME2 genes by RNA interference (RNAi) could reduce the AsA content by 40–60%, with increasing cell wall mannose content and fragility17. Suppressed expression of GME1 or GME2 separately revealed differ- ent functions in cell wall synthesis18, suggesting the divergence of functions of GME family members. In plants, the AsA content is affected by various factors, such as light19,20, temperature21, minerals22, and envir- onmental cues. AsA accumulation is also regulated by hormones such as auxin23, methyl jasmonate24, jasmonic acid25, and Abscisic Acid (ABA)26. Transcription factors or regulators purportedly modulate AsA biosynthesis. amr1 (Ascorbic acid Mannose Pathway Regulator 1), an activation-tagged Arabidopsis (Arabidopsis thaliana) ozone-sensitive mutant, showed decreased AsA content when the expression of genes in the D-Man/L-Gal pathway were negatively regulated27. Introduction The photomorphogenic fac- tor COP9 signalosome subunit 5B (CSN5B) could decrease the AsA content by modulating the protein abundance of the AsA biosynthetic enzyme GDP-Man- pyrophosphorylase (VTC1)28. Arabidopsis AtERF98 and the tomato HD-Zip I family transcription factor (SlHZ24) could bind to the VTC1 promoter and increased the AsA content by positively regulating VTC1 expression29,30. Two nucleotide sugar pyrophosphorylase-like proteins from Arabidopsis, KONJAC1 (KJC1) and KJC2, could stimulate GMP enzymatic activity and increase AsA accumulation31. Moreover, a Cys2/His2-type zinc-ﬁnger protein named SlZF3 could directly interact with CSN5B, preventing CSN5B binding to GMP and thus increasing the AsA content in tomato32. More recently, an upstream open reading frame (uORF) in the long untranslated region of GGP was demonstrated to encode a peptide that functions in the feedback regulation of AsA at the post- transcriptional level33. To date, the regulation of AsA biosynthesis largely occurs at the VTC1-catalyzed step28–30,32, while the regulation of other genes in the AsA synthesis pathway deserves further investigation. NFY factors have been known to function as hetero- trimer complexes during various processes of plant growth and development41–43, although only a single NFY transcription factor has been reported to be involved in the stress response in plants. However, the regulatory mechanism through which metabolites, e.g., AsA, accu- mulate has not been elucidated. In this study, an NFYA transcription factor was determined to negatively regulate AsA biosynthesis at multiple sites in the D-mannose/L- galactose pathway. Materials and methods Yeast one-hybrid A yeast one-hybrid assay was carried out via a Match- maker One-Hybrid Library Construction and Screening Kit (Clontech, http://www.clontech.com/) according to the manufacturer’s instructions. A 2-kb promoter frag- ment of SlGME1 from the initiation site, as well as a mutated fragment of the SlGME1 promoter were ampli- ﬁed from tomato genomic DNA and then cloned into a pAbAi vector (Clontech). The full-length and truncated NFYA CDSs were ampliﬁed from tomato complementary DNA (cDNA) via PCR and cloned into a pGADT7 vector (Clontech); the primer sequences used are listed in Sup- plementary Table S2. Both the pHIS2.0 bait vector and the pGADT7 prey vector were introduced into Y1H Gold yeast (Clontech) and cultured on SD/-Leu-Ura media. After 3 days, the positive yeast strains were selected and diluted in sterilized distilled water to an OD600 of 0.1, and 2.5 μl of suspension was spotted onto SD/-Leu-Ura media, with or without AbA (Clontech). The plates were subse- quently incubated for 3–7 days at 30 °C. NFYAs belong to the NFY (Nuclear Factor Y, also known as Heme activator protein or CCAAT-binding factor) complex, which contains NFYAs, NFYBs and NFYCs, and function as heterodimers. After dimerization between NFYB and NFYC in the cytoplasm, NFYA is further recruited to form a heterotrimer. NFYA binds to Page 3 of 12 Chen et al. Horticulture Research (2020) 7:200 HiScript II 1st Strand cDNA Synthesis Kit (Vazyme), and the gene expression in these tissues was measured. Quantitative PCR (qPCR) was performed using a SYBR Light Cycler 480 instrument in conjunction with SYBR Green I Master Kit (Roche, http://www.roche.com/) according to the manufacturer’s protocols. The RNA of the transgenic plants and wild type was extracted and reverse transcribed to cDNA to determine the expression of SlNFYA10, SlGME1, SlGME2, and other genes in the AsA biosynthesis pathway. The sequences of the primers used are listed in Supplementary Table S3. Three biolo- gical replicates for the transgenic lines and the wild type were evaluated. The relative expression of speciﬁc genes was quantiﬁed using the 2−ΔΔCt method, and the actin gene was used as a constitutive internal control. Transient expression in tobacco leaves by agro inﬁltration There are four CCAAT-boxes in the 2-kb promoter of SlGME1, and different 5’-deleted SlGME1 promoter fragments were ampliﬁed with speciﬁc primers (Supple- mentary Table S2). These fragments were cloned into a pGreenII 0800-LUC reporter vector. Ascorbic acid assays The AsA levels were measured in T1 transgenic lines, as well as wild-type plants as previously described47. Leaf samples were taken from the third or fourth leaf from the top of 1-month-old tomato plants. The fruit samples were taken at the red ripe stage. The samples were collected and ground to a ﬁne powder in liquid nitrogen. Approximately 0.2 g of frozen leaf tissue and 0.3 g of frozen fruit tissue were added to one milliliter of ice-cold 6% trichloroacetic acid in a 2-ml Eppendorf tube. Each homogenate was centrifuged at 16,000 × g for 10 min at 4 °C, after which the supernatant was transferred to a new clean Eppendorf tube. To assay total AsA levels, 20 μl of the supernatant was transferred to the wells of a micro- titer plate containing 20 μl of 5 mmol dithiothreitol (DTT). The plate was incubated for 20 min at 37 °C to convert the oxidized ascorbic acid into the reduced form. Ten microliters of N-ethylmaleimide (NEM; 0.5% w/v in water) was added to remove the excess DTT, followed by incubation for 1 min at room temperature (approximately 25 °C). Eighty microliters of the color reagent (see below) was then added to the mixture, followed by incubation for 1 h at 37 °C. The absorbance was subsequently recorded at 550 nm using an Inﬁnite M200 Pro instrument (Tecan; http://www.tecan.com/). The color reagents were pre- pared as follows: solution A consisted of 31% orthopho- sphoric acid, 4.6% (w/v) TCA and 0.6% (w/v) iron chloride (FeCl3); solution B consisted of 4% 2,2-dipyridyl (w/v in 70% ethanol); and solutions A and B were mixed at a ratio of 2.75:1 before use. To assay reduced AsA, the DTT and NEM were replaced with the same volume of 0.4 M potassium phosphate buffer (pH 7.4), while the rest of the procedure was the same as that for the total AsA assay. A 35S::UAS-GUS reporter system was used to char- acterize the transcriptional activity of SlNFYA10, as described by Wang et al.45. The CDS of SlNFYA10 was ampliﬁed and linked to a pYF503 vector, generating a GDBD-SlNFYA10 effector construct. The effector con- struct, empty control (pYF503, designated GDBD), and 35S::UAS-GUS reporter plasmid were then transformed into A. tumefaciens strain GV2260. The reporter and two effectors were mixed together, after which the mixture injected into tobacco leaves, followed by GUS staining after a 48 h incubation period. Materials and methods Yeast one-hybrid The full-length ORF of NFYA was ampliﬁed from tomato cDNA and inserted into a pGreenII 62-SK effector vector, which was subsequently introduced into A. tumefaciens strain GV3101 cells together with a pSoup helper vector. The Agrobacterium cells were subsequently activated by treatment with AI buffer (10 mmol/l MES, 10 mmol/l MgCl2, 150 μmol/l AS). The Agrobacterium cells with different vectors were mixed together and injected into young tobacco (Nicotiana benthamiana) leaves for tran- sient expression, after which they were were evaluated after a 2-day incubation period44. Luciferase reporter genes were assayed using a Dual-Luciferase Reporter Assay System (Promega, http://www.promega.com/) using an Inﬁnite M200 Pro instrument (Tecan). Vector construction and tomato transformation Full-length NFYA ORFs and RNAi fragments were ampliﬁed via PCR using Phanta polymerase (Vazyme, Nanjing, China) from tomato cDNA using gene-speciﬁc primers (Supplementary Table S2). The full-length SlNFYA10 ORF and RNAi fragment were then cloned into a pMV3 overexpression vector (modiﬁed from pHELLSGATE8) and pHELLSGATE8 (Invitrogen), respectively, via recombination using Exnase II. These vectors were transferred into A. tumefaciens strain C58 for tomato transformation. The constructs were subse- quently transformed into Ailsa Craig (AC) tomato, and transgenic plants were conﬁrmed via PCR using genomic DNA from the leaves; the primers used are shown in Supplementary Table S2. Ascorbic acid assays GUS staining was per- formed as described previously46. A CCAAT-box transcription factor, SlNFY10, is a candidate regulator of ascorbic acid biosynthesis GME1 and GME2 are two homologous genes in tomato. Both contain six exons and ﬁve introns, and the ﬁrst and fourth introns of GME2 are longer than those of GME1. GME1-overexpressing lines showed higher AsA levels than did GME2-overexpressing lines16. Therefore, we used the promoter of GME1 to perform a yeast one- hybrid assay in an equalized tomato cDNA library com- prising various tissues of roots, leaves, ﬂowers, and fruits at different stages to identify the candidate proteins binding to the promoter of GME1. The potential tran- scription factors or binding proteins involved in GME1 promoter binding and AsA regulation were isolated (Supplementary Table S1). Determination of POD activity Determination of POD activity the CaMV35S promoter to generate 35S::SlNFYA10-GFP constructs. Plasmids were extracted and transferred into tobacco protoplasts, and subcellular localization was observed as described previously48. Using H2O2 as an oxidant, POD (peroxidase) catalyzes redox reactions, scavenging H2O2 from cells. POD activity can reﬂect the antioxidant capacity and stress tolerance of plants. Leaves and ripe fruits from T2 transgenic lines and wild-type plants were harvested to measure POD activity. Samples were collected and ground to a ﬁne powder in liquid nitrogen, followed by the addition of 800 μl of precooled phosphate buffer (50 mmol/l, pH 7.8), mixing and centrifugation. The supernatant (20 μl) was subse- quently added to a 1.5 ml reaction mixture, which con- sisted of 50 ml of phosphate buffer (200 mmol/l, pH 6.0), 28 μl of 2-methoxyphenol and 19 μl of hydrogen peroxide (30%). The absorption value of the mixture was measured at a wavelength of 470 nm. The promoters of SlGME1 and SlNFYA10 were ampliﬁed and cloned into a pMV2 vector harboring the GUS reporter gene, and the resultant vectors were transferred into A. tumefaciens C58 for tomato transformation. GUS staining was performed on seedlings, ﬂowers and mature green fruits. Oxidative stress experiments To evaluate the oxidative resistance of SlNFYA10- overexpressing and knockdown lines and the wild type, 80 μl of methylviologen (MV, dissolved in water consist- ing of 0.1% Tween-20) or water consisting of 0.1% Tween- 20 (control) was sprayed onto one-month-old seedlings once a day for three consecutive days. The plant pheno- types were observed one week after spraying, and images were collected. Quantiﬁcation of MDA concentration Q Malondialdehyde (MDA), the ﬁnal product of lipid peroxidation of the cell membrane, reﬂects the plant response to oxidative stress. Leaves and ripe fruits from T2 transgenic lines and wild-type plants were utilized to measure the MDA concentration. Samples were collected and ground to a ﬁne powder in liquid nitrogen, followed by the addition of 3 ml of 5% TCA to 0.2 g of ground tissue; the extraction was performed at room temperature for 30 min. After centrifugation, 2 ml of the supernatant was transferred into a 10 ml tube, after which 2 ml of 0.67% TBA solution was added. After blending, the absorption value of the 200 μl mixture was measured at wavelengths of 450 nm, 532 nm and 600 nm. The super- natant in the standard solution was replaced with ddH2O. The concentration of MDA was calculated using the formula C = 6.45 × (OD532 – OD600) – 0.56 × OD450. DAB staining MV treatments induce cells to produce excessive ROS, resulting in oxidative damage to plants. Leaves from tomato T2 transgenic lines and wild-type plants were treated with MV (or water as a control) to investigate the tomato response to oxidative stress. The MV- and water- treated tomato leaves were soaked in 1 mg/ml DAB solution (pH = 3.8) and then incubated in darkness at room temperature for 24 h. The leaves were then trans- ferred to 96% ethanol and placed in a boiling water bath for 10 min to remove the chlorophyll. The ethanol solu- tion was discarded, and the leaves were soaked with 96% ethanol to remove the ﬂoating color (the decolorized leaves were stored in 96% ethanol). The reactive oxygen species of the decolorized leaves were evaluated visually according to the brownness of the leaves. Among the candidate binding proteins, the only anno- tated transcription factor, a CCAAT-box transcription factor (Solyc01g006930.2) that belong to the NFYA family and is a subunit of the NF-Y complex, was further investigated. The sequence of Solyc01g006930.2 was queried via BLAST against the SOL Genomics Network database (http://solgenomics.net/) and mapped onto chromosome 1. It contains an open reading frame of 936 bp, encoding 312 aa, with an NFYB/NFYC interaction domain (A1: 145–163 aa) and a CCAAT-binding domain (A2: 175–198 aa) (Fig. 1a), and its sequence is highly homologous with that of its counterparts from other plant species (Fig. 1b). According to phylogenetic evolution and annotation, the isolated CCAAT-box transcription factor is hereafter referred to as SlNFYA10. Subcellular localization and tissue-speciﬁc expression Total RNA was extracted from roots, stems, leaves, and ﬂowers as well as immature fruit, green fruit, breaker- stage fruit, and red ripe fruit using TRIzol reagent. The RNA was then reverse transcribed into cDNA using a The coding sequence without the terminal codon of SlNFYA10 was ampliﬁed and inserted into the 5’ terminus of GFP in a pCAMBIA-1302 vector under the control of Chen et al. Horticulture Research (2020) 7:200 Page 4 of 12 SlNFYA10 binds to the SlGME1 promoter in plants and yeast Four CCAAT elements are represented by black boxes and are named C1, C2, C3, and C4. b Dual-luciferase assay between the SlGME1 promoter and SlNFYA10. The full sequence and serial truncated versions of the SlGME1 promoter were ligated into pGreenII 0800-LUC as a reporter vector, and SlNFYA10 was ligated into pGreenII 62-SK as an effector vector. PG, the pGreenII 62-SK empty vector with serial SlGME1 truncated promoters, was used as a control. LUC luciferase activity. RLC Renilla luciferase. The data are means ± SEs (N = 3) Fig. 2 Interaction of SlNFYA10 with the SlGME1 promoter according to a dual-luciferase assay. a CCAAT-box distribution Fig. 2 Interaction of SlNFYA10 with the SlGME1 promoter according to a dual-luciferase assay. a CCAAT-box distribution within the SlGME1 2-kb promoter. There are four cis-elements in the 2-kb promoter region of SlGME1. Four CCAAT elements are represented by black boxes and are named C1, C2, C3, and C4. b Dual-luciferase assay between the SlGME1 promoter and SlNFYA10. The full sequence and serial truncated versions of the SlGME1 promoter were ligated into pGreenII 0800-LUC as a reporter vector, and SlNFYA10 was ligated into pGreenII 62-SK as an effector vector. PG, the pGreenII 62-SK empty vector with serial SlGME1 truncated promoters, was used as a control. LUC luciferase activity. RLC Renilla luciferase. The data are means ± SEs (N = 3) signalscan.html) and PlantCARE (http://bioinfor-matics. psb.ugent.be/webtools/plantcare/html/) databases49. We found four potential target elements, two elements of CCAAT and two of ATTGG, that were named C1 and C2 as well as C3 and C4, respectively (Fig. 2a). promoter containing native C1 and C2 boxes could be bound by SlNFYA10 and that fragments with mutant C1 boxes and native C2 boxes could be bound by SlNFYA10. However, the promoter fragments with native C1 and mutant C2 boxes or both mutant C1 and C2 boxes could not be bound by SlNFYA10. Taken together, these results indicated that SlNFYA10 may preferably bind to the C2 box of the SlGME1 promoter. NFYA proteins have been previously reported to recognize and bind to CCAAT-box cis-elements, thus regulating the expression of downstream genes. To verify whether SlNFYA10 recognizes and binds to one of these cis-elements, we generated ﬁve constructs containing serial truncated promoters (Fig. 2a). These constructs were then transiently expressed in tobacco (Nicotiana benthamiana) leaves by A. tumefaciens (GV2260)-medi- ated transformation. SlNFYA10 binds to the SlGME1 promoter in plants and yeast To test the hypothesis that SlNFYA10 could directly bind to the CCAAT-box in the promoter of GME1, we analyzed the 2-kb promoter fragment of GME1 through the PLACE (http://www.dna.affrc.go.jp/PLACE/ Page 5 of 12 Chen et al. Horticulture Research (2020) 7:200 Fig. 2 Interaction of SlNFYA10 with the SlGME1 promoter according to a dual-luciferase assay. a CCAAT-box distribution within the SlGME1 2-kb promoter. There are four cis-elements in the 2-kb promoter region of SlGME1. Four CCAAT elements are represented by black boxes and are named C1, C2, C3, and C4. b Dual-luciferase assay between the SlGME1 promoter and SlNFYA10. The full sequence and serial truncated versions of the SlGME1 promoter were ligated into pGreenII 0800-LUC as a reporter vector, and SlNFYA10 was ligated into pGreenII 62-SK as an effector vector. PG, the pGreenII 62-SK empty vector with serial SlGME1 truncated promoters, was used as a control. LUC luciferase activity. RLC Renilla luciferase. The data are means ± SEs (N = 3) Fig. 1 Alignment of the SlNFYA10 sequence with the sequences of its counterparts in plants. a Amino acid sequence of SlNFYA10. The NFYB-NFYC interaction (A1) domain and DNA-binding (A2) domain are underlined in black and gray, respectively. b Amino acid alignment of the conserved domains of SlNFYA10 and its counterparts from different species. The protein sequences are from tomato (SlNFYA10), potato (Solanum tuberosum: Sotub01g007510.1), pepper (Capsicum annuum: CA10g17560), tobacco (Nicotiana attenuata: Niben101Scf04921g00005.1, Niben101Scf00911g01003.1, and Niben101Scf05694g00003.1), Arabidopsis (AT5G06510.1 and AT3G05690.1), and rice (Oryza sativa L.: OS_03g44540.1 and OS_12g42400.1) Fig. 1 Alignment of the SlNFYA10 sequence with the sequences of its counterparts in plants. a Amino acid sequence of SlNFYA10. Fig. 1 Alignment of the SlNFYA10 sequence with the sequences Fig. 1 Alignment of the SlNFYA10 sequence with the sequences of its counterparts in plants. a Amino acid sequence of SlNFYA10. The NFYB-NFYC interaction (A1) domain and DNA-binding (A2) domain are underlined in black and gray, respectively. b Amino acid alignment of the conserved domains of SlNFYA10 and its counterparts from different species. The protein sequences are from tomato (SlNFYA10), potato (Solanum tuberosum: Sotub01g007510.1), pepper (Capsicum annuum: CA10g17560), tobacco (Nicotiana attenuata: Niben101Scf04921g00005.1, Niben101Scf00911g01003.1, and Niben101Scf05694g00003.1), Arabidopsis (AT5G06510.1 and AT3G05690.1), and rice (Oryza sativa L.: OS_03g44540.1 and OS_12g42400.1) Fig. 2 Interaction of SlNFYA10 with the SlGME1 promoter according to a dual-luciferase assay. a CCAAT-box distribution within the SlGME1 2-kb promoter. There are four cis-elements in the 2-kb promoter region of SlGME1. SlNFYA10 binds to the SlGME1 promoter in plants and yeast Plant agro inﬁltration showed that the truncation of the promoter successively reduced the promoter capability and luciferase (LUC) activity. When coinﬁltrated together with SlNFYA10, the SlGME1 pro- moter with four boxes (proGME1–1: C1-C4) and three boxes (proGME1–2: C2-C4) showed signiﬁcantly reduced LUC activity compared to that of the control. However, the promoter fragments with two boxes (proGME1–3: C3-C4), one box (proGME1–4: C4) or no box (proGME1–5) did not show signiﬁcant differences in LUC activity compared to that of the control (Fig. 2b). These results indicated that SlNFYA10 mainly binds to C1 and C2 boxes or only C2 boxes. To further investigate the interaction characteristics between SlNFYA10 and the core elements of the SlGME1 promoter, we analyzed the protein structure of SlNFYA10 via the NCBI database (https://www.ncbi.nlm.nih.gov/) and found that there are two domains: a DNA-binding domain and an NFYB/NFYC interaction domain. According to the protein structure of SlNFYA10, we divided the coding sequence into four segments (N1–N4) and inserted them into pGADT7 vectors. We used a yeast one-hybrid assay to test the interaction of these different SlNFYA10 segments with the C2 box of the SlGME1 promoter. The results demonstrated that N2, N3, and N4 could recognize and bind to the C2 box of the SlGME1 promoter, while the N1 fragment could not (Fig. 3b), indicating that the C-terminus of NFYA is crucial for binding to the SlGME1 promoter. We subsequently inserted the promoter of SlGME1 into pMV2 to generate proSlGME1::GUS and cloned SlNFYA10 into pHELLSGATE8 to generate a pro35S:: SlNFYA10 overexpression construct. We transferred the two vectors into A. tumefaciens (GV2260) and mixed them together for agro inﬁltration of tobacco leaves, after which GUS staining and GUS gene expression To identify the core sequence of the SlGME1 promoter bound by SlNFYA10, constructs with truncated pro- moters containing C1 and C2 boxes were used in a yeast one-hybrid assay (Fig. 3a). In addition, we artiﬁcially mutated the two boxes and transferred them into yeast Y1H Gold strains. The results showed that the SlGME1 Page 6 of 12 Chen et al. Horticulture Research (2020) 7:200 Fig. 3 SlNFYA10 binds to the SlGME1 promoter. a The SlGME1 promoter fragment harboring C1 and C2 elements interacts with SlNFYA10 according to a yeast one-hybrid assay. SlNFYA10 binds to the SlGME1 promoter in plants and yeast The C1 and C2 elements along with their mutated version constituted four combinations (the ﬁrst contained native C1 and C2 elements, the second contained mutated C1 and native C2 elements, the third contained native C1 elements and mutated C2 elements, and the fourth contained mutated C1 and C2 elements). These promoter segments were inserted into the pAbAi vector as bait, and SlNFYA10 was inserted into the pGADT7 vector as prey. The bait and prey vectors were cotransformed together along with a negative control (bait- pGADT7) or positive control (p53-AbAi/pGAD-p53) into yeast on selective media with or without antibiotics (25 ng/ml AbA). a The interaction between C2 elements and serial SlNFYA10 domains according to a yeast one-hybrid assay. On the basis of its structural characteristics, SlNFYA10 was divided into four segments and inserted into the pGADT7 vector to serve as prey, and the SlGME1 promoter contained the whole C2 element as bait. The transformed yeast cells were grown on selective media with or without antibiotics (25 ng/ml AbA) Fig. 3 SlNFYA10 binds to the SlGME1 promoter. a The SlGME1 promoter fragment harboring C1 and C2 elements interacts with SlNFYA10 according to a yeast one-hybrid assay. The C1 and C2 elements along with their mutated version constituted four combinations (the ﬁrst contained native C1 and C2 elements, the second contained mutated C1 and native C2 elements, the third contained native C1 elements and mutated C2 elements, and the fourth contained mutated C1 and C2 elements). These promoter segments were inserted into the pAbAi vector as bait, and SlNFYA10 was inserted into the pGADT7 vector as prey. The bait and prey vectors were cotransformed together along with a negative control (bait- pGADT7) or positive control (p53-AbAi/pGAD-p53) into yeast on selective media with or without antibiotics (25 ng/ml AbA). a The interaction between C2 elements and serial SlNFYA10 domains according to a yeast one-hybrid assay. On the basis of its structural characteristics, SlNFYA10 was divided into four segments and inserted into the pGADT7 vector to serve as prey, and the SlGME1 promoter contained the whole C2 element as bait. The transformed yeast cells were grown on selective media with or without antibiotics (25 ng/ml AbA) protein, which was then coexpressed together with 35S-UAS-GUS in tobacco leaves. Histochemical staining showed that GUS expression was prominently repressed in the leaves cotransformed with GDBD-SlNFYA10 compared with the leaves cotransformed with 35S- GDBD (Supplementary Fig. SlNFYA10 binds to the SlGME1 promoter in plants and yeast S1), suggesting that SlNFYA10 might act as a transcriptional repressor. were evaluated. The tobacco leaves inﬁltrated with proSlGME1::GUS and pHELLSGATE8 exhibited blue GUS activity, while replacement of pHELLSGATE8 with pro35S::SlNFYA10 led to signiﬁcantly reduced GUS expression. This was conﬁrmed by qPCR-based detection of GUS gene expression in the inﬁltrated leaves. Taken together, these results indicated that SlNFYA10 can interact with and negatively regulated the SlGME1 pro- moter (Fig. 4). SlNFYA10 regulates AsA biosynthesis by modulating SlGME1 expression A GUS reporter plus pHELLSGATE8 empty vector was utilized as a control. GUS staining (a) and GUS gene expression (b) were evaluated. The asterisks indicates signiﬁcant differences (P ≤0.01) Fig. 5 Relative expression of SlGME1 and SlNFYA10 in tomato. a Tissue expression of SlGME1 and SlNFYA10. SlGME1 and SlNFYA10 expression in different organs: R roots, S stems, L leaves, Fl ﬂowers, IMG immature fruits, MG mature green fruits, BR breaker-stage fruits, RR red ripe fruits. b Relative expression level of SlNFYA10 in transgenic lines. qPCR-based analysis was performed on two SlNFYA10-overexpressing lines (CO37–11, CO53–9) and two RNAi lines (CR6–3, CR16–4). The data are presented as the means ± SEs Fig. 5 Relative expression of SlGME1 and SlNFYA10 in tomato. a Tissue expression of SlGME1 and SlNFYA10. SlGME1 and SlNFYA10 expression in different organs: R roots, S stems, L leaves, Fl ﬂowers, IMG immature fruits, MG mature green fruits, BR breaker-stage fruits, RR red ripe fruits. b Relative expression level of SlNFYA10 in transgenic lines. qPCR-based analysis was performed on two SlNFYA10-overexpressing lines (CO37–11, CO53–9) and two RNAi lines (CR6–3, CR16–4). The data are presented as the means ± SEs than did the wild-type control, while the CR lines showed a decrease in expression. The expression of SlNFYA10 in the leaves of the CO-37–11 and CO-53–9 lines was 5.6- and 5.0-fold that in wild-type leaves, while the CR-6–3 and CR-16–4 lines showed decreased expression (4% and 16% lower than that of the wild-type control, respectively) (Fig. 5b). The AsA levels of the transgenic lines and the wild-type plants were assayed, and the results revealed a signiﬁcant decrease in the leaves and red ripe fruits of the overexpression lines (Fig. 6a, b), while the RNAi trans- genic lines showed no difference in AsA levels (Supple- mentary Fig. S2a, b). This might be due to the functional redundancy of the NFYA gene family members, for which suppression of a single gene member might be compen- sated by alternative gene members. consistently showed no interaction (Supplementary Fig. S3). The negative regulatory effects of SlNFYA10 on SlGME1 expression were also supported by the fact that SlNFYA10 and SlGME1 expression abundance exhibited opposite change patterns among the different tissues (Fig. 5a). In summary, SlNFYA10 exerts a speciﬁc regulatory effect on SlGME1 and subsequently negatively modulates AsA accumulation. SlNFYA10 regulates AsA biosynthesis by modulating SlGME1 expression A GAL4/UAS-based assay was also performed, where GDBD binds to six copies of UAS to activate GUS expression50,45. The SlNFYA10 coding sequence was fused to GDBD to generate a GDBD-SlNFYA10 fusion We constructed SlNFYA10-overexpressing (CO) and RNAi knockdown (CR) tomato lines. The overexpressing lines exhibited signiﬁcantly higher SlNFYA10 expression Page 7 of 12 Chen et al. Horticulture Research (2020) 7:200 Fig. 4 Transcriptional activity of SlNFYA10 in plants. Schematic diagram of the two vectors used for the transient expression assays. The GUS gene driven by the SlGME1 promoter (proSlGME1::GUS), along with the SlNFYA10-overexpressing vector as the effector (35S::SlNFYA10), were coinﬁltrated into tobacco leaves. A GUS reporter plus pHELLSGATE8 empty vector was utilized as a control. GUS staining (a) and GUS gene expression (b) were evaluated. The asterisks indicates signiﬁcant differences (P ≤0.01) Fig. 4 Transcriptional activity of SlNFYA10 in plants. Schematic diagram of the two vectors used for the transient expression assays. The GUS gene driven by the SlGME1 promoter (proSlGME1::GUS), along with the SlNFYA10-overexpressing vector as the effector (35S::SlNFYA10), were coinﬁltrated into tobacco leaves. A GUS reporter plus pHELLSGATE8 empty vector was utilized as a control. GUS staining (a) and GUS gene expression (b) were evaluated. The asterisks indicates signiﬁcant differences (P ≤0.01) Fig. 5 Relative expression of SlGME1 and SlNFYA10 in tomato. a Tissue expression of SlGME1 and SlNFYA10. SlGME1 and SlNFYA10 expression in different organs: R roots, S stems, L leaves, Fl ﬂowers, IMG immature fruits, MG mature green fruits, BR breaker-stage fruits, RR red ripe fruits. b Relative expression level of SlNFYA10 in transgenic lines. qPCR-based analysis was performed on two SlNFYA10-overexpressing lines (CO37–11, CO53–9) and two RNAi lines (CR6–3, CR16–4). The data are presented as the means ± SEs Fig. 4 Transcriptional activity of SlNFYA10 in plants. Schematic diagram of the two vectors used for the transient expression assays. The GUS gene driven by the SlGME1 promoter (proSlGME1::GUS), along with the SlNFYA10-overexpressing vector as the effector (35S::SlNFYA10), were coinﬁltrated into tobacco leaves. A GUS reporter plus pHELLSGATE8 empty vector was utilized as a control. GUS staining (a) and GUS gene expression (b) were evaluated. The asterisks indicates signiﬁcant differences (P ≤0.01) Fig. 4 Transcriptional activity of SlNFYA10 in plants. Schematic diagram of the two vectors used for the transient expression assays. The GUS gene driven by the SlGME1 promoter (proSlGME1::GUS), along with the SlNFYA10-overexpressing vector as the effector (35S::SlNFYA10), were coinﬁltrated into tobacco leaves. SlNFYA10 regulates multiple sites in the D-mannose/L- galactose pathway The SlGGP1 promoter was ligated into pGreenII 0800-LUC as a reporter vector, and SlNFYA10 was ligated into pGreenII 62-SK as an effector vector. PG, pGreenII 62-SK empty vector Fig. 7 SlNFYA10 binding to the SlGGP1 promoter. a Analysis of CCAAT elements in the 1.5 kb SlGGP1 promoter region. b Interaction between the SlGGP1 promoter and SlNFYA10 in a yeast one-hybrid assay. The SlGGP1 promoter was inserted into a pAbAi vector as bait, and SlNFYA10 was inserted into pGADT7 as prey. Cotransformed yeast cells were grown on selective media with or without 10 ng/ml AbA. c Transient expression in tobacco leaves. The SlGGP1 promoter was ligated into pGreenII 0800-LUC as a reporter vector, and SlNFYA10 was ligated into pGreenII 62-SK as an effector vector. PG, pGreenII 62-SK empty vector SlNFYA10 were inserted into GUS expression vectors separately. The two GUS constructs were subsequently transformed into the tomato AC cultivar. GUS staining was performed on the roots, stems, leaves, ﬂowers, and breaker fruits of proSlGME1::GUS and proSlNFYA10:: GUS transgenic lines. From these staining results, we found that both SlGME1 and SlNFYA10 are expressed in the roots, stems, ﬂowers and seeds (Fig. 8b). these gene promoters and SlNFYA10 by yeast one-hybrid assays. The yeast one-hybrid results showed that SlNFYA10 could bind to the promoter of SlGGP1 in yeast (Fig. 7a, b). The SlGGP1 promoter was then inserted into a pGreenII 0800-LUC vector for transient expression in tobacco. Compared with those inﬁltrated with proSlGGP1 and pGreenII 62-SK, tobacco leaves coinﬁltrated with proSlGGP1 and SlNFYA10 showed signiﬁcantly decreased LUC activity (Fig. 7c), suggesting negative regulatory effects of SlNFYA10 on SlGGP1 as well as SlGME1. To investigate the subcellular localization of SlNFYA10, a SlNFYA10::GFP fusion protein was constructed and transiently expressed in tobacco protoplasts, followed by observations via confocal microscopy. The results showed that SlNFYA10 was localized in the nucleus (Fig. 8a), which is consistent with the fact that it is a nuclear transcription factor. SlNFYA10 regulates multiple sites in the D-mannose/L- galactose pathway To identify whether SlNFYA10 regulates the expression of other biosynthetic genes in the D-Man/L-Gal pathway, we performed qPCR on the leaves and red ripe fruits of the SlNFYA10 transgenic lines. In the leaves and red ripe fruits, the expression of SlGGP1 was decreased in the CO lines and enhanced in the CR lines (Supplementary Fig. S4). The pattern of SlGGP1 expression modulated by SlNFYA10 in the trans- genic lines was similar to that of SlGME1 expression. These results indicate that SlNFYA10 might regulate AsA bio- synthesis at multiple sites. It was previously reported that overexpression of the AsA biosynthetic gene SlGME1 in tomato caused an increase in AsA levels16. We found that enhanced expression of SlNFYA10 modulated its target gene, SlGME1, in SlNFYA10-overexpressing plants. The expression of SlGME1 was signiﬁcantly decreased in the leaves and red ripe fruits of the overexpression lines compared with the wild type (Fig. 6c, d). However, SlGME1 expression was not altered in the RNAi lines (Supplementary Fig. S2c, d). In the SlNFYA10 transgenic lines, SlGME2 expression was unchanged (Fig. 6c, d and Supplementary Fig. S2c, d). Yeast one-hybrid assays between the SlGME2 promoter and SlNFYA10 We retrieved the sequences of the promoter fragments of AsA biosynthetic genes from the SOL Genomics Net- work database and analyzed their cis-elements using the PLACE program. Some genes (e.g., SlGGP, SlGME2, SlGLDH) were predicted to contain CCAAT-boxes that might be bound with NFYA proteins (Supplementary Table S4). We tested the potential interaction between Chen et al. Horticulture Research (2020) 7:200 Page 8 of 12 Fig. 6 Ascorbic acid levels and SlGME expression in SlNFYA10 transgenic tomato lines. a Total and reduced ascorbic acid content in young leaves of SlNFYA10-overexpressing lines. b Total and reduced ascorbic acid content in red ripe fruits of SlNFYA10-overexpressing lines. c Relative expression levels of SlGME1 and SlGME2 in young leaves of SlNFYA10-overexpressing lines. d Relative expression levels of SlGME1 and SlGME2 in red ripe fruits of SlNFYA10-overexpressing lines. The data are the means ± SEs (N = 3), and the asterisks indicate signiﬁcant differences (*P ≤0.01; P ≤0.05) Fig. 7 SlNFYA10 binding to the SlGGP1 promoter. a Analysis of CCAAT elements in the 1.5 kb SlGGP1 promoter region. b Interaction between the SlGGP1 promoter and SlNFYA10 in a yeast one-hybrid assay. The SlGGP1 promoter was inserted into a pAbAi vector as bait, and SlNFYA10 was inserted into pGADT7 as prey. SlNFYA10 regulates multiple sites in the D-mannose/L- galactose pathway Cotransformed yeast cells were grown on selective media with or without 10 ng/ml AbA. c Transient expression in tobacco leaves. The SlGGP1 promoter was ligated into pGreenII 0800-LUC as a reporter vector, and SlNFYA10 was ligated into pGreenII 62-SK as an effector vector. PG, pGreenII 62-SK empty vector Fig. 6 Ascorbic acid levels and SlGME expression in SlNFYA10 transgenic tomato lines. a Total and reduced ascorbic acid content in young leaves of SlNFYA10-overexpressing lines. b Total and reduced ascorbic acid content in red ripe fruits of SlNFYA10-overexpressing lines. c Relative expression levels of SlGME1 and SlGME2 in young leaves of SlNFYA10-overexpressing lines. d Relative expression levels of SlGME1 and SlGME2 in red ripe fruits of SlNFYA10-overexpressing lines. The data are the means ± SEs (N = 3), and the asterisks indicate signiﬁcant differences (*P ≤0.01; P ≤0.05) Fig. 6 Ascorbic acid levels and SlGME expression in SlNFYA10 transgenic tomato lines. a Total and reduced ascorbic acid content in young leaves of SlNFYA10-overexpressing lines. b Total and reduced ascorbic acid content in red ripe fruits of SlNFYA10-overexpressing lines. c Relative expression levels of SlGME1 and SlGME2 in young leaves of SlNFYA10-overexpressing lines. d Relative expression levels of SlGME1 and SlGME2 in red ripe fruits of SlNFYA10-overexpressing lines. The data are the means ± SEs (N = 3), and the asterisks indicate signiﬁcant differences (*P ≤0.01; P ≤0 05) Fig. 7 SlNFYA10 binding to the SlGGP1 promoter. a Analysis of CCAAT elements in the 1.5 kb SlGGP1 promoter region. b Interaction between the SlGGP1 promoter and SlNFYA10 in a yeast one-hybrid assay. The SlGGP1 promoter was inserted into a pAbAi vector as bait, and SlNFYA10 was inserted into pGADT7 as prey. Cotransformed yeast cells were grown on selective media with or without 10 ng/ml AbA. c Transient expression in tobacco leaves. The SlGGP1 promoter was ligated into pGreenII 0800-LUC as a reporter vector, and SlNFYA10 was ligated into pGreenII 62-SK as an effector vector. PG, pGreenII 62-SK empty vector Fig. 7 SlNFYA10 binding to the SlGGP1 promoter. a Analysis of CCAAT elements in the 1.5 kb SlGGP1 promoter region. b Interaction between the SlGGP1 promoter and SlNFYA10 in a yeast one-hybrid assay. The SlGGP1 promoter was inserted into a pAbAi vector as bait, and SlNFYA10 was inserted into pGADT7 as prey. Cotransformed yeast cells were grown on selective media with or without 10 ng/ml AbA. c Transient expression in tobacco leaves. Discussion Transcription factors or regulators of AsA biosynthesis have been reported, most of which focus on the regulation of SlGMP. Limiting SlGME1 can exert a large effect on AsA accumulation. Moreover, overexpression and knock- down of SlGME1 can affect AsA content17. In the present study, we used the promoter of SlGME1 to identify a CCAAT-binding transcription factor, SlNFYA10, which could collectively modulate SlGME1 and SlGGP1 expres- sion and alter the AsA content in tomato. Fig. 8 Nuclear localization of SlNFYA10 and its co-occurrence with SlGME1. a Subcellular localization of SlNFYA10 in tobacco protoplasts. A GFP tag was fused to SlNFYA10, and the fusion protein was transiently expressed in tobacco protoplasts followed by ﬂuorescence observations via confocal microscopy. CFP nuclear marker ﬂuorescence. b GUS staining of transgenic lines harboring proSlGME1::GUS and proSlNFYA10::GUS. The promoters of SlGME1 and SlNFYA10 were inserted into the pMV2 vector driving the GUS gene, and GUS staining was performed on the roots, stems, leaves, ﬂowers and breaker-stage fruits The CCAAT-binding transcription factor also named Nuclear Factor Y (NFY) and Heme activator protein con- tains three subunits: NFYA, NFYB and NFYC34,51. NFYAs bind to the CCAAT-box in promoters and, in conjunction with NFYB and NFYC subunits, form the NFY transcrip- tion complex to modulate the expression of downstream genes. SlNFYA10 belongs to the NFY family, whose members have conserved NFYB/NFYC interaction domains and CCAAT-binding domains. There are also conserved NFYB/NFYC interaction domains and DNA- binding domains in SlNFYA10 (Fig. 1a). NFYs have been demonstrated to regulate plant growth and development and stress responses: NFYA5 overexpression alleviates moisture loss and drought sensitivity39; overexpression of the wheat NFYA10 gene increases plant sensitivity to sali- nity, as judged from seed germination and root growth40; and NFYs can positively regulate ﬂowering by interacting with the FT (FLOWERING LOCUS) promoter41. SlNFYA10 modulates reactive oxygen species accumulation and the stress response Nuclear factor SlNFYA10 showed tissue-speciﬁc expression coincident with SlGME1 To observe the expression patterns of SlGME1 and SlNFYA10, 2-kb length promoters of SlGME1 and Page 9 of 12 Chen et al. Horticulture Research (2020) 7:200 Fig. 8 Nuclear localization of SlNFYA10 and its co-occurrence with SlGME1. a Subcellular localization of SlNFYA10 in tobacco protoplasts. A GFP tag was fused to SlNFYA10, and the fusion protein was transiently expressed in tobacco protoplasts followed by ﬂuorescence observations via confocal microscopy. CFP nuclear marker ﬂuorescence. b GUS staining of transgenic lines harboring proSlGME1::GUS and proSlNFYA10::GUS. The promoters of SlGME1 and SlNFYA10 were inserted into the pMV2 vector driving the GUS gene, and GUS staining was performed on the roots, stems, leaves, ﬂowers and breaker-stage fruits SlNFYA10 did not signiﬁcantly alter the ROS accumula- tion (Supplementary Fig. S6), MDA concentration or POD activity (Supplementary Fig. S5), suggesting func- tional redundancy among members of the NFYA gene family. However, the antioxidant activity of RNAi lines under stress conditions deserves further investigation. SlNFYA10 did not signiﬁcantly alter the ROS accumula- tion (Supplementary Fig. S6), MDA concentration or POD activity (Supplementary Fig. S5), suggesting func- tional redundancy among members of the NFYA gene family. However, the antioxidant activity of RNAi lines under stress conditions deserves further investigation. SlNFYA10 modulates reactive oxygen species accumulation and the stress response AsA is an antioxidant that scavenges reactive oxygen species and protects plants from oxidative stress. We measured the MDA concentration and POD activity in the leaves and fruits of transgenic and wild-type plants. The results showed that the MDA concentration in the overexpressing lines were signiﬁcantly higher than those in the wild type (Fig. 9a, b), while there was not much difference between the RNAi lines and the control (Sup- plementary Fig. S5a, b). POD activity was also evaluated in the leaves and fruits, and the results showed that the POD activity was signiﬁcantly lower in overexpression lines than in the wild-type and RNAi lines (Fig. 9c, d and Supplementary Fig. S5c, d). When the SlGME1 promoter was used as bait for yeast one-hybrid assays, a CCAAT-box transcription factor (SlNFYA10) was isolated. Among the four CCAAT-boxes predicted in the SlGME1 promoter, only the second CCAAT-box could interact with SlNFYA10, as revealed by luciferase and yeast one-hybrid assays (Figs. 2 and 3). GUS staining and expression quantiﬁcation through transient expression in tobacco (Fig. 4) and the expression patterns of SlNFYA10 and SlGME1 (Fig. 5) indicated that there may be a negative regulation effect between SlNFYA10 and SlGME1. Compared with the control plants, the SlNFYA10-overexpressing lines presented a lower AsA content in the leaves and fruits (Fig. 6a, b). These results indicated that SlNFYA10 exerts a repressive effect on SlGME1 transcription. The expression of alter- native enzymatic genes of the AsA biosynthesis pathway was also evaluated in SlNFYA10 transgenic lines, among which SlGGP1 expression pattern was altered in a manner similar to how the pattern of SlGME1 was altered To test whether alteration of SlNFYA10 expression exerts an effect on the plant stress response, transgenic lines and wild-type plants were treated with MV to induce oxidative stress. DAB staining was subsequently carried out to detect ROS in the leaves. We found that the color of the leaves of the SlNFYA10-overexpressing lines was darker than that of the RNAi lines and the control, indi- cating that the leaves of the SlNFYA10-overexpressing lines were more severely affected by oxidative stress (Supplementary Fig. S6). Thus, the AsA levels and ROS scavenging capability decreased in SlNFYA10-over- expressing lines. It should be noted that suppression of Chen et al. Horticulture Research (2020) 7:200 Page 10 of 12 Fig. 9 Antioxidant capacity of SlNFYA10 transgenic lines. SlNFYA10 modulates reactive oxygen species accumulation and the stress response The MDA concentration was determined in the leaves (a) and ripe fruits (b) of SlNFYA10-overexpressing lines and the wild-type control (AC). POD activity was determined in the leaves (a) and red ripe fruits (b) of SlNFYA10- overexpressing lines and the wild-type control. The data are the means ± SEs (N = 3), and the asterisks indicate signiﬁcant differences (*P ≤0.01; P ≤0.05) Fig. 9 Antioxidant capacity of SlNFYA10 transgenic lines. The MDA concentration was determined in the leaves (a) and ripe fruits (b) of SlNFYA10-overexpressing lines and the wild-type control (AC). POD activity was determined in the leaves (a) and red ripe fruits (b) of SlNFYA10- overexpressing lines and the wild-type control. The data are the means ± SEs (N = 3), and the asterisks indicate signiﬁcant differences (*P ≤0.01; P ≤0.05) unaltered phenotypes of the SlNFYA10 RNAi transgenic lines might be due to the functional redundancy of the members of the NFYA gene family. Unlike the NFYAs encoded by one or two genes in animals and yeast, plants tend to have evolved a relatively larger gene family; e.g., the NFY gene family has 10 members in Arabidopsis52, 8 in rice53, 10 in wheat54, and 10 in tomato55. Gene repli- cation may contribute to large numbers of gene families that allow functional compensation or buffering against individual mutations, subsequently leading to functional redundancy56. The F-box protein family also has a large amount of members, with almost 700 predicted genes in Arabidopsis. However, single mutations or combinations of mutations in pollen-speciﬁc F-box genes did not lead to gametophytic lethality, suggesting functional redundancy among the F-box protein-coding genes57. In plant viruses such as tobacco etch potyvirus, genotypes with genetic redundancy suffered less from the effects of deleterious mutations and showed relatively minor changes in pathogenicity58. Accordingly, the expression of alternative members of the NFYA gene family, e.g., SlNFYA1.1, SlNFYA3.1, and SlNFYA8, was signiﬁcantly higher in the SlNFYA10 RNAi lines than in the wild type (Supple- mentary Fig. S8). Thus, we hypothesized that the relative stability of plant performance in the SlNFYA10 RNAi transgenic lines might be in part due to functional redundancy of NFYA gene family members in tomato. (Supplementary Fig. S4). The LUC assay and yeast one- hybrid assay showed that SlNFYA10 could bind to the SlGGP1 promoter (Fig. 7). SlNFYA10 modulates reactive oxygen species accumulation and the stress response In addition, the subcellular localization results showed that SlNFYA10 is located in the nucleus, which is characteristic of nuclear transcrip- tion factors (Fig. 8a). The tissue distribution of SlNFYA10 and SlGME1 showed that they are mainly expressed in the same stem, ﬂower and seed tissues (Fig. 8b). Oxidative stress tests showed that, compared with the control, the SlNFYA10-overexpressing lines presented less ROS scavenging activity (Fig. 9 and Supplementary Fig. S6), presumably due to lower availability of AsA, which is the substrate required for ascorbate peroxidase function. Taken together, our results demonstrate that NFYA negatively regulates AsA biosynthesis by suppressing GME1 and GGP1 in tomato fruit. AsA biosynthesis has been widely investigated, and several transcription factors for biosynthetic genes such as GMP have been isolated30. In the present work, we conﬁrmed that NFYA could simultaneously regulate GME and GGP expression. NFY transcription factors have been reported to be involved in a wide range of biological processes39–41; however, this is the ﬁrst evidence that NFYA functions in the regulation of AsA metabolism. It is worth noting that the silencing of SlNFYA10 by RNAi did not cause signiﬁcant alterations in terms of AsA content, SlGME1 and SlGGP1 expression, ROS accumu- lation, MDA concentration or POD activity in the trans- genic lines (Supplementary Figs. S2, S4, S5, S6). Furthermore, fruit development or ripening was not sig- niﬁcantly altered in SlNFYA10 RNAi transgenic lines compared to the wild type (Supplementary Fig. S7). The References et al. myo-inositol oxygenase offers a possible entry point into plant ascorbate biosynthesis. Plant Physiol. 134, 1200–1205 (2004). 7. Lorence, A. et al. myo-inositol oxygenase offers a possible entry point into plant ascorbate biosynthesis. Plant Physiol. 134, 1200–1205 (2004). 32. Li, Y. et al. The C2H2 zinc-ﬁnger protein SlZF3 regulates AsA synthesis and salt tolerance by interacting with CSN5B. Plant Biotechnol. J. 16, 1201–1213 (2017). 8. Conklin, P. L. et al. 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https://openalex.org/W3193966792	https://link.springer.com/content/pdf/10.1007/s00586-021-06957-3.pdf	English	null	Trends of epidemiological characteristics of traumatic spinal cord injury in China, 2009–2018	European spine journal	2,021	cc-by	8,721	European Spine Journal (2021) 30:3115–3127 https://doi.org/10.1007/s00586-021-06957-3 European Spine Journal (2021) 30:3115–3127 https://doi.org/10.1007/s00586-021-06957-3 ORIGINAL ARTICLE ORIGINAL ARTICLE Abstract Objective We focus on providing the first comprehensive national dataset on the incidence, injury aetiology and mortality of TSCI in China.i Methods A multi-stage stratified cluster sampling method was used. We included TSCI cases from all hospitals in three regions, nine provinces and 27 cities in China via search of electronic medical records and retrospectively analysed the char- acteristics of TSCI in China from 2009 to 2018. We estimated the incidence of TSCI in the total population and subgroups. Results There were 5954 actual cases in 2009, corresponding to a total estimated TSCI incidence of 45.1 cases per million population (95% CI, 44.0–46.3). There were 10,074 actual cases in 2018, corresponding to a total estimated TSCI incidence of 66.5 cases per million population (95% CI, 65.2–67.8) (P < 0.001; annual average percentage change (AAPC), 4.4%). From 2009 to 2018, the incidence of almost all sex/age groups showed an increasing trend over time (P < 0.001; AAPC, 0.7–8.8%). The elderly population (aged 65–74) displayed the highest incidence of TSCI (with an average annual incidence of 127.1 cases per million [95% CI, 119.8–134.3]).i Conclusions The TSCI incidence increased significantly from 2009 to 2018. The incidence in the elderly populations was consistently high and continues to increase over time. The mortality of TSCI patients in hospitals is relatively low and con- tinues to decrease each year, but elderly individuals remain at a high risk of hospital death. Keywords Epidemiology · Incidence · Mortality · Spinal cord injury · China Trends of epidemiological characteristics of traumatic spinal cord injury in China, 2009–2018 Dingjun Hao1 · Jinpeng Du1 · Liang Yan1 · Baorong He1 · Xiao Qi2 · Shicheng Yu2 · Jiaojiao Zhang2 · Wenjing Zheng2 · Rongqiang Zhang3 · Da‑Geng Huang1 · Junsong Yang1 · Ming Zhu1 · Jiawei Ouyang1 · He Zhao1 · Keyuan Ding1 · Haodong Shi1 · Yang Cao1 · Ying Zhang1 · Qinghua Tang1 · Yuan Liu1 · Zilong Zhang1 · Yuhang Wang1 · Ye Tian1 · Hao Chen1 · Lulu Bai1 · Heng Li1 · Chenchen Mu1 · Youhan Wang1 · Xiaohui Wang1 · Chao Jiang1 · Jianhua Lin4 · Bin Lin5 · Shunwu Fan6 · Lin Nie7 · Jiefu Song8 · Xun Ma9 · Zengwu Shao10 · Yanzheng Gao11 · Zhong Guan12 · Yueming Song13 · Weihu Ma14 · Qixin Chen15 Received: 19 November 2020 / Revised: 12 June 2021 / Accepted: 6 August 2021 / Published online: 15 August 2021 © The Author(s) 2021 Received: 19 November 2020 / Revised: 12 June 2021 / Accepted: 6 August 2021 / Published online: 15 August 2021 © The Author(s) 2021 Dingjun Hao, Jinpeng Du, Liang Yan, and Baorong He these authors have contributed equally to this work. Statistical analysis We included TSCI cases from all hospitals in the sample cities and calculated the total incidence of TSCI per 1 mil- lion population based on the sum of population estimate data given by the Bureau of Statistics in each sample city. Then, we calculated the stratified incidence rate according to region, sex/age, urbanization and education level. The standardized incidence rates overall and by regional and sex/age stratifications were then calculated using the World Health Organization’s world standard population through a direct standardized method so that the results could be com- pared. The estimated number of cases in each stratification was then calculated. Additionally, we calculated the average annual incidence of each stratification by dividing the aver- age number of cases from 2009 to 2018 at each stratifica- tion by the average population at the corresponding level in 2009–2018. All incidence rates are shown with a 95% confidence interval (CI). Introduction of TSCI in China remains unknown and has only been reported in particular cities [3, 4]. The reported incidences vary widely, and there is significant error when extrapolat- ing the characteristics of national TSCI cases from survey data reported from a single region. Throughout the world, developed countries have identified the epidemic character- istics of TSCI through domestic large-scale epidemiological investigations [5–9] and then taken corresponding preven- tive and first-aid measures. We designed the Chinese TSCI Epidemiological Survey to create the first comprehensive and up-to-date national dataset of the incidence, injury aeti- ology, mortality and other epidemiological characteristics of TSCI in China. Traumatic spinal cord injury (TSCI) is a highly disabling condition. It places a heavy burden on individuals, families and society as a whole because of its labour loss, long-term rehabilitation, occupation of large medical resources and expensive medical costs [1, 2]. Due to the imperfections of the national registration system, the national epidemiology Dingjun Hao, Jinpeng Du, Liang Yan, and Baorong He these authors have contributed equally to this work. Extended author information available on the last page of the article :(0123 1 3456789) 3 European Spine Journal (2021) 30:3115–3127 3116 data. We classified the causes of TSCI into traffic injury, falling object injury, low falls (height < 1 m), high falls (height > 1 m), and other, as well as the classification of complications and surgical methods, which are often used in Chinese clinical practice and national health service surveys. Study design and participants This retrospective survey uses a multi-stage stratified cluster sampling method of the medical records of TSCI patients who were admitted to the hospital. According to the expected incidence rate reported in a previous literature review [10], the required sample size was cal- culated, and the number of sampling layers at all levels was determined. In the main sampling stage, the 32 prov- inces (autonomous regions) of China were divided into three regions (East, Central and West) according to their socio-economic development and geographical location, which is the same classification method often used by the China Bureau of Statistics. In the stratified random sam- pling stage, three provinces were randomly selected from each region for a total of nine provinces (Qinghai, Gansu, Shaanxi, Shanxi, Hubei, Sichuan, Shandong, Zhejiang and Fujian). Three cities were randomly selected from each province. The eligible TSCI cases in all hospitals in the sample cities were investigated. Similar to previous stud- ies, our study did not take into account those who died at the scene of the injury or in the emergency department [7, 8]. The ethics committee of Xi’an Honghui Hospital and the participating centres approved the study, and given the retrospective nature of the study, the requirement for informed consent was waived. Select trained investigation team members participated in this data collection. The relevant personnel were trained repeatedly before data collection and data entry and were provided detailed questionnaire management instructions. During the investigation stage, two members comprised an investigation team and performed the data collection and verification for each data site. We created nine quality con- trol teams (one for each province), and all the questionnaires collected were checked for omissions and errors by the qual- ity control team. The data in the medical records were inde- pendently interpreted by the attending physician to ensure the accuracy of the original data. Data collection To identify the rationality and comprehensiveness of the contents of the questionnaire, two cities were selected for pilot projects in Shaanxi Province in the pre-survey stage. After repeated argumentation and revision, the follow- ing survey indicators were finally confirmed: 1. general patient data (name, age, sex, occupation, urbanization, education level, medical insurance); 2, spinal cord injury index (cause of injury, injury level, severity of injury); 3, treatment measures (operation or rehabilitation treatment, operation procedure); 4, complications; and 5, death. i We calculated the proportion of the internal composi- tion of aetiology, hospital complications, hospital mortal- ity and other clinical and demographic characteristics of the included TSCI patients for 10 consecutive years; the unweighted calculation did not provide a nationally repre- sentative estimate. The CI of the internal composition ratio is calculated by using the asymptotic normal distribution of large samples. The trend analysis of the incidence and inter- nal constituent ratio was analysed with the Cochran–Armit- age trend test. P < 0.05 was considered statistically signifi- cant. If the trend test indicates that the incidence displays a changing trend over time, it is assumed that the trend is linear. In that case, the time term linear regression model is used for linear fitting, and the slope represents the speed of change, which can be simply used to compare the difference The investigators searched all cases in the digital archive of each target hospital (Qualified to treat patients with TSCI) from 2009 to 2018 that were diagnosed with TSCI as defined by the International Classification of Dis- eases, 10th edition (ICD-10) code (see Appendix). The ICD code for TCSI includes cauda equina injury but does not include spinal root and spinal plexus injury. The inves- tigators initially searched for qualified cases in the digital archives and then referred to the patients’ electronic medi- cal records after the initial screening to extract the relevant 1 3 3 European Spine Journal (2021) 30:3115–3127 3117 between linear trends. Simultaneously, the annual average percentage change (AAPC) of each stratification was calcu- lated to assist in the assessment of changes in incidence over the past 10 years. Data collection East, 2.5; Central, 4.4; West, 8.3%), with the highest annual average incidence rate of 49.4 cases (95% CI, 49.3–49.6) per million population in the eastern region (standardized) and the lowest annual incidence rate of 34.2 cases (95% CI, 34.0–34.3) per million population in the western region (standardized); however, the incidence rate has increased the fastest over time in the western region (slope, 3.0; R2, 0.9). See Fig. 1 and Appendix for details. between linear trends. Simultaneously, the annual average percentage change (AAPC) of each stratification was calcu- lated to assist in the assessment of changes in incidence over the past 10 years. We used Epidata software (version 3.1) for data entry and management and used Microsoft Office Excel 2019 to check and save the data. Analyses were performed with SAS (version 9.1). As shown in Table 1, except for the < 14-year-old age group of males, the incidence rates of the other sex/age groups increased over time (P < 0.001) (for example, the adolescent male age group [15–24 years old] [AAPC, 5.8%] increased from 17.5 cases/million people [202 cases] in 2009 to 29.0 cases/million people [255 cases] in 2018). The inci- dence rate of males was higher than that of females (stand- ardized annual average incidence rate, males: 66.4 cases/mil- lion people [95% CI, 66.2–66.7]; females: 23.6 cases/million people [95% CI, 23.4–23.8]). Among the male and female populations, the incidence of TSCI in the elderly popula- tion remained high from 2009 to 2018 (for example, the male 65–74 age group [AAPC, 2.55%] increased from 158.1 cases/million people [612 cases] in 2009 to 197.1 cases/ million people [1125 cases] in 2018, with an average annual incidence of 184.4/million people [95% CI, 172.1–196.8]). Incidence This study included a total of 78,061 patients with TSCI from 2009 to 2018 who were admitted to 200 hospitals (with the ability to treat TSCI) in 27 sample cities. The general data are detailed in the appendix. The number of cases in the dataset increased from 5954 in 2009 to 10,074 in 2018 (AAPC, 6.0%). The incidence rates in 2009 and 2018 were estimated at 45.1 (95% CI, 44.0–46.3) and 66.5 (95% CI, 65.2–67.8) cases per million population (P < 0.001; AAPC, 4.4%), respectively. The stratified incidence of each region showed an increasing trend (P < 0.001, for all trends; AAPC: Fig. 1 Traumatic spinal cord injury incidence rate per 1 million people by Region in China, 2009–2018 Fig. Incidence The number of cases stratified by age-sex in the database and the cor- responding case estimates are shown in the appendix.i Age groups Crude inci- dence Age standardized incidence* Year ≤ 14 15–24 25–34 35–44 45–54 55–64 65–74 ≥ 75 2016 9.9 (8.1–11.8) 13.7 (11.2– 16.3) 17.4 (15.0– 19.8) 31.4 (28.0– 34.7) 47.4 (43.5– 51.2) 62.0 (56.7– 67.3) 78.9 (71.3– 86.5) 40.6 (33.9– 47.3) 34.0 (32.7– 35.4) 27.8 (27.6–27.9) 2017 9.7 (7.9–11.5) 13.8 (11.2– 16.5) 18.1 (15.7– 20.5) 30.1 (26.8– 33.4) 48.4 (44.5– 52.3) 63.7 (58.4– 69.0) 78.5 (71.1– 85.9) 42.8 (35.9– 49.7) 34.7 (33.4– 36.1) 28.0 (27.8–28.1) 2018 10.2 (8.4–12.1) 14.4 (11.7– 17.1) 18.5 (16.1– 20.9) 30.3 (27.1– 33.5) 47.8 (44.0– 51.6) 61.8 (56.8– 66.9) 71.3 (64.5– 78.1) 39.9 (33.5– 46.4) 34.1 (32.8– 35.5) 27.6 (27.4–27.8) Annual Average 9.2 (7.3–11.0) 9.2 (7.2–11.1) 15.3 (13.0– 17.6) 23.5 (20.7– 26.3) 43.9 (40.0– 47.8) 52.0 (47.1– 57.0) 70.8 (63.1– 78.4) 35.4 (28.8– 42.0) 28.4 (27.2– 29.7) 23.6 (23.4–23.8) Rates standardized for age by the direct method using the WHO World Standard Population; data are incidence (95%CI), unless stated otherwise p g pp The TSCI incidence stratified by urbanization and educa- tion level shows an increasing trend linearly related to the time series (Fig. 2) (P < 0.001; for all trend). The incidence rate in the rural population was higher than that of the urban population (annual average IRR, 1.9 [95% CI, 1.8–2.0]), but the incidence rate in the urban population has increased faster over time (slope, 4.0; R2, 0.9). The incidence of the low-educated population (junior high school and below) was slightly higher than that in the higher educated population (senior high school and above) (annual average IRR, 1.1 [95% CI, 1.1–1.2]), and the incidence in low-educated popu- lation has increased faster over the past 10 years (slope, 3.2; R2, 0.9). Additionally, the incidence of urbanization and edu- cation showed a significant difference after regional strati- fication (appendix). For example, the incidence of the rural population in the eastern region shows a downward trend over time (P = 0.04; AAPC, − 1.0%; slope, − 0.5, R2, 0.1), whereas the incidence in the urban population increased faster in the eastern region than in other regions (AAPC, 9.0%; slope, 5.4, R2, 0.8). Incidence 1 Traumatic spinal cord injury incidence rate per 1 million people by Region in China, 2009–2018 1 3 European Spine Journal (2021) 30:3115–3127 3118 p p ( ) 1 3 Table 1 Age- and sex-stratified estimated traumatic spinal cord injury incidence rates per 1 million people Age groups Crude inci- dence Age standardized incidence Year ≤ 14 15–24 25–34 35–44 45–54 55–64 65–74 ≥ 75 Male 2009 3.2 (2.2–4.2) 17.5 (15.1– 20.0) 51.6 (47.1– 56.1) 57.4 (53.0– 61.8) 132.5 (125.3– 139.8) 144.8 (135.9– 153.6) 158.1 (145.5– 170.6) 70.5 (59.0– 82.0) 66.4 (64.5– 68.4) 57.1 (56.8–57.3) 2010 2.9 (1.9–3.9) 18.7 (16.3– 21.2) 45.1 (40.9– 49.2) 52.1 (48.0– 56.1) 132.7 (125.7– 139.8) 134.8 (126.7– 143.0) 152.6 (140.5– 164.6) 62.9 (52.3– 73.5) 64.0 (62.1– 65.9) 54.1 (53.9–54.4) 2011 3.5 (2.5–4.6) 14.9 (12.8– 17.1) 41.2 (37.4– 45.1) 56.6 (52.3– 60.9) 150.5 (142.8– 158.1) 158.3 (149.4– 167.2) 173.1 (160.1– 186.1) 70.3 (59.3– 81.4) 69.1 (67.2– 71.1) 59.0 (58.7–59.2) 2012 3.3 (2.3–4.3) 14.8 (12.6– 17.1) 41.7 (37.8– 45.5) 58.6 (54.2– 63.0) 148.0 (140.4– 155.5) 147.6 (139.2– 156.0) 169.0 (156.6– 181.5) 71.6 (60.6– 82.5) 68.9 (67.0– 70.9) 57.9 (57.6–58.1) 2013 3.4 (2.4–4.4) 18.0 (15.5– 20.6) 41.6 (37.8– 45.4) 65.7 (61.0– 70.4) 152.3 (144.8– 159.7) 153.2 (144.8– 161.6) 172.3 (160.1– 184.6) 74.7 (63.9– 85.6) 73.8 (71.8– 75.8) 60.6 (60.4–60.9) 2014 4.1 (2.9–5.2) 21.9 (19.0– 24.8) 47.5 (43.5– 51.5) 79.4 (74.3– 84.6) 189.7 (181.2– 198.4) 186.2 (177.0– 195.4) 198.0 (185.2– 210.9) 93.3 (81.3– 105.2) 88.6 (86.4– 90.8) 73.2 (72.9–73.4) 2015 3.4 (2.4–4.5) 22.3 (19.3– 25.3) 42.8 (39.1– 46.5) 77.5 (72.3– 82.6) 154.1 (146.9– 161.3) 183.5 (174.3– 192.6) 190.3 (177.9– 202.6) 78.8 (68.0– 89.7) 83.2 (81.1– 85.2) 66.9 (66.7–67.2) 2016 4.0 (2.9–5.0) 29.6 (26.1– 33.1) 56.9 (52.7– 61.2) 94.4 (88.7– 100.1) 172.5 (165.1– 179.9) 210.7 (200.9– 220.4) 209.7 (197.2– 222.2) 84.1 (73.3– 94.9) 98.0 (95.8– 100.3) 78.3 (78.0–78.6) 2017 3.7 (2.6–4.7) 30.0 (26.4– 33.7) 55.4 (51.2– 59.6) 95.5 (89.7– 101.2) 171.3 (164.0– 178.6) 210.2 (200.6– 219.9) 202.5 (190.4– 214.5) 94.2 (82.8– 105.6) 98.3 (96.0– 100.5) 78.0 (77.7–78.3) 2018 4.0 (3.0–5.1) 29.0 (25.4– 32.6) 55.0 (51.0– 59.1) 93.0 (87.4– 98.6) 171.2 (164.0– 178.5) 206.5 (197.3– 215.8) 197.1 (185.6– 208.6) 92.3 (81.3– 103.4) 97.4 (95.2– 99.6) 76.9 (76.6–77.1) Annual Average 3.6 (2.5–4.6) 21.0 (18.2– 23.8) 48.1 (44.1– 52.2) 72.7 (67.7– 77.6) 158.3 (150.8– 165.8) 175.1 (166.0– 184.1) 184.4 (172.1– 196.8) 80.3 (69.1– 91.5) 81.2 (79.1– 83.3) 66.4 (66.2–66.7) Female* 2009 8.0 (6.2–9.9) 6.7 (5.2–8.3) 15.1 (12.7– 17.6) 18.2 (15.8– 20.7) 35.7 (31.9– 39.4) 46.2 (41.1– 51.3) 60.3 (52.7– 67.9) 24.9 (18.9– 30.8) 22.8 (21.7– 24.0) 19.9 (19.7–20.0) 2010 7.1 (5.4–8.8) 6.6 (5.1–8.1) 13.5 (11.2– 15.7) 17.5 (15.1– 19.8) 34.4 (30.7– 38.0) 40.8 (36.3– 45.3) 59.6 (52.1– 67.2) 30.3 (23.8– 36.8) 21.8 (20.7– 22.9) 18.8 (18.6–18.9) 2011 8.8 (6.9–10.6) 5.9 (4.4–7.3) 13.8 (11.5– 16.0) 19.1 (16.6– 21.6) 44.7 (40.5– 48.9) 44.4 (39.7– 49.1) 69.7 (61.5– 78.0) 30.2 (23.8– 36.6) 24.5 (23.4– 25.7) 21.3 (21.2–21.5) 2012 8.7 (6.9–10.5) 6.8 (5.2–8.4) 12.0 (9.9–14.0) 18.9 (16.4– 21.4) 42.5 (38.4– 46.5) 42.8 (38.3– 47.3) 65.7 (57.9– 73.4) 31.6 (25.2– 38.1) 24.3 (23.1– 25.4) 20.6 (20.5–20.8) 2013 8.9 (7.1–10.7) 8.6 (6.7–10.5) 13.5 (11.3– 15.6) 21.1 (18.4– 23.8) 41.7 (37.8– 45.6) 46.3 (41.6– 50.9) 65.7 (58.1– 73.3) 34.2 (27.6– 40.7) 26.0 (24.8– 27.2) 21.8 (21.6–21.9) 2014 10.3 (8.4–12.3) 9.5 (7.5–11.5) 14.9 (12.7– 17.2) 25.8 (22.8– 28.8) 47.3 (43.2– 51.4) 53.2 (48.3– 58.1) 78.8 (70.7– 86.9) 36.6 (30.0– 43.3) 30.3 (29.0– 31.6) 25.2 (25.0–25.3) 2015 9.3 (7.5–11.2) 9.9 (7.8–12.0) 15.4 (13.2– 17.6) 24.4 (21.5– 27.3) 45.9 (41.9– 49.8) 54.4 (49.4– 59.4) 72.5 (64.9– 80.0) 38.1 (31.4– 44.8) 29.9 (28.6– 31.2) 24.5 (24.3–24.6) 1 3 European Spine Journal (2021) 30:3115–3127 3119 The incidence rate in the male 55–64 age group increased the fastest over time (from 144.8 cases/million people [1025 cases] in 2009 to 206.5 cases/million people [1914 cases] in 2018; [AAPC, 4.0%]; slope, 9.0; R2, 0.9). Incidence The incidence rate in the rural population in the western region has increased faster than that in the other regions over time (AAPC, 11.8%; slope, 7.2, R2, 0.9), whereas the increase in incidence in the urban population was the slowest in the western region (AAPC, 4.2%; Slope, 1.4, R2, 0.6). In addition, the incidence rate of the higher educated population in the eastern region has increased faster than that of the other regions (AAPC, 4.1%; slope, 4.7, R2, 0.7), and the incidence rate of the low-edu- cated population in the western region has increased faster than that in the other regions (AAPC, 11.3%; slope, 5.1, R2, 0.9). WHO World Standard Population; data are incidence (95%CI), unless stated otherwise Rates standardized for age by the direct method using the WHO World Standard Population; da Cause of injury From 2009 to 2018, the three leading causes of injury included traffic injuries, low falls and high falls (Fig. 3). Over the past 10 years, the largest proportion of TSCIs has always been caused by traffic injuries (from 2009 [30.7%; 95% CI, 29.5–31.9%] to 2018 [32.4%; 95% CI, 31.5–33.3%]), and the overall trend has remained stable (P = 0.071). The proportion of TSCIs caused by low falls was correlated with the time series (P = 0.015) and ranged from 27.8% (95% CI, 26.7–29.0%) in 2009 to 26.6% (95% CI, 25.8–27.5%) in 2018. The proportion of TSCIs caused by high falls tended to increase over time (P < 0.001), from 2009 [23.0%; 95% CI, 22.0–24.1%] to 2018 [25.8%; 95% CI, 24.9–26.6%]. After stratifying by region, the 1 3 3120 European Spine Journal (2021) 30:3115–3127 Fig. 2 Urbanization and education level stratified national traumatic spinal cord injury incidence rates per 1 million people in China, 2009–2018 tion and education level stratified national traumatic spinal cord injury incidence rates per 1 million people in China, Fig. 2 Urbanization and education level stratified national traumatic spinal cord injury incidence rates per 1 millio Fig. 2 Urbanization and education level stratified national traumatic spinal cord injury incidence rates per 1 million people in China, 2009–2018 Fig. 3 Major aetiology of acute traumatic spinal cord injury by Region in China, 2009–2018 Fig. 3 Major aetiology of acute traumatic spinal cord injury by Region in China, 2009–2018 Results of general clinical indicators proportion of TSCIs caused by traffic injuries was always the highest in the eastern region and increased linearly along the time series (P < 0.001) whereas it decreased line- arly along the time series in the central region (P < 0.015); however, traffic injuries were still the main cause of injury in the central region. The proportion of TSCIs caused by high falls decreased linearly along the time series in the eastern region (P < 0.022) but increased linearly along the time series in the central and western regions (P < 0.001, for all trends). High falls have always been the main cause of injury in patients with TSCIs in the western region. Detailed data are given in Appendix. The proportions of these clinical indexes such as cervical spinal cord injury, complete spinal cord injury, rehabilitation treatment, surgical treatment, and complications showed a linear increasing trend over time (P < 0.05) (Table 2). How- ever, the proportion of patients with bedsores decreased over time (P < 0.001). Most TSCI patients received decom- pression and fixation fusion; this proportion increased from 60.3% (95% CI, 58.9–61.8%) in 2009 to 63.4% (95% CI, 62.3–64.5%) in 2018 (P < 0.001). Detailed data for more indicators are given in Appendix. Results of general clinical indicators 1 3 European Spine Journal (2021) 30:3115–3127 3121 1 Table 2 Details of selected secondary outcomes in included inpatient samples with traumatic spinal cord injury in China, 2009–2018 Years P value 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 Injury level Cervical spinal cord 3729 3856 4101 4139 4625 5428 5450 6428 6699 6946 62.6 (61.4– 63.9) 64.9 (63.6– 66.1) 62.7 (61.6– 63.9) 63.3 (62.2– 64.5) 65.8 (64.7– 66.9) 64.7 (63.7– 65.7) 67.0 (66.0– 68.0) 66.5 (65.5– 67.4) 68.4 (67.5– 69.3) 68.9 (68.0– 69.9) < 0.001 Thoracic spinal cord 785 755 945 825 885 1032 993 1081 1182 1208 13.2 (12.3– 14.0) 12.7 (11.9– 13.5) 14.5 (13.6– 15.3) 12.6 (11.8– 13.4) 12.6 (11.8– 13.4) 12.3 (11.6– 13.0) 12.2 (11.5– 12.9) 11.2 (10.5– 11.8) 12.1 (11.4– 12.7) 12.0 (11.4– 12.6) < 0.001 Lumbosacral spinal cord 1440 1334 1491 1571 1515 1926 1694 2163 1915 1920 24.2 (23.1– 25.3) 22.4 (21.4– 23.5) 22.8 (21.8– 23.8) 24.0 (23.0– 25.1) 21.6 (20.6– 22.5) 23.0 (22.1– 23.9) 20.8 (19.9– 21.7) 22.4 (21.5– 23.2) 19.5 (18.8– 20.3) 19.1 (18.3– 19.8) < 0.001 Severity of injury, Com- plete SCI 1015 1179 1107 1285 1577 1614 1998 2372 2721 2763 17.0 (16.1– 18.0) 19.8 (18.8– 20.8) 16.9 (16.0– 17.8) 19.7 (18.7– 20.6) 22.4 (21.5– 23.4) 19.2 (18.4– 20.1) 24.6 (23.6– 25.5) 24.5 (23.7– 25.4) 27.8 (26.9– 28.7) 27.4 (26.6– 28.3) < 0.001 Rehabilitation, Yes 1073 1181 1707 1783 2070 2973 3802 4742 5808 6322 18.0 (17.0– 19.0) 19.9 (18.9– 20.9) 26.1 (25.0– 27.2) 27.3 (26.2– 28.4) 29.5 (28.4– 30.5) 35.5 (34.4– 36.5) 46.7 (45.6– 47.8) 49.0 (48.0– 50.0) 59.3 (58.3– 60.3) 62.8 (61.8– 63.7) < 0.001 Surgery, Yes 4246 4240 4653 4657 5005 6008 5866 6979 7079 7258 71.3 (70.2– 72.5) 71.3 (70.2– 72.5) 71.2 (70.1– 72.3) 71.3 (70.2– 72.4) 71.2 (70.2– 72.3) 71.6 (70.7– 72.6) 72.1 (71.1– 73.1) 72.2 (71.3– 73.1) 72.3 (71.4– 73.2) 72.0 (71.2– 72.9) 0.022 Surgical procedures Simple spinal cord decom- pression 256 224 267 252 275 315 311 374 362 382 6.0 (5.3–6.7) 5.3 (4.6–6) 5.7 (5.1–6.4) 5.4 (4.8–6.1) 5.5 (4.9–6.1) 5.2 (4.7–5.8) 5.3 (4.7–5.9) 5.4 (4.8–5.9) 5.1 (4.6–5.6) 5.3 (4.7–5.8) 0.067 Decompres- sion and fixation 1183 1217 1262 1303 1388 1623 1621 1872 1857 1900 27.9 (26.5– 29.2) 28.7 (27.3– 30.1) 27.1 (25.8– 28.4) 28.0 (26.7– 29.3) 27.7 (26.5– 29.0) 27.0 (25.9– 28.1) 27.6 (26.5– 28.8) 26.8 (25.8– 27.9) 26.2 (25.2– 27.3) 26.2 (25.2– 27.2) 0.001 Decompres- sion, fixation and fusion 2562 2565 2865 2845 3089 3773 3639 4364 4500 4604 60.3 (58.9– 61.8) 60.5 (59.0– 62.0) 61.6 (60.2– 63.0) 61.1 (59.7– 62.5) 61.7 (60.4– 63.1) 62.8 (61.6–64) 62.0 (60.8– 63.3) 62.5 (61.4– 63.7) 63.6 (62.4– 64.7) 63.4 (62.3– 64.5) < 0.001 Other 245 234 259 257 253 297 295 369 360 372 5.8 (5.1–6.5) 5.5 (4.8–6.2) 5.6 (4.9–6.2) 5.5 (4.9–6.2) 5.1 (4.4–5.7) 4.9 (4.4–5.5) 5.0 (4.5–5.6) 5.3 (4.8–5.8) 5.1 (4.6–5.6) 5.1 (4.6–5.6) 0.050 Complica- tions, Yes 1542 1494 1663 1713 1880 2248 2210 2611 2615 2702 25.9 (24.8– 27.0) 25.1 (24.0– 26.2) 25.4 (24.4– 26.5) 26.2 (25.1– 27.3) 26.8 (25.7– 27.8) 26.8 (25.9– 27.8) 27.2 (26.2– 28.1) 27.0 (26.1– 27.9) 26.7 (25.8– 27.6) 26.8 (26.0– 27.7) 0.002 Urinary system 446 404 435 462 553 557 644 669 674 739 7.5 (6.8–8.2) 6.8 (6.2–7.4) 6.7 (6.1–7.3) 7.1 (6.4–7.7) 7.9 (7.2–8.5) 6.6 (6.1–7.2) 7.9 (7.3–8.5) 6.9 (6.4–7.4) 6.9 (6.4–7.4) 7.3 (6.8–7.8) 0.798 1 3 European Spine Journal (2021) 30:3115–3127 3122 Years P value* 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 Respiratory system 614 533 710 697 725 776 925 1108 1089 1190 10.3 (9.5– 11.1) 9.0 (8.2–9.7) 10.9 (10.1– 11.6) 10.7 (9.9– 11.4) 10.3 (9.6– 11.0) 9.3 (8.6–9.9) 11.4 (10.7– 12.1) 11.5 (10.8– 12.1) 11.1 (10.5– 11.7) 11.8 (11.2– 12.4) < 0.001 Electrolyte disorder 355 353 393 418 465 577 543 614 641 581 6.0 (5.4–6.6) 5.9 (5.3–6.5) 6.0 (5.4–6.6) 6.4 (5.8–7.0) 6.6 (6.0–7.2) 6.9 (6.3–7.4) 6.7 (6.1–7.2) 6.3 (5.9–6.8) 6.5 (6.1–7.0) 5.8 (5.3–6.2) 0.592 Bedsore 261 246 266 244 269 361 325 355 352 340 4.4 (3.9–4.9) 4.1 (3.6–4.6) 4.1 (3.6–4.5) 3.7 (3.3–4.2) 3.8 (3.4–4.3) 4.3 (3.9–4.7) 4.0 (3.6–4.4) 3.7 (3.3–4.0) 3.6 (3.2–4.0) 3.4 (3.0–3.7) < 0.001 Venous thrombosis 134 126 169 161 186 227 202 272 232 292 2.3 (1.9–2.6) 2.1 (1.8–2.5) 2.6 (2.2–3.0) 2.5 (2.1–2.8) 2.6 (2.3–3.0) 2.7 (2.4–3.1) 2.5 (2.1–2.8) 2.8 (2.5–3.1) 2.4 (2.1–2.7) 2.9 (2.6–3.2) 0.011 Infection, Yes 848 875 947 972 1092 1253 1305 1437 1514 1528 14.2 (13.4– 15.1) 14.7 (13.8– 15.6) 14.5 (13.6– 15.3) 14.9 (14.0– 15.7) 15.5 (14.7– 16.4) 14.9 (14.2– 15.7) 16.0 (15.2– 16.8) 14.9 (14.1– 15.6) 15.5 (14.7– 16.2) 15.2 (14.5– 15.9) 0.028 Upper respiratory tract 36 34 34 33 43 48 52 63 72 64 0.6 (0.4–0.8) 0.6 (0.4–0.8) 0.5 (0.3–0.7) .5 (0.3–0.7) 0.6 (0.4–0.8) 0.6 (0.4–0.7) 0.6 (0.5–0.8) 0.7 (0.5–0.8) 0.7 (0.6–0.9) 0.6 (0.5–0.8) 0.111 Lower respiratory tract 228 251 224 263 305 346 373 455 496 501 3.8 (3.3–4.3) 4.2 (3.7–4.7) 3.4 (3.0–3.9) 4.0 (3.5–4.5) 4.3 (3.9–4.8) 4.1 (3.7–4.6) 4.6 (4.1–5.0) 4.7 (4.3–5.1) 5.1 (4.6–5.5) 5.0 (4.5–5.4) < 0.001 Urinary system 352 317 352 376 468 430 513 520 548 603 5.9 (3.3–4.3) 5.3 (4.8–5.9) 5.4 (4.8–5.9) 5.8 (5.2–6.3) 6.7 (6.1–7.2) 5.1 (4.7–5.6) 6.3 (5.8–6.8) 5.4 (4.9–5.8) 5.6 (5.1–6.0) 6.0 (5.5–6.4) 0.662 Data are n or % (95%CI), unless stated otherwise; P value for Cochran–Armitage trend test. Discussion y g y We found that elderly individuals (65–74 age group) had the highest incidence of TSCI (with an average annual inci- dence of 127.1 cases/million people) and that the incidence increased slightly over time (AAPC, 2.3%); moreover, the incidence of TSCI in people aged 55–64 increased sig- nificantly over time (AAPC, 3.8%; with an AAPC of 9.0% for men). The peak age at which TSCI occurs in Beijing, Shanghai, Tianjin and other cities is 40–60 years old[3, 4, 17], but the time range of the report is earlier (2002–2010), indicating that with the ageing of the population in recent years, the proportion of cases of TSCI in elderly individuals is increasing. The results of studies from many countries indicate that young people are the second most vulnerable population to TSCI; thus, the age of TSCI patients shows a bimodal distribution [6, 7, 18]. However, our study found that the incidence in young people was much lower than that in elderly individuals (unimodal distribution). In addi- tion, countries such as the USA and Australia [6, 8] have found that the incidence of TSCI among young people has decreased significantly over time. Conversely, the incidence of TSCI in young people aged 15–24 in China has increased significantly over time, which may be due to the relative lag in public education, the improvement of motor vehicle safety functions, traffic safety laws and their enforcement despite the rapid development of the economy. We assessed the national trends in the incidence, aetiology, general clinical indicators and mortality of TSCI in China as reflected by hospitalized patients from 2009 to 2018. Our data show that the incidence of TSCI in China increased rapidly from 2009 to 2018 (that is, from 45.1 cases per mil- lion population in 2009 to 66.5 cases per million population in 2018). There were an estimated 59,685 and 92,841 cases in China in 2009 and 2018, respectively (Appendix). The incidence in the western region was lower than that in other areas. In the past 10 years, the incidence of TSCIs in the elderly population has always been high and continues to increase. The incidence in the rural population was higher than that in the urban population, but the incidence in the urban population is increasing faster over time. Mortality The hospital mortality rates in 2009 and 2018 were 2.5% (95% CI, 2.1–2.9%) and 1.9% (95% CI, 1.6–2.1%) (P < 0.001), respectively. Although the mortality rate increased with age (Table 3), among people 55 or older, it tended to decrease over time. For example, in the age group ≥ 75, the mortality rate decreased from 6.2% (95% CI, 2.9–9.4%) in 2009 to 3.9% (95% CI, 2.0–5.7%) in 2018 (P = 0.032). Results of general clinical indicators SCI means spinal cord injury Bold means that the internal constituent ratio of secondary results shows a linear trend over time 1 European Spine Journal (2021) 30:3115–3127 3123 cases/million people). The imbalance of regional economic development may be the main reason for this difference [2]. Previous literature has made it clear that the difference in the level of socio-economic development between devel- oped and developing countries is a potential factor for dif- ferences in the incidence of TSCI [12, 16]. The economic development of the three regions in China is not coordinated, and the economic level of the western region has always lagged far behind that of the eastern and central regions. The rapid increase in the incidence in the western region may be related to the continuous strong governmental support of the economy of the western region in recent years. Discussion The inci- dence in the low-educated population was slightly higher than that of the high-educated population, and the incidence in low-educated people has increased faster over time. The hospital mortality rate of TSCI in China is low and continues to decline (from 2.5% in 2009 to 1.9% in 2018), particularly among patients aged 55 and older.i This is the first time that we have reported the results of an epidemiological survey of TSCIs at the national level. The annual incidence of TSCI in the world is estimated to range from 10.4 to 83.0 cases per 1 million people [11, 12]. The incidence of TSCI is 20.7–83.0 cases per 1 million in North America, 8.0–130.6 cases per 1 million in Europe [13, 14], and 14.0 cases per 1 million people in Australia [6]. A review of studies focusing on developing countries found that the incidence of TSCI was 25.5 cases per 1 million peo- ple [15]. The USA has a similar reported incidence (crude incidence rate of 54 cases/million people) of TSCI compared to that of China (crude incidence rate of 55.3 cases/million people, standardized incidence rate of 44.9 cases/million people) [8].iif The incidence of TSCI can also vary with the progress of urbanization and the education level. We found that the inci- dence was higher in the rural population, but the incidence in the urban population has increased each year. The sum- mary study by Yuan et al. [19] classified the occupations of TSCI patients in China and found that farmers, workers and unemployed people had a significantly higher risk of TSCI. In China, these occupations are mainly concentrated in the rural population, and the educational level is generally low. However, with the acceleration of urbanization, these people gradually gather to work or even settle in cities. As a result, the incidence in the urban population and the incidence in the low-educated population increased significantly with the passage of time. Migrant workers are more likely to gather in the eastern and central economically developed regions, which may be responsible for the large differences in the We identified significant differences in the characteris- tics and trends of the TSCI incidence among the eastern, central and western regions. Discussion The incidence in the western region was significantly lower than that in the other areas (the standardized annual average incidence rate was 34.2 1 3 3 3124 European Spine Journal (2021) 30:3115–3127 3 Table 3 Temporal trends in mortality of traumatic spinal cord injury in China Data are n or % (95%CI), unless stated otherwise; P value for Cochran–Armitage trend test Bold means that the mortality of age subgroup shows a linear trend over time Years P value* Age group (year) 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 ≤ 14 0.633 No. died/total 1/114 1/103 0/131 0/126 2/132 3/159 3/144 1/162 2/159 1/177 % (95%CI) 0.9 (− 0.8–2.6) 1.0 (− 0.9–2.9) 0 0 1.5 (− 0.6–3.6) 1.9 (− 0.2–4.0) 2.1 (− 0.2–4.4) 0.6 (− 0.6–1.8) 1.3 (− 0.5–3.0) 0.6 (− 0.5–1.7) 15–24 0.948 No. died/total 5/274 2/292 2/246 2/240 4/274 3/307 4/295 6/375 5/364 3/365 % (95%CI) 1.8 (0.2–3.4) 0.7 (− 0.3–1.6) 0.8 (− 0.3–1.9) 0.8 (− 0.3–2.0) 1.5 (0.0–2.9) 1.0 (− 0.1–2.1) 1.4 (0.0–2.7) 1.6 (0.3–2.9) 1.4 (0.2–2.6) 0.8 (− 0.1–1.7) 25–34 0.829 No. died/total 12/652 7/590 6/586 7/582 10/609 6/715 9/703 14/902 12/891 15/934 % (95%CI) 1.8 (0.8–2.9) 1.2 (0.3–2.1) 1.0 (0.2–1.8) 1.2 (0.3–2.1) 1.6 (0.6–2.7) 0.8 (0.2–1.5) 1.3 (0.4–2.1) 1.6 (0.7–2.4) 1.3 (0.6–2.1) 1.6 (0.8–2.4) 35–44 0.503 No. died/total 11/872 14/835 12/899 15/914 11/994 14/1203 15/1136 18/1395 16/1386 18/1394 % (95%CI) 1.3 (0.5–2.0) 1.7 (0.8–2.5) 1.3 (0.6–2.1) 1.6 (0.8–2.5) 1.6 (0.5–1.8) 1.2 (0.6–1.8) 1.3 (0.7–2.0) 1.3 (0.7–1.9) 1.2 (0.6–1.7) 1.3 (0.7–1.9) 45–54 0.511 No. died/total 26/1633 26/1685 24/1922 35/1903 36/2024 35/2358 32/2274 34/2659 39/2688 41/2752 % (95%CI) 1.6 (1.0–2.2) 1.5 (1.0–2.1) 1.2 (0.8–1.7) 1.8 (1.2–2.4) 1.8 (1.2–2.4) 1.5 (1.0–2.0) 1.4 (0.9–1.9) 1.3 (0.9–1.7) 1.5 (1.0–1.9) 1.5 (1.0–1.9) 55–64 < 0.001 No. died/total 44/1345 48/1372 45/1558 43/1538 51/1667 47/2020 47/1994 49/2317 53/2377 50/2489 % (95%CI) 3.3 (2.3–4.2) 3.5 (2.5–4.5) 2.9 (2.1–3.7) 2.8 (2.0–3.6) 3.1 (2.2–3.9) 2.3 (1.7–3.0) 2.4 (1.7–3.0) 2.1 (1.5–2.7) 2.2 (1.6–2.8) 2.0 (1.5–2.6) 65–74 < 0.001 No. died/total 39/854 42/850 37/954 41/976 43/1039 41/1274 44/1265 40/1489 45/1522 44/1548 % (95%CI) 4.6 (3.2–6.0) 4.9 (3.5–6.4) 3.9 (2.7–5.1) 4.2 (2.9–5.5) 4.1 (2.9–5.3) 3.2 (2.2–4.2) 3.5 (2.5–4.5) 2.7 (1.9–3.5) 3.0 (2.1–3.8) 2.8 (2.0–3.7) ≥ 75 0.032 No. died/total 13/210 14/218 14/241 12/256 16/286 14/350 15/327 15/373 18/409 16/415 % (95%CI) 6.2 (2.9–9.4) 6.4 (3.2–9.7) 5.8 (2.9–8.8) 4.7 (2.1–7.3) 5.6 (2.9–8.3) 4.0 (1.9–6.1) 4.6 (2.3–6.9) 4.0 (2.0–6.0) 4.4 (2.4–6.4) 3.9 (2.0–5.7) Overall < 0.001 No. Discussion When vigorously developing infrastructure construction in the western region, construction units must be required to strengthen personal safety awareness and focus on supervis- ing work safety. The increasing proportions of cervical spinal cord inju- ries and complete spinal cord injuries may be related to the increase in traffic injuries and high falls. With the expansion and upgrading of Chinese medical institutions and the full The hospital mortality rate of TSCI reported in the USA is very high and has increased from 6.6% in 1993–1996 to 7.5% in 2010–2012 [8]. The standardized mortality rate of TSCI reported in Australia increased from 2.34 per million people in 2002 to 3.57 per million people [17]. Although we reported that the hospital mortality of TSCI patients is low and has decreased over time, in fact, we only reported the proportion of hospital deaths. Recently, through a survey of the China Disease Surveillance Points system covering 161 surveillance sites in China, Li et al. [23] found that the mortality of TSCI increased from 1.9 cases/million people in 2006 to 3.4 cases/million people in 2016. Many pre-hospital deaths can be monitored by using the system, which may be a potential cause of the difference in incidence trends. f The changes of population ageing, economic develop- ment, urbanization and education level that we mentioned earlier affect the incidence of TSCI in China. However, we cannot ignore the impact of utilization/accessibility of healthcare on the changing trend of the incidence of TSCI in China, because with the significant expansion of health care in China over the decade, utilization/accessibility of healthcare continues to improve. For example, it is possible that 10 years ago, many patients with TSCI gave up treat- ment before hospitalization because of the limited medical resources and heavy financial burden. At present, due to the comprehensive coverage of China’s medical insurance and rich medical resources, the hospitalization rate of patients with TSCI is higher than that of 10 years ago, which may be a confounding factor contributing to the increase in the incidence of TSCI over the decade. However, we believe that economic development and so on are the main factors.fi Conclusions p In developed countries, traffic accidents and falls are the main causes of TSCIs [8, 20, 21]. Traffic injuries are also the main cause of TSCIs in China, particularly in the eastern and central regions. With the rapid development of China’s economy, most people own private cars, which has led to a significant increase in car accidents. In the west- ern region, the proportion of TSCIs caused by high-altitude falls remains high and has increased over time. In recent years, the acceleration of urbanization and infrastructure construction in the western region with the support of the government may be potential factors for this increase. The government must formulate corresponding prevention strate- gies according to the aetiological characteristics of injuries in different regions. For example, the eastern and central regions must further standardize driver behaviour, improve road conditions, strengthen the crackdown on drunk driving, etc. High falls are more likely to occur in construction sites. When vigorously developing infrastructure construction in the western region, construction units must be required to strengthen personal safety awareness and focus on supervis- ing work safety. The incidence of TSCI increased significantly from 2009 to 2018, particularly in the western region. The incidence in the elderly population was always high and has contin- ued to increase over time. The incidence is higher in the rural population, but the incidence in the urban population has increased significantly in recent years. The incidence in the low-educated population is slightly higher than that in the high-educated population, and the incidence of the low- educated population has increased rapidly in recent years. TSCI caused by traffic accidents is the focus of prevention work in the future, but the proportion of high falls is the highest in the western region, so it is necessary to formulate corresponding preventive measures according to the char- acteristics of regional injury causes. The death rate of TSCI patients in the hospital is relatively low and continues to decrease over time, but elderly individuals are still at a high risk of hospital death. Supplementary Information The online version contains supplemen- tary material available at https://doi.org/10.1007/s00586-021-06957-3. Discussion died/total 151/5954 154/5945 140/6537 155/6535 173/7025 163/8386 169/8137 177/9672 190/9796 188/10074 % (95%CI) 2.5 (2.1–2.9) 2.6 (2.2–3.0) 2.1 (1.8–2.5) 2.4 (2.0–2.7) 2.5 (2.1–2.8) 1.9 (1.6–2.2) 2.1 (1.8–2.4) 1.8 (1.6–2.1) 1.9 (1.7–2.2) 1.9 (1.6–2.1) 1 3 3 European Spine Journal (2021) 30:3115–3127 3125 a higher probability of complications and infection [22], which should be the most reasonable explanation. stratified incidence of urbanization and education level in different regions. different regions. The changes of population ageing, economic develop- ment, urbanization and education level that we mentioned earlier affect the incidence of TSCI in China. However, we cannot ignore the impact of utilization/accessibility of healthcare on the changing trend of the incidence of TSCI in China, because with the significant expansion of health care in China over the decade, utilization/accessibility of healthcare continues to improve. For example, it is possible that 10 years ago, many patients with TSCI gave up treat- ment before hospitalization because of the limited medical resources and heavy financial burden. At present, due to the comprehensive coverage of China’s medical insurance and rich medical resources, the hospitalization rate of patients with TSCI is higher than that of 10 years ago, which may be a confounding factor contributing to the increase in the incidence of TSCI over the decade. However, we believe that economic development and so on are the main factors. In developed countries, traffic accidents and falls are the main causes of TSCIs [8, 20, 21]. Traffic injuries are also the main cause of TSCIs in China, particularly in the eastern and central regions. With the rapid development of China’s economy, most people own private cars, which has led to a significant increase in car accidents. In the west- ern region, the proportion of TSCIs caused by high-altitude falls remains high and has increased over time. In recent years, the acceleration of urbanization and infrastructure construction in the western region with the support of the government may be potential factors for this increase. The government must formulate corresponding prevention strate- gies according to the aetiological characteristics of injuries in different regions. For example, the eastern and central regions must further standardize driver behaviour, improve road conditions, strengthen the crackdown on drunk driving, etc. High falls are more likely to occur in construction sites. Declarations 9. Pickett GE, Campos-Benitez M, Keller JL, Duggal N (2006) Epidemiology of traumatic spinal cord injury in Canada. Spine 31:799–805 Conflict of interest The authors declare that there are no conflicts of interest. 10. Chen XY, Chen D, Chen CH (2018) The epidemiology and dis- ease burden of traumatic spinal cord injury in China: a systematic review. Chin J Evid-Based Med 18:143–150 Ethical approval The study was approved by the appropriate institu- tional review board for each study site, and given the retrospective nature of the study, the requirement for informed consent was waived. review. Chin J Evid-Based Med 18:143–150 11. Jackson AB, Dijkers M, Devivo MJ, Poczatek RB (2004) A demo- graphic profile of new traumatic spinal cord injuries: change and stability over 30 years. Arch Phys Med Rehabil 85:1740–1748. https://doi.org/10.1016/j.apmr.2004.04.035 Availability of data and materials Except for the personal data of all included patients with spinal cord injury such as ID number and place of residence cannot be displayed because of privacy protection, the epidemiological raw data of other key variables (cause of injury, injury level, severity of injury) and supplementary materials used to calculate the results are published on Figshare (https://doi.org/10.6084/m9.figsh are.12782834) and can be obtained. 12. Seyed Behzad J, Sara B, Farhad S, Ellen Merete H, Vafa RM (2015) Incidence of traumatic spinal cord injury worldwide: a systematic review. Eur Spine J 24:905–918 13. Cripps RA, Lee BB, Wing P, Weerts E, Mackay J, Brown D (2011) A global map for traumatic spinal cord injury epidemiol- ogy: towards a living data repository for injury prevention. Spinal Cord 49:493–501. https://doi.org/10.1038/sc.2010.146 Standard statement We have no personal or institutional financial interest in drugs, materials, or devices described in our submissions. p g 14. Wyndaele M, Wyndaele JJ (2006) Incidence, prevalence and epi- demiology of spinal cord injury: what learns a worldwide litera- ture survey? Spinal Cord 44:23–29 15. Rahimi-Movaghar V, Sayyah MK, Akbari H, Khorramirouz R, Rasouli MR, Moradi-Lakeh M, Shokraneh F, Vaccaro AR (2013) Epidemiology of traumatic spinal cord injury in developing coun- tries: a systematic review. Neuroepidemiology 41:65–85. Supplementary Information The online version contains supplemen- tary material available at https://doi.org/10.1007/s00586-021-06957-3. The increasing proportions of cervical spinal cord inju- ries and complete spinal cord injuries may be related to the increase in traffic injuries and high falls. With the expansion and upgrading of Chinese medical institutions and the full implementation of the medical insurance policy, the propor- tion of patients receiving rehabilitation treatment and surgi- cal treatment continues to increase each year. Despite the obvious improvement in medical conditions and the rapid improvement in the treatment level in China in recent years, the proportion of hospitalized complications and infection in TSCI patients reported in our study still increased over time. Previous studies have shown that patients with cervi- cal spinal cord injury and complete spinal cord injury have Authors’ contributions DH, JD, and LY searched the literature, con- ceived the study, designed the study, analysed the data, interpreted the results, and drafted the report. XQ, SY, JZ, and WZ designed the methods of the study and analysed the data. D-GH, JY, MZ, JO, HZ, KD, HS, YC, YZ, QT, YL, ZZ, YW, YT, HC, LB, HL, CM, YW, XW, and CJ collected the data. RZ, JL, BL, SF, LN, JS, XM, ZS, YG, ZG, YS, WM, and QC revised the report. DH, JD, LY, and BH obtained funding. DH and BH conceived the study, organized, and supervised the study, interpreted the results, and revised the report. Funding This study was supported by the Key Program from the National Natural Science Funds (81830077); the General Program from the National Natural Science Funds (81772357); the General Project from the Science and Technology Commission of Shaanxi (2020SF- 187); the Scientific and Technological Achievements Transformation 1 3 3 3126 European Spine Journal (2021) 30:3115–3127 Incidence trends of traumatic spinal cord injury and traumatic brain injury in Spain, 2000–2009. Accid Anal Prev 46:37–44 Project from the Science and Technology Commission of Shaanxi (2018HJCG-08); and the General Project from the Xi’an Municipal Health Committee (2020yb34). Project from the Science and Technology Commission of Shaanxi (2018HJCG-08); and the General Project from the Xi’an Municipal Health Committee (2020yb34). 8. Jain NB, Ayers GD, Peterson EN, Harris MB, Leslie M, O’Connor KC, Eric G (2015) Traumatic spinal cord injury in the United States, 1993–2012. JAMA 313:2236–2243 Declarations https:// doi.org/10.1159/000350710 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/. 16. Lee BB, Cripps RA, Fitzharris M, Wing PC (2014) The global map for traumatic spinal cord injury epidemiology: update 2011, global incidence rate. Spinal Cord 52:110–116 17. Chang FS, Zhang Q, Sun M, Yu HJ, Hu LJ, Wu JH, Chen G, Xue LD, Lu J (2018) Epidemiological study of Spinal Cord Injury indi- viduals from halfway houses in Shanghai, China. J Spinal Cord Med 41:450–458. https://doi.org/10.1080/10790268.2017.13673 57 18. Kang Y, Ding H, Zhou HX, Wei ZJ, Liu L, Pan DY, Feng SQ (2018) Epidemiology of worldwide spinal cord injury: a literature review. 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Pérez K, Novoa AM, Santamariña-Rubio E, Narvaez Y, Arrufat V, Borrell C, Cabeza E, Cirera E, Ferrando J, García-Altés A (2012) 1 3 3127 European Spine Journal (2021) 30:3115–3127 Authors and Affiliations Dingjun Hao1 · Jinpeng Du1 · Liang Yan1 · Baorong He1 · Xiao Qi2 · Shicheng Yu2 · Jiaojiao Zhang2 · Wenjing Zheng2 · Rongqiang Zhang3 · Da‑Geng Huang1 · Junsong Yang1 · Ming Zhu1 · Jiawei Ouyang1 · He Zhao1 · Keyuan Ding1 · Haodong Shi1 · Yang Cao1 · Ying Zhang1 · Qinghua Tang1 · Yuan Liu1 · Zilong Zhang1 · Yuhang Wang1 · Ye Tian1 · Hao Chen1 · Lulu Bai1 · Heng Li1 · Chenchen Mu1 · Youhan Wang1 · Xiaohui Wang1 · Chao Jiang1 · Jianhua Lin4 · Bin Lin5 · Shunwu Fan6 · Lin Nie7 · Jiefu Song8 · Xun Ma9 · Zengwu Shao10 · Yanzheng Gao11 · Zhong Guan12 · Yueming Song13 · Weihu Ma14 · Qixin Chen15 n Hao1 · Jinpeng Du1 · Liang Yan1 · Baorong He1 · Xiao Qi2 · Shicheng Yu2 · Jiaojiao Zhang2 · 2 3 1 1 1 Dingjun Hao1 · Jinpeng Du1 · Liang Yan1 · Baorong He1 · Xiao Qi2 · Shicheng Yu2 · Jiaojiao Zhang2 · Wenjing Zheng2 · Rongqiang Zhang3 · Da‑Geng Huang1 · Junsong Yang1 · Ming Zhu1 · Jiawei Ouyang1 · He Zhao1 · Keyuan Ding1 · Haodong Shi1 · Yang Cao1 · Ying Zhang1 · Qinghua Tang1 · Yuan Liu1 · Zilong Zhang1 · Yuhang Wang1 · Ye Tian1 · Hao Chen1 · Lulu Bai1 · Heng Li1 · Chenchen Mu1 · Youhan Wang1 · Xiaohui Wang1 · Chao Jiang1 · Jianhua Lin4 · Bin Lin5 · Shunwu Fan6 · Lin Nie7 · Jiefu Song8 · Xun Ma9 · Zengwu Shao10 · Yanzheng Gao11 · Zhong Guan12 · Yueming Song13 · Weihu Ma14 · Qixin Chen15 9 Department of Orthopaedic, Shanxi Bethune Hospital, Taiyuan, China 1 Department of Spine Surgery, Honghui Hospital, Xi’an Jiao Tong University, Youyidong Road, Xi’an 710000, Shaanxi Province, China 1 Department of Spine Surgery, Honghui Hospital, Xi’an Jiao Tong University, Youyidong Road, Xi’an 710000, Shaanxi Province, China 9 Department of Orthopaedic, Shanxi Bethune Hospital, Taiyuan, China 10 Department of Orthopaedic Union Hospital Tongji Medic 10 Department of Orthopaedic, Union Hospital, Tongji Medical College of Huazhong University of Science and Technology, Wuhan, China 2 Office of Epidemiology, Chinese Center for Disease Control and Prevention, Beijing, China 11 Department of Orthopaedic, Henan Provincial People’s Hospital, Zhengzhou, China 3 Office of Epidemiology, School of Public Health, Shaanxi University of Traditional Chinese Medicine, Xianyang, China 12 Department of Spine Surgery, Qinghai University Affiliated Hospital, Xining, China 4 Department of Spine Surgery, The First Affiliated Hospital of Fujian Medical University, Fujian, China 13 Department of Spine Surgery, West China Hospital of Sichuan University, Chengdu, China 5 Department of Orthopaedic, The No. 1 Department of Spine Surgery, Honghui Hospital, Xi’an Jiao Tong University, Youyidong Road, Xi’an 710000, Shaanxi Province, China Authors and Affiliations 909 Hospital of the People’s Liberation Army, Zhangzhou, China 14 Department of Spine Surgery, Ningbo No. 6 Hospital, Ningbo, China 6 Department of Orthopaedic, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Zhejiang, China 15 Department of Orthopaedic, The Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, China 7 Department of Spine Surgery, Qilu Hospital of Shandong University, Shandong, China 8 Department of Orthopaedic, Shanxi Provincial People’s Hospital, Taiyuan, China 1 3 1 3
https://openalex.org/W2116499371	https://www.spiedigitallibrary.org/journals/journal-of-photonics-for-energy/volume-5/issue-1/057204/Modeling-approach-to-derive-the-anisotropic-complex-refractive-index-of/10.1117/1.JPE.5.057204.pdf	English	null	Modeling approach to derive the anisotropic complex refractive index of polymer:fullerene blends for organic solar cells utilizing spectroscopic ellipsometry	Journal of photonics for energy	2,015	cc-by	9,129	Modeling approach to derive the anisotropic complex refractive index of polymer:fullerene blends for organic solar cells utilizing spectroscopic ellipsometry Michael F. G. Klein Gustavo Q. Glasner de Medeiros Panagiota Kapetana Uli Lemmer Alexander Colsmann Modeling approach to derive the anisotropic complex refractive index of polymer:fullerene blends for organic solar cells utilizing spectroscopic ellipsometry Michael F. G. Klein, Gustavo Q. Glasner de Medeiros, Panagiota Kapetana, Uli Lemmer, and Alexander Colsmann* Karlsruhe Institute of Technology (KIT), Light Technology Institute (LTI), Engesserstrasse 13, Karlsruhe, 76131, Germany Abstract. The knowledge of the complex refractive indices of all thin layers in organic solar cells (OSCs) is a prerequisite for comprehensive optical device simulations that are particularly important for sophisticated device architectures, such as tandem OSCs. Therefore, refractive indices are often determined via spectroscopic ellipsometry and subsequent time-consuming modeling. Here, we investigate a modeling approach that allows for the determination of com- plex refractive indices of bulk-heterojunctions by superimposing the optical models of the respective fullerenes and polymers. The optical constants of neat [6,6]-phenyl C71-butyric acid methyl ester (PC71BM), poly{[4,4’-bis(2-ethylhexyl)dithieno(3,2-b;2’,3’-d)silole]- 2,6-diyl-alt-(2,1,3-benzothidiazole)-4,7-diyl} (PSBTBT) and poly[2,6-(4,4-bis(2-ethylhexyl)- 4H-cyclopenta[2,1-b;3,4-b′′]dithiophene)-alt-4,7-(2,1,3-benzothiadiazole) (PCPDTBT) are determined, covering the OSC relevant spectral region from 250 to 1,000 nm. Then the blends PSBTBT∶PC71BM and PCPDTBT∶PC71BM are described within an effective medium approxi- mation. From this approximation, the mass density ratio of polymer and fullerene can be derived. This approach furthermore allows for a uniaxial anisotropic optical description of the polymers and provides insight into thin-film morphology. In contrast to x-ray diffraction experiments, this method also allows for probing amorphous materials. Spectroscopic ellipsometry can be a valuable tool for the investigation of bulk-heterojunction morphologies of the latest high- performance OSC materials that exhibit a low degree of crystallinity. © The Authors. Published by SPIE under a Creative Commons Attribution 3.0 Unported License. Distribution or repro- duction of this work in whole or in part requires full attribution of the original publication, including its DOI. [DOI: 10.1117/1.JPE.5.057204] Keywords: spectroscopic ellipsometry; organic solar cells; morphology; optical anisotropy; material densities. Paper 14054SS received Aug. 18, 2014; accepted for publication Nov. 10, 2014; published on- line Jan. 6, 2015. *Address all correspondence to: Alexander Colsmann, E-mail: alexander.colsmann@kit.edu Klein et al.: Modeling approach to derive the anisotropic complex refractive index. . . Unfortunately, optical constants from the literature often cannot be used to describe certain experiments since the optical properties strongly depend on the substrate, the underlying inter- face, the intrinsic polymer properties (molecular weight, polydispersity), the polymer:fullerene ratio, and the process conditions, such as the solvent system, the drying kinetics, and the postprocessing treatments.13–24 Therefore, an urgent need for flexible optical descriptions of bulk-heterojunctions emerged, which use parametric descriptions and allow for individual adjustments. If carried out purposefully, the optical model further reveals information about anisotropy within the bulk-heterojunction.25 Such models were recently utilized to determine the vertical gradient in the active layer of OSCs13,25 and to investigate the shape of [6,6]-phenyl C61-butyric acid methyl ester (PC61BM) inclusions in poly(3-hexylthiophene-2,5-diyl) (P3HT) solar cells.26 y 61 p y y p y In this work, we present a modeling approach for spectroscopic ellipsometry (SE) data analysis, which facilitates the determination of the complex refractive indices of various poly- mer:fullerene blends. Therefore, in a first step, the relative dielectric functions of the fullerene [6,6]-phenyl C71-butyric acid methyl ester (PC71BM) and the polymers poly{[4,4’-bis(2-ethyl- hexyl)dithieno(3,2-b;2’,3’-d)silole]-2,6-diyl-alt-(2,1,3-benzothidiazole)-4,7-diyl} (PSBTBT) and poly[2,6-(4,4-bis(2-ethylhexyl)-4H-cyclopenta[2,1-b;3,4-b’’]dithiophene)-alt-4,7-(2,1,3- benzothiadiazole)] (PCPDTBT) are described by the superposition of Gaussian oscillators. In a second step, the relative dielectric functions of the polymer:fullerene blends are determined by superimposing the optical models of both constituents. PSBTBT and PCPDTBT are well- known donor-acceptor copolymers with good power conversion efficiencies when blended with PC71BM.27–31 Their similar structures allow a direct comparison. For the active layer dep- osition and blend ratios, we follow literature-known process protocols, which are optimized for high-performance solar cells.27–29 Optical data are presented in the spectral region from 250 to 1,000 nm. We further compare isotropic and anisotropic optical models of neat polymers. The results of this comparison serve as input parameters for anisotropic optical descriptions of the blends, which are then analyzed with respect to the predominant polymer orientation and the degree of aggregation. Journal of Photonics for Energy 1 Introduction Organic solar cells (OSCs) combine the advantages of low-cost and large-area fabrication with the use of nonhazardous and environmentally friendly materials. Over the last few years, power conversion efficiencies improved continuously, now exceeding 10% in single-junction solar cells.1 Improvements often originated from the synthesis of new absorber materials that allowed for an enhanced spectral coverage of the solar spectrum or enabled higher internal quantum efficiencies.2–7 In the lab, screening and optimization of new materials and cell architectures often follow a trial and error approach, although only very little amounts of material are avail- able. An alternative and material saving route to optimized OSCs is a comprehensive optoelec- tronic device simulation that reduces the experimental parameter space. This becomes particularly important for sophisticated device architectures, such as tandem solar cells.8 However, in order to carry out meaningful optical simulations, a profound knowledge of the refractive indices is mandatory.5,9–12 057204-1 Vol. 5, 2015 Vol. 5, 2015 057204-1 Journal of Photonics for Energy Journal of Photonics for Energy Klein et al.: Modeling approach to derive the anisotropic complex refractive index. . . Klein et al.: Modeling approach to derive the anisotropic complex refractive index. . . εr;2;nðEÞ ¼ Ane − 2 ﬃﬃﬃﬃﬃ lnð2Þ p ðE−EnÞ Brn 2 −Ane − 2 ﬃﬃﬃﬃﬃ lnð2Þ p ðEþEnÞ Brn 2 ; (2) (2) εr;1;nðEÞ ¼ 2 π P Z∞ 0 ξ ε2;nðξÞ ξ2 −E2 dξ; (3) (3) where An is the amplitude, En is the center energy, Brn is the width, and P is the principal value.33 Higher-energy transitions outside the measurement range are summarized in a high- energy pole function, i.e., an oscillator with zero-width. Further, a real constant εr;∞is added to εr;1. ; Based on this parameterized model, the optical response of the sample is calculated and compared to the measured Ψ and Δ data and the transmission. The parameters for the individual layers (thickness, oscillator parameters, etc.) are fitted in order to achieve a good match between model-generated and measured values. The difference between these values is quantified by the mean squared error (MSE).34 MSE is used by the analysis software as weighted test function during the fitting procedure to further reduce the deviation between the three values by making use of the Marquardt-Levenberg algorithm.35,36 We verify the results of the fit and ensure physical significance of the calculated optical constants by, first, measuring the thicknesses of thin-films deposited on glass substrates in a transparent energy band by applying the Cauchy dispersion relation and by comparing them to profilometry results. (We note that the Cauchy model does not fulfill the Kramers-Kronig relation but is a valid approximation for transparent spectral regions.) Second, we perform a multisample analysis: several samples with varying thin-film thicknesses are characterized. All respective datasets are described with the same optical model, hence enabling a general and robust optical description and reducing parameter correlation.37,38 In the end, the oscillator model will guarantee full Kramers-Kronig consistency. Further, we include a wide range of angles of incidence in the analysis, typically 25 deg around the Brewster angle (step width: 5 deg). To perform the multisample analysis, the datasets are measured on several samples with different layer thicknesses. All SE investigations cover the spectral region from 250 to 1,700 nm (1,000 nm for PC71BM) and layer thicknesses are determined in the transparent (k ¼ 0) wavelength regime from 1,000 to 1,700 nm. For reasons of clarity, we will show a reduced dataset in the following figures. Klein et al.: Modeling approach to derive the anisotropic complex refractive index. . . In particular, we skip data in the infrared region beyond 1,000 nm, restrict to some representative angles of incidence, and only show typical data for one sample of the multisample analysis. 3.1 Spectroscopic Ellipsometry Ψ and Δ were measured with a variable angle spectroscopic ellipsometer (VASE®, J.A. Woollam Co., Inc.), equipped with a rotating analyzer and two detectors. A rotating retarder plate (MgF2 Berek waveplate) in the beam path behind the fixed polarizer allows for a precise determination of Δ.39 Datasets were recorded between 250 and 1,700 nm (1,000 nm for PC71BM). Transmission data of the samples were collected at normal incidence and by moving the VASE detector arm into the optical path behind the sample. Thus, the transmission spectra were determined on the very same spot as the ellipsometric data, allowing for their incorporation into the analysis. The datasets were analyzed with the software WVASE32® (J. A. Woollam, version 3.774). Journal of Photonics for Energy 2 Spectroscopic Ellipsometry SE is a contactless and nondestructive measurement technique that allows for deriving the complex refractive indices of thin-films and their thicknesses simultaneously. The complex refractive index ˜n, given by ˜n ¼ n −ik ¼ ﬃﬃﬃﬃ ˜εr p ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ εr;1 −iεr;2 p (1) (1) comprises the refractive index n (real part) and the extinction coefficient k (imaginary part) and is directly related to the complex relative dielectric function ˜εr, assuming a relative permeabil- ity ˜μr ¼ 1. The basic ellipsometry working principle relies on measuring the change of the light beam polarization state after interacting with the sample. This change is described by the two ellipso- metric values Ψ and Δ, which represent the amplitude ratio and phase difference between p- and s-polarization.32 In this study, various wavelengths and angles of incidence were investigated. In addition, transmission spectra were recorded at normal incidence on the very same spot and incorporated into the analysis. We use vendor-supplied software (WVASE32®, J. A. Woollam) to analyze the datasets. First, a multilayer model is constructed, where each individual layer describes a certain subset of physical properties of the sample, e.g., the level of interface intermixing, surface roughness, and the relative dielectric function of the material. In the following, “layer” will refer to a layer in the optical model and not necessarily to a physical layer. The relative dielectric functions are defined by oscillator models, i.e., the description of each optical transition by an appropriate oscillator. Then the relative dielectric function ˜εr of a material can be described by the sum of these oscillators. To describe the transitions within the meas- urement range, we choose an oscillator type generating a Gaussian line shape in εr;2, Eq. (2), with a Kramers-Kronig consistent counterpart in εr;1, Eq. (3). 057204-2 057204-2 Vol. 5, 2015 Vol. 5, 2015 Klein et al.: Modeling approach to derive the anisotropic complex refractive index. . . 3.3 Materials and Sample Preparation Soda-lime glass substrates were cleaned with acetone and isopropyl alcohol and then dried in a nitrogen stream. All materials were used as received and were deposited under inert conditions in a nitrogen glovebox (O2 and H2O < 1 ppm). PC71BM (Solenne, >99%) was dissolved in DCB (anhydrous, 99%, Sigma-Aldrich®) at a concentration of 60 mg∕mL, stirred overnight at 80°C, and spin cast on substrates at room temperature with different spin coater settings to achieve thicknesses between 85 and 120 nm. p p g PSBTBT and PCPDTBT were both dissolved in DCB with a concentration of 13 mg∕mL, stirred overnight at 70 or 90°C, respectively, and were deposited from hot solution on hot soda-lime glass substrates. Different spin coater settings allowed for tuning the layer thicknesses between 40 and 60 nm for PSBTBT or 15 and 30 nm for PCPDTBT. The blends PSBTBT∶PC71BM (1∶2, layer thicknesses between 90 and 115 nm) and PCPDTBT∶PC71BM (1∶3.4, layer thicknesses between 90 and 130 nm) with a polymer con- centration of 13 mg∕mL in DCB were spin coated following the same conditions used for neat films. The blend ratios were chosen according to optimized process protocols.27,29 SE data of the neat polymer films and the blend films were recorded between 250 and 1,700 nm at different angles of incidence α between 30 and 80 deg in 5 deg steps. Datasets were analyzed over the entire spectral range. For reasons of clarity, we only show a reduced dataset in the figures, i.e., we skip data in the infrared beyond 1,000 nm, restrict to some representative angles of incidence, and only show data for one sample of the multisample analysis. Thin-film thicknesses were cross-checked with a stylus profiler (DektakXT, Bruker AXS GmbH) and compared with ellipsometry results. 3.2 Transmission Spectroscopy Transmission spectra of diluted solutions and of solid films spin cast onto quartz-glass substrates were recorded with a spectrophotometer (Lambda 1050, Perkin Elmer), enabling a lower noise level than the measurements carried out with the ellipsometer. 057204-3 Vol. 5, 2015 Vol. 5, 2015 Klein et al.: Modeling approach to derive the anisotropic complex refractive index. . . Journal of Photonics for Energy 4.2 PC71BM Solar cells based on the polymers PCPDTBT and PSBTBT in combination with the electron acceptor PC71BM exhibit enhanced power conversion efficiencies. We first analyze PC71BM. Therefore, the fullerene is dissolved in 1,2-dichlorobenzene (DCB) and then spin cast on the glass substrates as described in Sec. 3.3. A multisample SE analysis is performed on six samples with varying thicknesses. Figures 2(a) and 2(b) show the experimental data of a typical sample. Transmission spectra of PC71BM in DCB solution and of a solid PC71BM thin- film are recorded with a spectrophotometer, Fig. 2(c). From the absorption features present in all spectra, the starting values En;input for the center energies of the Gaussian oscillators are derived, Table 1. Based on this estimation of En, an optical model is set up comprising nine Gaussian oscillators Gaun, a pole taking into account higher-energy transitions, and a real constant εr;∞. A stepwise fit (MSE ¼ 11.14), starting from the infrared and progressing toward higher energies, results in the oscillator parameters given in Table 1. The fitted center energies En differ slightly from their respective starting values En;input. In this multisample analysis, all measured datasets are taken into account. If the fit is restricted to a single sample, lower MSEs can be achieved. As the multisample analysis only differs slightly from the results of the single-sample analysis and as we aim, here and in the following, at finding a widely applicable optical model, we utilize the multisample approach. The model-generated data Ψmod, Δmod, and the optical constants are depicted in Figs. 2(a), 2(b), and 2(d), respectively. The good agreement with the optical constants in Ref. 42, supporting information, verifies the chosen approach. The good match between simu- lated and experimental transmission, which was measured on the same spot as the ellipsometry data, proves the fit quality, Fig. 3(a). All thin-film thicknesses tStylus as determined by profilometry are well within the 90% confidence limit of the thickness tSE as determined by SE. (a) (b) (d) (c) Fig. 2 (a) and (b) Spectroscopic ellipsometry data of an 87 nm [6,6]-phenyl C71-butyric acid methyl ester (PC71BM) thin-film spin cast on a glass substrate for five angles of incidence. The colored solid lines correspond to the measured data; the dashed black lines correspond to the model- generated data. (c) Transmission spectra of PC71BM in diluted dichlorobenzene (DCB) solutions and of a solid film on fused silica measured with a spectrophotometer. 4.1 Glass Substrate Soda-lime glass is used as substrate for all thin-films.37 Therefore, we first set up an optical model for the glass substrate. As the substrate is ∼1 mm thick, unwanted backside reflections are expected. They can either be suppressed by roughening the substrate’s backside or be accounted for in the analysis software. Here we use the latter approach as this allows measuring the transmission spectra on the very same spot after the ellipsometry characterization. p y p p y The glass substrate is described with a Cauchy dispersion relation40 with an Urbach-tail41 and a surface roughness layer of ∼1.3 nm. The derived complex refractive indices ðn; kÞ of the soda-lime glass substrates are depicted in Fig. 1 versus wavelength. In the following, we keep the parameters of the model constant and treat the surface roughness layer as an intermixed layer of the subsequently applied thin-film and the glass substrate. Fig. 1 Optical constants n and k of the soda-lime glass, which is used as substrate for all thin-films discussed in this study. The optical model includes a surface roughness layer of ∼1.3 nm . Fig. 1 Optical constants n and k of the soda-lime glass, which is used as substrate for all thin-films discussed in this study. The optical model includes a surface roughness layer of ∼1.3 nm . 057204-4 Vol. 5, 2015 Journal of Photonics for Energy 057204-4 Vol. 5, 2015 057204-4 057204-4 Journal of Photonics for Energy Vol. 5, 2015 Journal of Photonics for Energy 4.3 Polymers Both polymers, PCPDTBT and PSBTBT, exhibit strong structural similarities. They differ only in the 5-position, where PCPDTBT comprises a carbon atom, whereas PSBTBT features a silicon atom, Fig. 4(a). It was shown that the silicon substitution in PSBTBT reduces the steric hindrance from the bulky alkyl groups and improves molecular packing.43 The main absorption features of both polymers are still comparable though. The measured datasets Ψ and Δ for a PSBTBT thin-film spin cast on a glass substrate are shown in Figs. 5(a) and 5(b), respectively. The isotropic optical model is constructed in analogy to the procedure described for PC71BM. The transmission spectra of PSBTBT in DCB solution and of a solid PSBTBT thin-film are recorded, Fig. 5(c). The analysis of the absorption features leads to the starting values En;input for the center energies En of the Gaussian oscillators. Then, all oscillator parameters, a pole for the high-energy transitions and εr;∞are fitted. The respective values are summarized in Table 2. Based on this model, the isotropic optical constants are derived, Fig. 6(a). PCPDTBT is investigated accordingly. The respective isotropic datasets are shown in Fig. 7, Table 3, and Fig. 6(b). The spectral shape of the complex indices of refraction is comparable with the isotropic data presented by Guerrero et al.42 As they used PCPDTBT from a different supplier, the amplitudes are different though. Both polymer models are derived independently. However, the derived optical models utilize very similar sets of Gaussian oscillators, which reflect their strong structural similarity. A com- parison of the isotropic optical constants further shows that the overall optical density of PSBTBT is somewhat higher than the optical density of PCPDTBT. This can be either attributed to a higher intrinsic absorption or to a denser packing of the polymer chains.43 For both polymers, we assign the transitions Gau1 and Gau2 to the 0–0 and 0–1 absorption peaks, while Gau3 comprises higher vibronic progressions and an amorphous background. The nature of Gau4 remains unclear, but may be attributed to transitions involving more localized states.44 A more detailed model has to take into account the anisotropy of the films. 4.2 PC71BM The gray shaded area indicates the absorption of the cuvettes. The center energies of the absorption features Gau1 to Gau9 are used as starting values for the Gaussian oscillator center energies in the optical model. (d) Optical constants n and k are derived from a multisample analysis. (b) (a) (c) (d) (d) Fig. 2 (a) and (b) Spectroscopic ellipsometry data of an 87 nm [6,6]-phenyl C71-butyric acid methyl ester (PC71BM) thin-film spin cast on a glass substrate for five angles of incidence. The colored solid lines correspond to the measured data; the dashed black lines correspond to the model- generated data. (c) Transmission spectra of PC71BM in diluted dichlorobenzene (DCB) solutions and of a solid film on fused silica measured with a spectrophotometer. The gray shaded area indicates the absorption of the cuvettes. The center energies of the absorption features Gau1 to Gau9 are used as starting values for the Gaussian oscillator center energies in the optical model. (d) Optical constants n and k are derived from a multisample analysis. 057204-5 Vol. 5, 2015 057204-5 Vol. 5, 2015 Journal of Photonics for Energy Klein et al.: Modeling approach to derive the anisotropic complex refractive index. . . Table 1 From the transmission spectra in Fig. 1(c), the starting values E n;input for the fit of the Gaussian oscillators’ center energies are derived. The optical model of [6,6]-phenyl C71-butyric acid methyl ester (PC71BM) comprises nine Gaussian oscillators (Gaun with center energy En, amplitude An, and width Br n,33 a pole, and the real constant εr;∞¼ 2.00 eV. Table 1 From the transmission spectra in Fig. 1(c), the starting values E n;input for the fit of the Gaussian oscillators’ center energies are derived. The optical model of [6,6]-phenyl C71-butyric acid methyl ester (PC71BM) comprises nine Gaussian oscillators (Gaun with center energy En, amplitude An, and width Br n,33 a pole, and the real constant εr;∞¼ 2.00 eV. Oscillator En;input (eV) En (eV) An (1) Br n (eV) Gau1 1.83 1.85 0.10 0.16 Gau2 2.01 2.10 0.36 0.35 Gau3 2.23 2.26 0.46 0.38 Gau4 2.62 2.59 1.44 0.55 Gau5 2.98 3.02 0.81 0.36 Gau6 3.28 3.30 0.92 0.35 Gau7 3.48 3.59 0.41 0.30 Gau8 3.78 3.80 0.05 0.12 Gau9 4.77 4.71 3.42 1.63 Pole n/a 7.00 23.27 n/a Journal of Photonics for Energy 4.3 Polymers OSC polymers often exhibit elongated structures and, hence, form mostly flat two-dimensional molecules.45–48 Therefore, we probe the thin-films for anisotropy, assuming no preferential order in the in-plane direction as indicated by the invariance of Ψ under in-plane sample rotation.32,49,50 This assumption allows us to utilize a uniaxial optical description, only distinguishing between Vol. 5, 2015 Vol. 5, 2015 Klein et al.: Modeling approach to derive the anisotropic complex refractive index. . . (a) (b) (c) (d) (e) Fig. 3 Measured (red lines) and simulated transmission (black dashed lines) of (a) PC71BM, (b) poly{[4,4’-bis(2-ethylhexyl)dithieno(3,2-b;2’,3’-d)silole]-2,6-diyl-alt-(2,1,3-benzothidiazole)-4,7- diyl} (PSBTBT), (c) poly[2,6-(4,4-bis(2-ethylhexyl)-4H-cyclopenta[2,1-b;3,4-b′′]dithiophene)-alt- 4,7-(2,1,3-benzothiadiazole)] (PCPDTBT), (d) PSBTBT∶PC71BM, and (e) PCPDTBT∶PC71BM. The measured transmission spectra were determined at the same spot as the ellipsometry data by rotating the ellipsometer’s detector straight behind the sample. The noise levels are higher than in the spectrophotometer measurements that were described in Sec. 3.2 and that are depicted in Figs. 2(c), 5(c), and 7(c). The simulated data in Figs. 3(b)–3(e) rely on anisotropic models. Klein et al.: Modeling approach to derive the anisotropic complex refractive index. . . (a) (c) (b) (d) (e) Fig. 3 Measured (red lines) and simulated transmission (black dashed lines) of (a) PC71BM, (b) poly{[4,4’-bis(2-ethylhexyl)dithieno(3,2-b;2’,3’-d)silole]-2,6-diyl-alt-(2,1,3-benzothidiazole)-4,7- diyl} (PSBTBT), (c) poly[2,6-(4,4-bis(2-ethylhexyl)-4H-cyclopenta[2,1-b;3,4-b′′]dithiophene)-alt- 4,7-(2,1,3-benzothiadiazole)] (PCPDTBT), (d) PSBTBT∶PC71BM, and (e) PCPDTBT∶PC71BM. The measured transmission spectra were determined at the same spot as the ellipsometry data by rotating the ellipsometer’s detector straight behind the sample. The noise levels are higher than in the spectrophotometer measurements that were described in Sec. 3.2 and that are depicted in Figs. 2(c), 5(c), and 7(c). The simulated data in Figs. 3(b)–3(e) rely on anisotropic models. Fig. 3 Measured (red lines) and simulated transmission (black dashed lines) of (a) PC71BM, diyl} (PSBTBT), (c) poly[2,6-(4,4-bis(2-ethylhexyl)-4H-cyclopenta[2,1-b;3,4-b′′]dithiophene)-alt- 4,7-(2,1,3-benzothiadiazole)] (PCPDTBT), (d) PSBTBT∶PC71BM, and (e) PCPDTBT∶PC71BM. The measured transmission spectra were determined at the same spot as the ellipsometry data by rotating the ellipsometer’s detector straight behind the sample. The noise levels are higher than in the spectrophotometer measurements that were described in Sec. 3.2 and that are depicted in Figs. 2(c), 5(c), and 7(c). The simulated data in Figs. 3(b)–3(e) rely on anisotropic models. the xy- and the z-contribution. Journal of Photonics for Energy 4.3 Polymers [The xy-component is sometimes referred to as k (in-plane) and z as ⊥(out-of-plane).] For this orientation, the off-diagonal elements of the Jones matrix vanish.32 In the uniaxial configuration, the optical constants nxy and kxy are determined by the xy-layer, and the z-component by the z-layer. The xy-optical constants describe light-matter interaction for light under normal incidence with its electrical field vector E parallel to the sample surface. For light impinging at grazing incidence, E is almost normal to the sample surface and the interaction with the polymer is described with nz and kz. z z For the anisotropic model, the isotropic oscillator parameters serve as input parameters for both the xy- and z-layer, consequently doubling the number of fit parameters. Since doubling of the fit parameters does hamper finding a unique solution, we simplify our model by assuming that the same optical transitions will be observed in both directions (xy and z), changing their relative amplitude only. Accordingly, the oscillator amplitudes An;xy and An;z are defined as fit parameters. Vol. 5, 2015 Vol. 5, 2015 Klein et al.: Modeling approach to derive the anisotropic complex refractive index. . . Klein et al.: Modeling approach to derive the anisotropic complex refractive index. . . (c) (a) (e) (d) (b) (f) ,kxy nxy ,kz nz PCPDTBT PSBTBT x b y c z a PSBTBT PCPDTBT PSBTBT:PC BM 71 PCPDTBT:PC BM 71 Fig. 4 (a) Chemical structure of PSBTBT and PCPDTBT and definition of the polymer-chain refer- ence frame (a, b, and c). The c axis is oriented along the polymer backbone. (b) The optical con- stants nxy and kxy describe light-polymer interaction for polymers with their c axes being aligned parallel to the substrate surface, while the z-component describes vertically oriented polymers. (x, y, and z) is the substrate reference frame. Schemes of the morphology of (c) neat PSBTBT, (d) neat PCPDTBT, (e) PSBTBT∶PC71BM, and (f) PCPDTBT∶PC71BM showing different degrees of aggregate alignment relative to the surface. Black dots represent PC71BM. (b) ,kxy nxy Fig. 4 (a) Chemical structure of PSBTBT and PCPDTBT and definition of the polymer-chain refer- ence frame (a, b, and c). The c axis is oriented along the polymer backbone. (b) The optical con- stants nxy and kxy describe light-polymer interaction for polymers with their c axes being aligned parallel to the substrate surface, while the z-component describes vertically oriented polymers. (x, y, and z) is the substrate reference frame. Schemes of the morphology of (c) neat PSBTBT, (d) neat PCPDTBT, (e) PSBTBT∶PC71BM, and (f) PCPDTBT∶PC71BM showing different degrees of aggregate alignment relative to the surface. Black dots represent PC71BM. (a) (c) (b) (d) Fig. 5 (a) and (b) Spectroscopic ellipsometry data Ψ and Δ of a typical neat PSBTBT thin-film spin cast on a glass substrate. The colored solid lines correspond to the measured data; the dashed black lines correspond to data generated from a uniaxial optical model. (c) Transmission spectra of PSBTBT in diluted DCB solutions and of a solid film on fused silica. The starting values for the Gaussian oscillator center energies En;input are derived from the center energies of the absorption features, Table 2. (d) The optical constants comprise an in-plane (index xy) and out-of-plane component (index z) and are derived from a multisample analysis with an overall mean squared error ðMSEÞ ¼ 6.37. (b) (a) (d) (c) (d) Fig. 5 (a) and (b) Spectroscopic ellipsometry data Ψ and Δ of a typical neat PSBTBT thin-film spin cast on a glass substrate. Journal of Photonics for Energy 057204-7 Klein et al.: Modeling approach to derive the anisotropic complex refractive index. . . Klein et al.: Modeling approach to derive the anisotropic complex refractive index. . . The colored solid lines correspond to the measured data; the dashed black lines correspond to data generated from a uniaxial optical model. (c) Transmission spectra of PSBTBT in diluted DCB solutions and of a solid film on fused silica. The starting values for the Gaussian oscillator center energies En;input are derived from the center energies of the absorption features, Table 2. (d) The optical constants comprise an in-plane (index xy) and out-of-plane component (index z) and are derived from a multisample analysis with an overall mean squared error ðMSEÞ ¼ 6.37. 057204-8 Vol. 5, 2015 057204-8 057204-8 Journal of Photonics for Energy Vol. 5, 2015 Klein et al.: Modeling approach to derive the anisotropic complex refractive index. . . Table 2 Isotropic, parameterized description of poly{[4,4’-bis(2-ethylhexyl)dithieno(3,2-b;2’,3’-d) silole]-2,6-diyl-alt-(2,1,3-benzothidiazole)-4,7-diyl} (PSBTBT). The starting values E n;input are derived from the transmission spectra in Fig. 5(c). The center energies E n change only slightly upon performing the fit. The parameters An and Br n are determined defining the Gaussian oscil- lators Gaun. The offset was found to be εr;∞¼ 2.46 eV. Table 2 Isotropic, parameterized description of poly{[4,4’-bis(2-ethylhexyl)dithieno(3,2-b;2’,3’-d) silole]-2,6-diyl-alt-(2,1,3-benzothidiazole)-4,7-diyl} (PSBTBT). The starting values E n;input are derived from the transmission spectra in Fig. 5(c). The center energies E n change only slightly upon performing the fit. The parameters An and Br n are determined defining the Gaussian oscil- lators Gaun. The offset was found to be εr;∞¼ 2.46 eV. Oscillator En;input (eV) En (eV) An (1) Br n (eV) Gau1 1.63 1.62 0.10 0.16 Gau2 1.77 1.79 0.36 0.35 Gau3 2.04 2.08 0.46 0.38 Gau4 2.90 2.92 1.44 0.55 Gau5 4.01 4.01 0.81 0.36 Gau6 4.86 4.87 0.92 0.35 Pole n/a 5.52 4.46 n/a (b) (a) Fig. 6 Optical constants of (a) PSBTBT and (b) PCPDTBT within an isotropic description. We note the different scaling of the y axes. Oscillator En;input (eV) En (eV) An (1) Br n (eV) Gau1 1.63 1.62 0.10 0.16 Gau2 1.77 1.79 0.36 0.35 Gau3 2.04 2.08 0.46 0.38 Gau4 2.90 2.92 1.44 0.55 Gau5 4.01 4.01 0.81 0.36 Gau6 4.86 4.87 0.92 0.35 Pole n/a 5.52 4.46 n/a Oscillator En;input (eV) En (eV) An (1) Br n (eV) Gau1 1.63 1.62 0.10 0.16 Gau2 1.77 1.79 0.36 0.35 Gau3 2.04 2.08 0.46 0.38 Gau4 2.90 2.92 1.44 0.55 Gau5 4.01 4.01 0.81 0.36 Gau6 4.86 4.87 0.92 0.35 Pole n/a 5.52 4.46 n/a (b) (a) Fig. Journal of Photonics for Energy Klein et al.: Modeling approach to derive the anisotropic complex refractive index. . . Klein et al.: Modeling approach to derive the anisotropic complex refractive index. . . (b) (d) (a) (c) Fig. 7 (a) and (b) Spectroscopic ellipsometry data Ψ and Δ of a typical neat PCPDTBT thin-film spin cast on a glass substrate. The solid lines correspond to the measured data; the dashed black lines correspond to the model-generated data within a uniaxial description. (c) Transmission spec- tra of PCPDTBT in dilute DCB solutions and of a film on fused silica. The center energies E n;input of the absorption features Gaun are given in Table 3. (d) Anisotropic optical constants derived from a multisample analysis with an overall MSE ¼ 7.51. (a) (b) (c) (d) (c) (d) Fig. 7 (a) and (b) Spectroscopic ellipsometry data Ψ and Δ of a typical neat PCPDTBT thin-film spin cast on a glass substrate. The solid lines correspond to the measured data; the dashed black lines correspond to the model-generated data within a uniaxial description. (c) Transmission spec- tra of PCPDTBT in dilute DCB solutions and of a film on fused silica. The center energies E n;input of the absorption features Gaun are given in Table 3. (d) Anisotropic optical constants derived from a multisample analysis with an overall MSE ¼ 7.51. For PCPDTBT, the xy- and z-extinction coefficients also split up and show anisotropic behavior in the low-wavelength regime, Fig. 7(d). The low-energy shoulder, which is visible in the kxy data, does not appear in the kz data. In analogy to PSBTBT, the shoulder in kxy can be attributed to aggregates of polymers with c axes aligned parallel to the xy-plane, whereas the interpretation of kz leads to ambiguous results. This can either be attributed to nonaggregated standing polymers or an isotropic amorphous background, Fig. 4(d). Table 3 Poly[2,6-(4,4-bis(2-ethylhexyl)-4H-cyclopenta[2,1-b;3,4-b′′]dithiophene)-alt-4,7-(2,1,3- benzothiadiazole] (PCPDTBT). Starting values E n;input as derived from Fig. 4(c). The parameters E n, An, and Br n determine the optical model. εr;∞¼ 2.28 eV. Gau7 is outside the ellipsometer’s measurement range; the pole function covers its contribution. Table 3 Poly[2,6-(4,4-bis(2-ethylhexyl)-4H-cyclopenta[2,1-b;3,4-b′′]dithiophene)-alt-4,7-(2,1,3- benzothiadiazole] (PCPDTBT). Starting values E n;input as derived from Fig. 4(c). The parameters E n, An, and Br n determine the optical model. εr;∞¼ 2.28 eV. Gau7 is outside the ellipsometer’s measurement range; the pole function covers its contribution. Klein et al.: Modeling approach to derive the anisotropic complex refractive index. . . 6 Optical constants of (a) PSBTBT and (b) PCPDTBT within an isotropic description. We note the different scaling of the y axes. (b) (b) (a) Fig. 6 Optical constants of (a) PSBTBT and (b) PCPDTBT within an isotropic description. We note the different scaling of the y axes. Fig. 6 Optical constants of (a) PSBTBT and (b) PCPDTBT within an isotropic description. We note the different scaling of the y axes. The fitted datasets Ψ and Δ for PSBTBT are shown in Figs. 5(a) and 5(b) and the derived anisotropic optical constants in Fig. 5(d). The MSE improves from 7.23 to 6.37 upon utilizing an anisotropic model. The respective datasets for PCPDTBT are shown in Fig. 7. For PCPDTBT, the MSE improves from 7.77 to 7.51. Evidence of the fit quality is provided in Figs. 3(b) and 3(c), where we compare the experimental transmission data that were measured on the same spot as the ellipsometry data, with the simulated transmission based on the ellipsometric model. Both neat polymers exhibit pronounced uni- axial anisotropy. Since the polymer π →π transitions, here modeled with Gau1—Gau3, are excited by an electrical field that is polarized parallel to the polymer backbone, i.e., the poly- mer c axis as defined in Fig. 4(a), a detailed analysis of the anisotropic optical constants can further reveal morphological details.51,52,53 The optical constants nxy and kxy describe light-polymer interaction for polymers with their c axes aligned parallel to the substrate surface, while the z-component describes upright polymers with their c axes oriented in z-direction, Fig. 4(b). y y light-polymer interaction for polymers with their c axes aligned parallel to the substrate surface, while the z-component describes upright polymers with their c axes oriented in z-direction, Fig. 4(b). The extinction coefficient of PSBTBT splits up in an xy- and a z-component between ∼540 and 800 nm, Fig. 5(d). Toward lower wavelengths, no splitting is observed, indicating a different nature of the involved transitions. According to similar observations in the literature, we attribute the shoulder around 761 nm (Gau1) to polymer aggregation.43 As the contribution of the xy- component is more pronounced than the contribution of the z-component, we conclude that aggregates of polymers with a c axis aligned in-plane prevail, Fig. 4(c). Vol. 5, 2015 Vol. 5, 2015 057204-9 Klein et al.: Modeling approach to derive the anisotropic complex refractive index. . . Klein et al.: Modeling approach to derive the anisotropic complex refractive index. . . g Oscillator En;input (eV) En (eV) An (1) Br n (eV) Gau1 1.66 1.66 1.21 0.21 Gau2 1.81 1.84 1.42 0.34 Gau3 2.06 2.17 1.00 0.57 Gau4 2.98 3.06 0.72 0.62 Gau5 3.80 3.89 0.16 1.75 Gau6 4.34 4.34 0.15 2.20 Gau7 4.98 n/a n/a n/a Pole n/a 5.83 5.28 n/a Journal of Photonics for Energy 057204-10 Vol. 5, 2015 4.4 Blends t i l d it f t i l d k th b l t t i l d it f th th Table 4 The normalized volume ratios in the blends as derived from ellipsometry fittings show excellent agreement with the volume ratio calculated from the material densities. ρPC71BM ¼ 1.52 g∕cm3, ρPSBTBT ¼ 1.17 g∕cm3, and ρPCPDTBT ¼ 1.10 g∕cm3 as determined by helium pycnometry in the group of Dadmun et al.54,55 Quantity Initial ratio Normalized volume ratio Normalized volume ratio Method By weight Pycnometry54,55 Ellipsometry fit PSBTBT∶PC71BM 1∶2.0 39.4∶60.6 39.2∶60.8 PCPDTBT∶PC71BM 1∶3.4 28.9∶71.1 29.0∶71.0 Table 4 The normalized volume ratios in the blends as derived from ellipsometry fittings show excellent agreement with the volume ratio calculated from the material densities. ρPC71BM ¼ 1.52 g∕cm3, ρPSBTBT ¼ 1.17 g∕cm3, and ρPCPDTBT ¼ 1.10 g∕cm3 as determined by helium pycnometry in the group of Dadmun et al.54,55 Quantity Initial ratio Normalized volume ratio Normalized volume ratio Method By weight Pycnometry54,55 Ellipsometry fit PSBTBT∶PC71BM 1∶2.0 39.4∶60.6 39.2∶60.8 PCPDTBT∶PC71BM 1∶3.4 28.9∶71.1 29.0∶71.0 material density of one component is already known, the absolute material density of the other component can be determined. material density of one component is already known, the absolute material density of the other component can be determined. In the next step, we investigate whether the polymer’s anisotropy is preserved upon blending with PC71BM. Therefore, the polymer oscillator amplitudes are fitted leading to the anisotropic optical constants shown in Fig. 8(e) for PSBTBT∶PC71BM and in Fig. 8(f) for PCPDTBT∶PC71BM. As indicated in Figs. 8(a) and 8(c) and in Figs. 8(b) and 8(d), both multi- sample fits describe the experimental data very well (MSE ¼ 3.90 and 4.64). Again, the com- parison of the simulated and the experimental transmission in Figs. 3(d) and 3(e) confirms the good fit quality. Further, the anisotropic optical constants of PSBTBT∶PC71BM match the shape of the isotropic optical constants known in the literature.56 When performing the fits, only the amplitudes of the oscillators Gau1 to Gau3 change considerably. In analogy to the analysis of the neat polymers, those oscillators are an indicator for the degree of polymer aggregation. For PSBTBT∶PC71BM, a pronounced uniaxial anisotropy becomes visible in the optical constants, Fig. 8(e). Similar to neat PSBTBT, both the in-plane and the out-of-plane extinction coefficients exhibit shoulders around 760 nm, where the xy-contribution dominates the z- contribution. 4.4 Blends The dominance of the xy-contribution can be attributed to polymer aggregates with their c axes aligned in-plane as illustrated in Fig. 4(e). This interpretation of the polymer orientation from SE experiments fits well to the time-resolved x-ray analysis that we published earlier, where we found that PSBTBT nucleation takes place in defined orientation at an inter- face, while randomly oriented aggregates form in the bulk during solvent evaporation.29 As the degree of PSBTBT∶PC71BM anisotropy is similar to the anisotropy of neat PSBTBT, the poly- mer orientation remains mostly unaffected upon blending with PC71BM. For PCPDTBT∶PC71BM, the optical constants in xy- and in z-directions differ slightly, Fig. 8(f). The PCPDTBT low-energy shoulder appears in both directions. This can be attributed to randomly oriented PCPDTBT aggregates as depicted in Fig. 4(f). In the blend model, the low- energy shoulder is more pronounced than in the model for neat PCPDTBT, indicating a higher degree of aggregation in the blend. The higher degree of the c axis in-plane alignment within the PSBTBT∶PC71BM blend as compared to the PCPDTBT∶PC71BM blend, probably induced by the substrate surface, may be explained by the lower PC71BM volume fraction, Table 4. Journal of Photonics for Energy 4.4 Blends The measured spectroscopic ellipsometry data Ψ and Δ of a PSBTBT∶PC71BM and a PCPDTBT∶PC71BM thin-film are depicted in Figs. 8(a) and 8(c) and in Figs. 8(b) and 8(d), respectively. The optical properties of the polymer:fullerene blends are modeled within an effec- tive medium approximation (EMA). Therefore, the optical constants of polymer and fullerene constituents are superimposed. As the analysis of neat polymers in Sec. 4.3 revealed a preferred orientation of the PSBTBT and PCPDTBT c axes parallel to the substrate, we choose uniaxial models for the blends as well. The oscillator parameters in Tables 1, 2, and 3 are used as starting parameters. After determining the layer thicknesses in the transparent spectral regime, the volume ratio of polymer to PC71BM is fitted. The resulting ratios are listed in Table 4 showing excellent agree- ment with volume ratios being calculated on the basis of material density as determined by helium pycnometry in the literature.54,55 This result shows that SE can be an alternative and contactless metrology approach to determine material density ratios in blend films. If the (a) (c) (e) (b) (d) (f) Fig. 8 Spectroscopic ellipsometry data Ψ and Δ of [(a) and (c)] a typical PSBTBT∶PC71BM (1∶2) thin-film and [(b) and (d)] a typical PCPDTBT∶PC71BM (1∶3.4) thin-film. The solid lines correspond to the measured data; the dashed black lines correspond to the model-generated data within a uniaxial description. Anisotropic optical constants of (e) PSBTBT∶PC71BM and (f) PCPDTBT∶PC71BM are derived from a multisample analysis with an overall MSE ¼ 3.90 and MSE ¼ 4.64, respectively. (b) (a) (d) (c) (f) (e) Fig. 8 Spectroscopic ellipsometry data Ψ and Δ of [(a) and (c)] a typical PSB Fig. 8 Spectroscopic ellipsometry data Ψ and Δ of [(a) and (c)] a typical PSBTBT∶PC71BM (1∶2) thin-film and [(b) and (d)] a typical PCPDTBT∶PC71BM (1∶3.4) thin-film. The solid lines correspond to the measured data; the dashed black lines correspond to the model-generated data within a uniaxial description. Anisotropic optical constants of (e) PSBTBT∶PC71BM and (f) PCPDTBT∶PC71BM are derived from a multisample analysis with an overall MSE ¼ 3.90 and MSE ¼ 4.64, respectively. 057204-11 Vol. 5, 2015 057204-11 Journal of Photonics for Energy Vol. 5, 2015 Journal of Photonics for Energy Klein et al.: Modeling approach to derive the anisotropic complex refractive index. . . Journal of Photonics for Energy 057204-12 Vol. 5, 2015 5 Conclusion In conclusion, we present a modeling approach for the analysis of ellipsometry data, which facilitates the precise determination of the optical constants of various polymer:fullerene blends. The optical constants of the neat constituents PC71BM, PSBTBT, and PCPDTBT are deter- mined, first, by parameterizing the constituents’ relative dielectric functions, where each optical excitation is described by a Gaussian oscillator. The center energies of the oscillators are derived phenomenologically from their respective transmission spectra. Based on this set of optical constants, the blends PSBTBT∶PC71BM and PCPDTBT∶ PC71BM are described within an EMA. An initial fit on the polymer-to-fullerene ratio allows to derive the volume density ratio of both constituents. Our approach further allows for considering a uniaxial anisotropic optical description of the neat polymers and their blends. The detailed analysis of the anisotropy provides deep insight into the PSBTBT, PCPDTBT, and blend morphology. Since many of the latest high-performance materials exhibit a lower degree of crystallinity than the vastly studied P3HT,62 we propose SE as a nondestructive bulk-heterojunction probing technique for future polymer:fullerene bulk-heterojunction investigations. Klein et al.: Modeling approach to derive the anisotropic complex refractive index. . . Klein et al.: Modeling approach to derive the anisotropic complex refractive index. . . The determination of the anisotropic blend models benefits from the weak spectral overlap of the polymer and fullerene extinction coefficients, which allows separating both components and performing a precise fit. Fortunately, this applies to the vast majority of polymer:fullerene blends. Even more stable fit results can be achieved by taking into account advanced photo- physical descriptions.59 So far, such a detailed description has been elaborated for P3HT only.60,61 A description of the complex absorption spectra of the low-band gap polymers is still lacking. Therefore, we suggest utilizing the phenomenological derived values of the oscil- lator center energies and robust multisample fits as discussed in this work. Acknowledgments The authors acknowledge funding by the Federal Ministry of Education and Research (BMBF) within the project Taurus, contract 03EK3504, the Deutsche Forschungsgemeinschaft (DFG) priority program 1355 “Elementary Processes of Organic Photovoltaics,” and the State of Baden-Württemberg through the DFG Center for Functional Nanostructures (CFN). Poly [2,6-(4,4-bis(2-ethylhexyl)-4H-cyclopenta[2,1-b;3,4-b′′]dithiophene)-alt-4,7-(2,1,3-benzothia- diazole)] and poly{[4,4’-bis(2-ethylhexyl)dithieno(3,2-b;2’,3’-d)silole]-2,6-diyl-alt-(2,1,3- benzothidiazole)-4,7-diyl} were supplied from Konarka. The authors thank Professor M. Dadmun, Professor G. Bazan, Dr. H. Chen, Dr. J. Peet, Dr. S. Hu, and Dr. J. Azoulay for the communication of their results54,55 prior to publication, Dr. B. Schmidt-Hansberg (Karlsruhe Institute of Technology) for fruitful discussions, and Dr. T. Wagner (LOT- QuantumDesign) for technical support. M.F.G.K. was supported by the Karlsruhe School of Optics and Photonics (KSOP). 4.5 Limitations The EMA approach requires negligible cross-coupling of optical transitions between the blend’s components. For example, charge transfer (CT) absorption could cause such a new excitation channel. Since CT absorption in state-of-the-art OSCs is some magnitudes lower than the π →π transition,57 we disregard CT absorption. Both polymers that are investigated in this study exhibit a lower degree of crystallinity than other semicrystalline materials, such as P3HT.58 Upon blending more crystalline materials, the degree of crystallinity may be reduced significantly. Therefore, in order to generate a suitable model for highly crystalline materials, care should be taken that the degree of crystallinity between the neat material and the blend is comparable. 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https://openalex.org/W4379196836	https://www.researchsquare.com/article/rs-2959416/latest.pdf	English	null	The Silent Threat of a Non-native Oligochaete Species in Iran's freshwater: Assessment of the Diversity and Origin of Eiseniella tetraedra (Savigny, 1826) and its Response to Climate Change	Research Square (Research Square)	2,023	cc-by	8,188	Research Article Research Article Keywords: Eiseniella tetraedra, non-native species, genetic origin, SDMs Posted Date: May 30th, 2023 DOI: https://doi.org/10.21203/rs.3.rs-2959416/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/21 Page 1/21 Abstract Oligochaetes are the most abundant benthic taxa in aquatic ecosystems that play an important role in food webs and energy transmission. The aim of the current study is to assess the origin and diversity of Eiseniella tetraedra a non-native species of Lar National Park in Alborz Mountain ranges of Iran and also its response to current and future climate change. To this, we collected the specimen (23 samples) from Lar National Park Rivers and performed the mitochondrial gene, mitochondrial Cytochrome c oxidase subunit I (COI) combining them with 117 sequences from Jajroud, Karaj River in Iran and native regions from GenBank (NCBI). We also used an ensemble model approach for Species Distribution Modelings (SDMs) that it was estimated according to two Shared Socio-economic Pathways (SSPs): 126 and 585 of the MRI-ESM2 based on CMIP6. According to our results, all the samples examined in the current study originated from Spanish rivers and there was no unique haplotype found in Lar National Park. Moreover, The results also show high haplotype diversity that can have a positive effect on the success of its non- native in the different freshwater of Iran. As well as, the result of SDMs maps illustrated that climate change would significantly affect the distribution of E. tetraedra and it showed a sharp tendency to expand and verified the invasion power of the E. tetraedra in Iran's freshwater ecosystems over time. Introduction Oligochaete species belonging to annelid worms occur in marine and terrestrial ecosystems. About one- third of the almost 5,000 known Oligochaete species is aquatic and semiaquatic (Suriani et al. 2007; Krieger et al. 2010). With some exceptions, these groups of oligochaetes are small in size, ranging from 1 mm to a few centimetres in length (Martin et al. 2008). Regarding the idea that earthworms have a limited capacity to autonomously disperse (Sakai et al. 2001), it has been thought that the cosmopolitan dissemination of some oligochaetes (about 110 species) will often be due to human activities or animal- mediated transportation (Costa 2013). The occurrence of introduced species may cause ecological changes in the ecosystem if they can adapt to new habitats, leading to potential negative interaction with the native species (Gozlan and Newton 2009). Several researches demonstrated that introduced Oligochaete worms have remarkable impacts on the ecosystem. Once introduced earthworms become invasive, and they can cause changes in microorganisms fauna of soil, competition with native species and loss of biodiversity resulting in economic losses and detrimental effects on habitats (Eisenhouer 2010; Blouinet al. 2013; Craven et al. 2017). Furthermore, management options for controlling invasive species are generally troublesome and presumably threaten native species. Hence, clarifying the population structure of exotic species is essential to decrement their harmful effects on the ecosystem. Parallel to the worldwide status, Iranian inland waters are exposed to habitat degradation and species introductions (either intentionally (e.g., for soil remediation or commercial applications) or inadvertently (e.g., in soil associated with horticultural and agricultural products) likely decrease endemic populations (Abdoli et al. 2022). Page 2/21 Page 2/21 So far, 20 species of Oligochaetes have been identified from Iran (Jablonska and Pesic 2014; Nazarhaghighi et al. 2015). No molecular surveys have been conducted to clarify whether these species are native or exotic. Just recently, Javidkar et al (2020) reported a non-native Oligochaete Eiseniella tetraedra (Savigny, 1826) from two protected rivers in the southern Alborz Mountains. They deduced European origin for Iranian populations that were transported to this country by anthropogenic activities. Population genetic structure is the distribution of genotypes in space and time and is determined by both historical and current evolutionary processes (Hewitt and Butlin 1997). The previous study (Javidkar et al. 2020) reported genetic variation in E. tetraedra populations in Iran that may be related to the successful establishment and colonization of the species in new habitats. Taxon Sampling and Laboratory Procedures A total of 23 specimens of E. tetradra from 12 stations were collected from the rivers of Lar National Park in Iran (Fig. 1). A small piece of the end of the tail was dissected for each specimen then all tissues for DNA extraction were preserved in 96% ethanol and at -20°C. Locality and collection data for E. tetraedra are explained in additional file: Table S1. Introduction However, the origin and distribution paths have not fully been investigated. For example, whether current populations are derived from a single introduction or are the result of several successive waves. Besides molecular analysis, Species Distribution Models (SDMs) can act as a useful tool to predict the distribution of species according to climate change. In fact, SDMs significantly relate species presence points with climate and topographical data, unveiling species-to-environment connections that are responsible for shaping species distribution patterns (Ashrafzadeh et al. 2022). However, for non-native species, it has been firmly documented that SDMs approach could be a valuable proactive tool to distinguish invasion potential (Guisan and Thuiller 2005; Reshetnikov and Ficetola 2011; Gallien et al. 2012; Banha et al. 2017; Godefroid et al. 2019; Nunez et al. 2019). To this end, the aim of the current study is to integrate the mitochondrial marker and SDMs method to investigate the origin and differentiation of E. tetraedra populations from two protected rivers (Jajroud and Karaj) and one national park (Lar) in Southern Alborz Mountains and predict potential E. tetradra distribution based on climate change scenarios to find how invasion potential. DNA Amplification and Sequencing Page 3/21 The DNA was extracted from tissues using a standard high-salt method (Sambrook et al. 1989). The partial mitochondrial Cytochrome c Oxidase subunit I (COI) was amplified for all specimens using the universal primers forward LCO1490 (5´-TACTC-AACAA-ATCAC-AAAGA-TATTG-G-3´) and reverse HCO2198 (5´-TAAAC-TTCAGGGTGA-CCAAAAAATC-A-3´) (Shekhovtsov et al. 2016). Polymerase Chain Reactions (PCRs) were conducted with 1 µl template DNA (50–100 ng), 0.5 µl of each primer, 12.5 µl Master Mix Page 3/21 Red (Ampliqon) and 10.5 µl of ddH2O up to 25 µl of reaction mix. The amplification of DNA was performed with an initial denaturation period of 3 min at 94°C followed by 34 cycles of 94°C for 30 s, primer-specific annealing temperature of 48°C (Additional file 2: Table S2) for 30 s, 72°C for 1 min and a single final extension at 72°C for 5min. The quality of PCR products were assessed with agarose gel 1% stained with Safe-Red™. The suitable amplicons were sent to Pishgam Inc, for purification and sequencing. Genetic Diversity and Demographic Analysis The number of haplotypes (H), number of polymorphic sites(s), haplotype diversity (h) and nucleotide diversity (π) of each population were extracted by DnaSP v5 (Librado and Rozas 2009). To demographic history analyses, we used selective neutrality test of Fu’s Fs statistics (Fu 1997) and Tajima's D (Tajima 1989) based on COI to find evidence of recent expansion for each lineage using Arlequin v.3.5 (Excoffier and Lischer 2010). A Mismatch Distribution (MMD) analysis was separately performed for each population to estimate the frequency distribution of the pairwise nucleotide differences, assuming a sudden expansion with spatial parameters. The test was performed using Arlequin v.3.5. Also, we used NETWORK v.10.2 (Bandelt et al. 1999) to construct a median-joining network for COI. Also, we used NETWORK v.10.2 (Bandelt et al. 1999) to construct a median-joining network for COI. Phylogenetic Analyses and Haplotype Netw Phylogenetic analyses of E. tetraedra of the COI data were reconstructed using maximum-likelihood (ML) and Bayesian inference (BI) approaches. Hermodice carunculata downloaded from GenBank was used as outgroup. The Akaike information criterion was used to select Nucleotide substitution models (GTR + I + G) in MrModeltest v 2 (Nylander 2004). For BI and ML analyses, two independent runs and four Markov chains (three heated chains and a single cold chain) using the best-fit models were performed in MrBayes v 3.1.2 (Huelsenbeck and Ronquist 2001). Each run was conducted with Markov Chain Monte Carlo (MCMC) sampling for six million generations and parameters were saved every 100 iterations, which produced 6001 trees during the analysis. Finally, 10% of the trees were discarded as burn-in and the remaining trees were used to reconstruct the consensus tree. Tracer v.1.7 (Rambaut et al. 2009) was implemented to the performance of each run and evaluate convergence. To edit and visualis phylogenetic tree, FigTree v.1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/) was used. Sequence Analyses Initially, the nucleotide sequences were edited by BioEdit V.2.34 (Hall 1999), aligned by Geneious Prime® V. 2021.0.0 (Biomatters, www.geneious.com) and optimized by eye using MEGA X (Kumar et al. 2018). We extracted 117 sequences from Genbank which were added to our dataset (Table S1). The final gene dataset was 528 bp and MAFFT v. 7 (https://mafft.cbrc.jp/alignment/server/) was used for alignment. Occurrence Data For Species Distribution Modeling (SDMs), a total of 127 E. tetraedra locations were compiled between 2019 e2023 through multiple sources (a) direct field surveys (b) the Global Biodiversity Information Facility (GBIF) website, and (c) distribution recorded by published papers. We visually checked occurrence points with those collected from the literature review in ArcGIS and to reduce spatial autocorrelation in the occurrence points all them filtered with inaccurate spatial information using the package “CoordinateCleaner” (Zizka et al. 2019) in the R v 4.1.3. This process reduced our presence records to 98 points that were available for the habitat modeling approach. Phylogenetic Analyses and Haplotype Network The datasets, with 528 bp length included partial sequences of COI to investigate the position of individuals belonging to the E. tetraedra in a phylogeny tree was constructed. The tree based on 140 sequences examinaed in this research contain Lar National Park (23 sequences), Jajroud (40 sequences) and Karaj river (40 sequences) and also 37 sequences from the native distribution area of the species was drawn. The constructed intraspecies phylogenetic trees based on COI showed similar topologies for both ML and BI trees and revealed well-supported monophyletic lineages (only BI tree shown, Fig. 2). In association with the outgroup, the Hermodice carunculata clustered as sister monophyletic lineages. Based on the phylogenetic tree, the E. tetradra group was considered a well-supported monophyletic group as expected and the Iranian samples were well placed together with other samples of the population of origin. (Fig. 2). The phylogenetic trees depict that the E. tetraedra group are well separated with high support values. The samples of this study belong to the Spanish catchment clade, which indicates the origin of the individuals studied in Iran. The phylogenetic trees depict that the E. tetraedra group are well separated with high support values. The samples of this study belong to the Spanish catchment clade, which indicates the origin of the individuals studied in Iran. The parsimony haplotype NETWORK for 140 E. tetraedra specimens (103 specimens from Iran and 37 specimens from the native distribution area) showed separate haplogroups and different haplotypes for COI by recognizable mutations (Fig. 3). It showed five haplogroups and 40 haplotype for 140 E. tetraedra from the distribution area. Also, according to the results, four haplotypes were verified for Lar National Park as they were shared with Jajroud and Karaj, and Lar National Park region did not have a specific haplotype. Environmental Variables We used 19 environmental variables that were used to affect the spatial range of a species (Hermes et al. 2018). All bioclimatic variables (Bio1-Bio19) were downloaded from the WorldClim-Global Climate Database (https://www.worldclim.org/) with a resolution ~ 1 km2 (30-arc second) for both current (1970– 2000) and future (2061–2080) climatic scenarios. Slope data was extracted from the Digital Elevation Model (DEM, http://www.worldclim.org) as an additional geographical input to provide a measure of topographic heterogeneity. In addition, Human Footprint Model offered by Sanderson et al (2002) to evaluate quantifies anthropogenic effects on the E. tetraedra habitat. All layers with WGS1984 datum were projected onto a UTM grid and resampled resolution at 1 km2. A principal Component Analysis (PCA) was estimated for multicollinearity among predictors by calculating coefficients (r < 0.75) and criteria to select which essential variables in the distribution models for the present study. Eventually, the remaining input variables for the modelling were as follows: Annual Mean Temperature (Bio1), Temperature Annual Range (Bio7), Mean Temperature of Wettest Quarter (Bio 8), Mean Temperature of Driest Quarter (Bio 9), Precipitation of Wettest Month (Bio 13), Precipitation of Driest Month (Bio 14), Precipitation of Wettest Quarter (Bio 16), Human Footprint and Slope. For future mapping of the suitable climate of E. tetradra under future climate change, we extracted the bioclimatic data from MRI-CGCM3 (Meteorological Research Institute, Japan) and used two scenarios Shared Socio-economic Pathways (SSPs): 126 and 585 based on CMIP6. An ensemble model approach was applied to E. tetraedra distribution model (Thuiller et al. 2009) using BIOMOD2 package (Thuiller et al. 2016) in R v. 4.1.3 (R Development Core Team. 2014). The ensemble model was formed using nine modelling techniques: Generalized Boosting Method (GBM), the Generalized Linear Model (GLM), Maximum Entropy (MaxEnt), Flexible Discriminant Analysis (FDA), Random Forest (RF), Classification Tree Analysis (CTA), Multivariate Adaptive Regression Splines (MARS), Surface Range Envelops (SRE) and Artificial Neural Network (ANN). To provide more accurate predictions we created many pseudo- absences (n = 220 points) with five replicates per model (Hamid et al. 2018; Dar et al. 2021). We also evaluated model performance using Area Under the receiver operating Curve (AUC = ROC), the True Skill Statistic (TSS) and Cohen’s Kappa (KAPPA) metrics. Page 5/21 Page 5/21 p yp Genetic Diversity and Demographic Analysis Demographic analyses for four localities from all regions were considered. The result of molecular diversity indices depicts the number of haplotypes (H), haplotype diversity (h), number of polymorphic sites (s) and nucleotide diversity (π) based on COI in Table 1. Page 6/21 Table 1 Molecular diversity indices based on Cyt b for E. tetraedra and its regional populations, including Number of sequences (N), the number of haplotypes (H), haplotype diversity (h), nucleotide diversity (π), and the number of polymorphic sites (S) Locality Number of sequences(N) Number of haplotypes (H) Haplotype diversity (h) Nucleotide diversity (π) Number of polymorphic sites (S) Lar River 23 4 0.597 0.05502 68 Jajroud 34 7 0.770 0.05614 70 Karaj river 30 10 0.867 0.05953 86 Spain 40 19 0.868 0.05148 85 Tajima's D and Fu's fs analysis were not significant for each population (Lar National Park population p < 0.88, Jajroud population p < 0.98, Karaj river population p < 0.89 and Spanish population p < 0.64 ) (Table Table 1 Tajima's D and Fu's fs analysis were not significant for each population (Lar National Park population p < 0.88, Jajroud population p < 0.98, Karaj river population p < 0.89 and Spanish population p < 0.64 ) (Table 2). In addition, The MMD diagrams for all population indicated a multimodal pattern (Additional file: Fig. 1). Table 2 Tests for population expansion for proposed subspecies of E. tetraedra Tajima's D Tajima's D p-value FS FS p-value Lar River 1.04 0.88 2.91 0.88 Jajroud 2.73 0.98 11.96 0.99 Karaj river 1.06 0.89 -0.31 0.49 Spain 0.11 0.64 -1.3 0.37 Table 2 Table 3 Evaluation of nine applied models predicting E. tetraedra distribution in Iran freshwater using AUC, TSS and KAPPA. SRE MARS FDA GLM MaxEnt ANN CTA GBM RF AUC 0.98 0.91 0.97 1 0.99 1 0.95 0.96 0.98 TSS 0.74 0.71 0.74 0.88 0.86 0.89 0.71 0.76 0.88 KAPPA 0.82 0.77 .079 0. 89 0.94 0.82 0.88 .089 .091 Table 3 Page 7/21 Modelling the Present and Future Distribution In all pattern predictions of models for E. tetraedra AUC (0.90-1.00), TSS (0.70–0.89) and KAPPA (0.78– 0.92) were good to excellent predictive capacity. Also, the best performing models for E. tetraedra were MaxEnt, GLM, ANN, and RF with AUC, TSS and KAPPA > 0.80 (Table. 3). In among variables BIO 1 (34.3), BIO 9 (24.8), BIO 14 (16.6) and Footprint (10.7) were the greatest contribution to model performance (Table. 4). Table 4. Uncorrelated predictors and mean of their contributions (%) in nine E. tetraedra distribution models in Iran freshwater. Table 4. Uncorrelated predictors and mean of their contributions (%) in nine E. tetraedra distribution models in Iran freshwater. Discussion This study clearly reports the role of genetic data in the identification of the origin of E. tetraedra a non- native species in Iran freshwater ecosystem. In fact, we investigated the origin of the introduction of the E. tetraedra in Iran and tried to explain how the suitable habitat will change using the SDMs approach. The beginning of studies on aquatic Oligochaeta back to 1920 (Stephenson 1920) and after about 100 years the Iranian fauna of aquatic Oligochaeta is inadequately known and limited to just a few studies (Egglishaw 1980; Ahmadi et al. 2012; Javidkar et al. 2019). Based on previous studies, 20 Oligochaeta species have been verified in Iran until 2015 (Jabłon´ska and Pesˇic´ 2014). Considering the area, mountainous landscapes, geographical features and specific hydrological characteristics of Iran, it seems that there will be an increase in the number of these species in the future. Latif et al (2009) identified E. tetraedra based on morphology as a non-native species with European and Palearctic origin from Haraz and Chalus rivers in Iran. Then Javidkar et al (2019) reported the first molecular attempts to discover the aquatic oligochaetes in Iran and they confirmed the non-native of the species by combining samples from Jajroud and Karaj with sequences from NCBI from studies elsewhere in the world for the species. Until this study and Javidkar et al (2020), the name of this species has not been listed in the native aquatic oligochaetes of Iran and our results reported E. tetraedra as a non-native species in Lar national park freshwater ecosystem. In line with our results, a number of researchers have reported E. tetraedra as a non-native species in other regions (Brinkhurst et al. 1960; Wood and James 1993; Martinsson et al. 2015; Sosa et al. 2017; Kim et al. 2017; Javidkar et al. 2020). According to the future climate change scenarios (SSP126 and 585) in our models projected the increase of suitable areas under all climate scenarios over time and suitable habitats will sharply increase over time across most of the E. tetraedra range (Fig. 4), as it tends to shift a wide range in the Western and almost the Southern of Iran. According to the future climate change scenarios (SSP126 and 585) in our models projected the increase of suitable areas under all climate scenarios over time and suitable habitats will sharply increase over time across most of the E. tetraedra range (Fig. 4), as it tends to shift a wide range in the Western and almost the Southern of Iran. According to the future climate change scenarios (SSP126 and 585) in our models projected the increase of suitable areas under all climate scenarios over time and suitable habitats will sharply increase over time across most of the E. tetraedra range (Fig. 4), as it tends to shift a wide range in the Western and almost the Southern of Iran. Page 8/21 Page 8/21 Haplotype and Genetic Diversity The results of the haplotype network clearly showed that the samples of Lar National Park, Jajroud and Karaj rivers did not have a specific haplotype and haplotypes of the current study are shared with Jajroud and Karaj rivers. To explain this phenomenon three hypotheses can be suggested; (a): E. tetraedra was independently introduced into all three habitats in Iran, (b) initially, E. tetraedra was introduced in Lar National Park, and then transferred to Jajrud and Karaj river, and its diversity and abundance decreased over time in Lar National Park; considering the Karaj river has the highest haplotype diversity and specific haplotype and also from Karaj river to Jajroud and Lar National Park, the haplotype diversity decreases, the third hypothesis is proposed, (c) E. tetraedra was initially introduced the Karaj river and was transferred to Jajroud and then to Lar National Park. However, based on the evidence and results, the third hypothesis is stronger. As well as, according to studies, altitude is one of the important limiting factors of distribution for the species as the abundance and diversity of Oligochaeta decrease with increasing altitude (Salome et al. 2011). Considering that the Lar National Park is located at an altitude of about 3000 meters, it seems that the species was not native to the region and accidentally transferred to the area. Page 9/21 Page 9/21 The molecular diversity indices depicted the haplotype and genetic diversity within the species were almost high (Table. 1). Also according to Table 1, the π of E. tetraedra in the three Iranian populations were 0.05502e0.05953 and in the Spanish population was 0.05148 that it showed almost no different genetic diversity in all populations and the Iranian population has not low genetic diversity than the Spanish population. Therefore, not having low genetic diversity compared to the origin population, can express the invasivation of the species in the introduced areas. Xu et al (2001) mentioned that for non- native species genetic diversity is so necessary to adapt to new habitats and maintain new population sizes. In addition, having high haplotype diversity one of the most important features affecting the success of invasivation of the species (Kolbe et al. 2004). The result (Table. 1) showed that E. tetraedra haplotype diversity in Iran's freshwater is increasing and it will have invasive success in Iran's freshwaters. Species Distribution Modeling (Present–Future) 2022) and it results in lowering the species diversity and degrading the sustainability of native freshwater species (Shi et al. 2010). One of the main drivers of worldwide biodiversity loss is biotic exchange in ecosystems by invasive species (Butchart et al. 2010). Although we did not appraise the effects of E. tetraedra as a non-native on other non-oligochaete species, studies e.g Migge-Kleian et al (2006) and Ziemba et al (2016) have shown the negative effects of non-native earthworms across trophic levels. According to the evidence in the present study and the identification of the success of Oligochaetes species in terms of being invasive in the river systems of Iran, it is assumed that freshwater ecosystems may be quite vulnerable to Oligochaetes of Western Palearctic origin and taking into account the negative consequences on native species, careful management strategies and regulations can help to mitigate these risks. It is essential that governments and individuals alike take a proactive approach to preventing the spread of invasive species and work to protect native ecosystems. Additionally, quarantine policies must be strictly enforced to help ensure that no potentially damaging organisms are imported into new environments. Species Distribution Modeling (Present–Future) Our study showed the impact of climate change on the distribution range of non-native E. tetrahedra in Iran's freshwater ecosystems. Carosi et al (2019) believed that species can experience four reactions under climate-change effects (i.e. expansion, reduction, both, or stable) in their habitats. The current map for E. tetrahedra clearly shows the suitable distribution for the species, that could occur in a wider distribution range especially in some regions out of the recorded areas (Fig. 4). Based on the outcome future maps of climate change modelling under SSPs scenarios, it will be predicted that climate change would significantly affect the distribution of E. tetrahedra as maps showed a sharp tendency to expand over time in its distribution areas (Fig. 4). In connection with our results, Mamun et al (2018) predicted future climate-change effects on an invasive alien species Micropterus salmoides in the Korean peninsula for 2050 and 2100. According to their results, the potentially suitable habitats for M. salmoides are most likely to increase by 2050 and 2100. Moreover, regarding the output of the modelling, it seems where human population density is high, these areas are probably more affected by the species in the future. This is maybe due to the high human activities, travel and trade in these areas. The temperature increase is an effective factor in the expansion of E. tetrahedra in Iran's freshwater ecosystems as expected climate change would benefit the species. In fact, temperature and precipitation were the most important role in model predictions. Based on the studies, E. tetraedra is expanding in most regions of the world and usually prefers humid habitats (Latif et al. 2009; Ezzatpanah et al. 2010; Mirmonsef et al. 2011; Yousefi et al. 2009). Therefore, it may be possible to justify their distribution in the humid regions of the country, including the northern and southern regions. Hong et al (2022) with SDMs tools mentioned temperature as the most reason for the expansion of range shifts in two invasive alien species under future climate-change scenarios. However, the result of SDMs explicitly illustrated the invasion power of the E. tetrahedra in Iran's freshwater ecosystems over time. It is mentioned that with expansions of alien species the vulnerability Page 10/21 Page 10/21 Page 10/21 of native species will probably be more significant (Hansen et al. 2017; Abdoli et al. 2022; Kim et al. Funding not applicable. Availability of data and materials The accession numbers for the datasets that were analyzed in this study c data. The accession numbers for the datasets that were analyzed in this study can be found in supplementary data. Ethics approval and consent to participate Not applicable. CONCLUSIONS E. tetraedra as an Oligochaete species is an interesting example of non-native species in Iran's freshwater. Integrating the phylogeny method and Species Distribution Models (SDMs) allowed us to unveil the successful biological invasion of E. tetraedra in the Iran's freshwater. Our results provided the origin of specimens of this study and supplied a novel approach to assessing the biological invasion of E. tetraedra under climate change. Although, the current study presented evidence for the invasion E. tetraedra, the information can help establish strategic and priority area data for ecosystem conservation. Authors’ contributions MB: Conceptualization (supporting); formal analysis (lead); methodology (lead); project administration (equal); software (lead); visualization (supporting); writing–original draft (lead); MA: Conceptualization (supporting); formal analysis (lead); software (lead); visualization (supporting); writing–original draft (lead); HK: Conceptualization (supporting); formal analysis (lead); software (lead); visualization (supporting); writing–original draft (lead); FA: Conceptualization (lead); data curation (lead); formal analysis (equal); methodology (equal); supervision (lead); writing–original draft (equal); writing–review and editing (equal) and A A:Conceptualization (lead); data curation (lead); formal analysis (equal); methodology (equal); supervision (lead); review and editing (equal). Acknowledgments We thank Najmeh Nazarian and Sarina Saljoughi for providing expert comments on current study. Additional accessibility data is provided as Additional file. 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J of Appli Sci 9 (20): 3746- 3751. doi:10.3923/JAS.2009.3746.3751. 71. 2527-2532. doi:10.1007/s10530-015-0910-7 Hong S, Jang I, Kim D, Kim S, Park HS, Lee K (2022) Predicting Potential Habitat Changes of Two Invasive Alien Fish Species with Climate Change at a Regional Scale, Sustain 14: 6093. doi:10.3390/su14106093 71. Hong S, Jang I, Kim D, Kim S, Park HS, Lee K (2022) Predicting Potential Habitat Changes of Two Invasive Alien Fish Species with Climate Change at a Regional Scale, Sustain 14: 6093. doi:10.3390/su14106093 72. Hansen GJA, Read JS, Hansen JF, Winslow LA (2017) Projected shifts in fish species dominance in Wisconsin lakes under climate change, Glo Chan Biol 23: 1463–1476. doi:10.1111/gcb.13462. 72. Hansen GJA, Read JS, Hansen JF, Winslow LA (2017) Projected shifts in fish species dominance in Wisconsin lakes under climate change, Glo Chan Biol 23: 1463–1476. doi:10.1111/gcb.13462. 73. Abdoli A, Valikhani H, Nejat N, Khosravi M (2022) Non-native freshwater fishes of Iran (Identification, Impacts, Management), ISBN: 978-964-479-203-8. 73. Abdoli A, Valikhani H, Nejat N, Khosravi M (2022) Non-native freshwater fishes of Iran (Identification, Impacts, Management), ISBN: 978-964-479-203-8. 74. Kim Z, Shim T, Ki SJ, An KG, Jung J (2022) Prediction of three-dimensional shift in the distribution of largemouth bass (Micropterus salmoides) under climate change in South Korea, Ecol Indic 137: 108731. doi:10.1016/j.ecolind.2022.108731 Figures Figures Page 17/21 Figure 1 Locations of sampling stations E. tetraedra in current study. Page 18/21 Figure 2 Phylogenetic tree reconstructed for E. tetraedra based on the COI. For each node, no BI posterior probabilities (top) and ML bootstrap support (in percent, base). Figure 2 Phylogenetic tree reconstructed for E. tetraedra based BI posterior probabilities (top) and ML bootstrap supp Figure 2 Phylogenetic tree reconstructed for E. tetraedra based on the COI. For each node, nodal supports indicate BI posterior probabilities (top) and ML bootstrap support (in percent, base). Figure 2 Figure 2 Phylogenetic tree reconstructed for E. tetraedra based on the COI. For each node, nodal supports indicate BI posterior probabilities (top) and ML bootstrap support (in percent, base). Page 19/21 Page 19/21 Figure 3 Figure 3 Figure 3 Median-joining (MJ) haplotype network. Each circle represents a unique haplogroup, and its size reflects the number of individuals expressing that haplotype. Crosshatches indicate the number of nucleotide differences between haplotypes. Median-joining (MJ) haplotype network. Each circle represents a unique haplogroup, and its size reflects the number of individuals expressing that haplotype. Crosshatches indicate the number of nucleotide differences between haplotypes. Figure 4 Figure 4 Page 20/21 Dynamic changes in the suitable habitat of E. tetraedra in the freshwaters of Iran under current and two future climate scenarios 2061-2080 (ssp 126 and ssp 585) based on MRI-CGCM3 model. Dynamic changes in the suitable habitat of E. tetraedra in the freshwaters of Iran under current and two future climate scenarios 2061-2080 (ssp 126 and ssp 585) based on MRI-CGCM3 model. suplement.docx Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. suplement.docx Page 21/21
https://openalex.org/W4321597200	https://jneuroengrehab.biomedcentral.com/counter/pdf/10.1186/s12984-023-01151-6	English	null	Predicting patient-reported outcome of activities of daily living in stroke rehabilitation: a machine learning study	Journal of neuroengineering and rehabilitation	2,023	cc-by	9,333	Journal of NeuroEngineering and Rehabilitation Journal of NeuroEngineering and Rehabilitation Journal of NeuroEngineering and Rehabilitation Chen et al. Journal of NeuroEngineering and Rehabilitation (2023) 20:25 https://doi.org/10.1186/s12984-023-01151-6 Chen et al. Journal of NeuroEngineering and Rehabilitation https://doi.org/10.1186/s12984-023-01151-6 Chen et al. (2023) 20:25 Open Access Yu‑Wen Chen1,4, Keh‑chung Lin1,2, Yi‑chun Li1,3 and Chia‑Jung Lin1 Yu‑Wen Chen1,4, Keh‑chung Lin1,2, Yi‑chun Li1,3 and Chia‑Jung Lin1 Abstract Keywords Stroke rehabilitation, Prognosis, Prediction, Patient-reported outcome measures, Activities of daily living, Machine learning Keywords Stroke rehabilitation, Prognosis, Prediction, Patient-reported outcome measures, Activities of daily living, Machine learning 3 Department of Occupational Therapy, I-Shou University College of Medicine, Kaohsiung, Taiwan 4 Department of Speech Language Pathology and Audiology, National Taipei University of Nursing and Health Sciences, Taipei, Taiwan *Correspondence: Keh‑chung Lin kehchunglin@ntu.edu.tw 1 School of Occupational Therapy, College of Medicine, National Taiwan University, 17, F4, Xuzhou Rd., Taipei, Taiwan 2 Division of Occupational Therapy, Department of Physical Medicine and Rehabilitation, National Taiwan University Hospital, 7 Chung‑Shan S. Rd., Taipei, Taiwan Abstract Background Machine Learning is increasingly used to predict rehabilitation outcomes in stroke in the context of precision rehabilitation and patient-centered care. However, predictors for patient-centered outcome measures for activities and participation in stroke rehabilitation requires further investigation. Methods This study retrospectively analyzed data collected for our previous studies from 124 participants. Machine Learning models were built to predict postintervention improvement of patient-reported outcome measures of daily activities (i.e, the Motor Activity Log and the Nottingham Extended Activities of Daily Living) and participation (i.e, the Activities of Daily Living domain of the Stroke Impact Scale). Three groups of 18 potential predictors were included: patient demographics, stroke characteristics, and baseline assessment scores that encompass all three domains under the framework of International Classification of Functioning, Disability and Health. For each target variable, classifica‑ tion models were built with four algorithms, logistic regression, k-nearest neighbors, support vector machine, and random forest, and with all 18 potential predictors and the most important predictors identified by feature selection. Results Predictors for the four target variables partially overlapped. For all target variables, their own baseline scores were among the most important predictors. Upper-limb motor function and selected demographic and stroke characteristics were also among the important predictors across the target variables. For the four target variables, prediction accuracies of the best-performing models with 18 features ranged between 0.72 and 0.96. Those of the best-performing models with fewer features ranged between 0.72 and 0.84. Conclusions Our findings support the feasibility of using Machine Learning for the prediction of stroke rehabilita‑ tion outcomes. The study was the first to use Machine Learning to identify important predictors for postintervention improvement on four patient-reported outcome measures of activities and participation in chronic stroke. The study contributes to precision rehabilitation and patient-centered care, and the findings may provide insights into the iden‑ tification of patients that are likely to benefit from stroke rehabilitation. Background The other recent trend in medicine and rehabilitation, patient-centered care, aims at engaging the patients, family, and caregivers in the clinical decision-making process. To achieve this goal, patient-reported out- come measures (PROMs) for activities and participation should be incorporated in the assessment in addition to therapist-rated and impairment-level measures. How- ever, most of the existing ML predictive studies on stroke rehabilitation outcomes investigated therapist-rated out- come measures such as the Barthel Index [5, 6] and the FIM [4] for the acute and subacute stages. In the chronic stage, earlier reports studied the FMA-UE [13, 14], and one recent study investigated SIS [15] as the concept of PROMs emerges. There is still a need to expand our knowledge of the relevance of ML predictive models to include more commonly used PROMs of activities and participation. Stroke is a leading cause of disability that requires long- term post-stroke care and rehabilitation [1]. Along the course, patients and family and the care team are required to make multiple clinical decisions. Clinical decision making in rehabilitation benefits from accu- rate predictions of prognosis, which prompts research that investigates predictors for stroke-rehabilitation outcomes. Two recent trends in rehabilitation are precision rehabilitation and patient-centered care. Clinical deci- sion making in the context of precision rehabilita- tion involves identifying the characteristics of patients who would likely benefit from rehabilitation programs. Machine learning (ML) is increasingly used for the task of understanding predictors for rehabilitation out- comes by the construction of models that can predict outcomes when given new data. ML is a branch of arti- ficial intelligence that uses algorithms to find patterns in the input data and generate models to predict target variables. Through pattern-finding, the models identify the most important “features,” or potential predictors, for the “target,” or the predicted variable. The advan- tages of ML include its ability to take a large amount of features at once, to conduct multidimensional data analyses, and to learn from the data without substantial a priori knowledge about the features [2]. Another common practice found in the literature has been the inclusion of only one predicted outcome meas- ure. However, given the heterogeneous nature of the stroke population, including multiple predicted outcome measures in research studies was recommended [4]. In fact, most therapists use multiple assessment tools to quantify related but distinct aspects of body functions, activities, and participation in clinical practice. Background For exam- ple, the Motor Activity Log (MAL) [17] and the Not- tingham Extended Activities of Daily Living (NEADL) [18, 19] are commonly used patient-reported assessment tools of activities, and the SIS [20] has been widely used to measure function of participation. In stroke rehabilitation, studies have investigated the feasibility of ML models for the prediction of postinter- vention outcomes. Most studies focused on patients in the subacute stage. The predicted outcome measures in these studies represent the three domains of the World Health Organization’s International Classification of Functioning, Disability and Health (ICF) [3], and range from measures of motor function, including the Ten-Meter Walk Test, Six-Minute Walk Test, and Berg Balance Scale [4], to measures of activities and partici- pation, including the Barthel Index [5, 6], the modi- fied Rankin Scale [7–10], the Functional Independence Measure (FIM) [4], and patients’ discharge placement [11, 12]. However, few ML predictive studies on chronic stroke investigated the postintervention outcomes [13– 16]. To our knowledge, two studies investigated postin- tervention improvements in upper-limb (UL) motor function measured by the Fugl-Meyer Assessment Upper Extremity subscale (FMA-UE) [13, 14] or lower- limb motor function measured by step threshold [16]. One study used the Stroke Impact Scale (SIS), a meas- ure in the ICF domain of activities and participation. Studies using ML remains scarce on the prediction of postintervention improvements, especially in measures of the ICF domains of activities and participation, for chronic stroke. The MAL was designed to measure the use of the affected upper-limb in basic activities of daily living (ADL). Patients are asked to rate how much (amount of use; MAL-AOU) and how well (quality of movement; MAL-QOM) they use the affected arm for a number of given ADL. The NEADL measures instrumental ADL and assesses functional independence in community living. The SIS measures patients’ health-related quality of life and includes items for participation; one of its domains is Activities of Daily Living (SIS-ADL). Assessing multiple outcome measures to provide multifaceted clinical infor- mation about potential prognosis could empower the patients and their families to make appropriate decisions that are most relevant and meaningful to the patient. However, most predictive studies only reported one out- come measure. There is a need to expand the repertoire of outcome measures in research studies to meet clinical applications. © 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. Chen et al. Journal of NeuroEngineering and Rehabilitation (2023) 20:25 Page 2 of 12 Chen et al. Journal of NeuroEngineering and Rehabilitation (2023) 20:25 Data analysish The potential predictors and the target variables were used to build ML models. The objective of the ML pro- grams was to find patterns to classify the samples into responders and non-responders. For each PROM, four ML algorithms were used to find the patterns: logistic regression (LR), k-nearest neighbors (KNN), support vector machine (SVM), and random forest (RF). KNN and SVM were selected because they were frequently reported to yield high performance in existing predic- tive studies of stroke rehabilitation outcomes [5, 8, 9, 11, 13, 27]. LR was selected as a baseline model to test the predicting capability of a simpler algorithm for our data set. RF was selected to test whether its higher model complexity would benefit the predictions. Using multi- ple algorithms to construct models and compare perfor- mances is also common. One previous study specifically recommended the use of multiple algorithms [13]. Background This study used ML to build predictive models to pre- dict postintervention outcomes and identify the most important predictors for these outcome measures in stroke rehabilitation. We have expanded on previ- ous findings to use multiple PROMs for activities and Page 3 of 12 Chen et al. Journal of NeuroEngineering and Rehabilitation (2023) 20:25 participation in consideration of clinical applications and recent trends in stroke rehabilitation. participation in consideration of clinical applications and recent trends in stroke rehabilitation. Wolf Motor Function Test-Time (WMFT-Time), Wolf Motor Function Test-Quality (WMFT-Quality), Chedoke Arm and Hand Activity Inventory (CAHAI), MAL-AOU, MAL-QOM, NEADL, FIM, SIS-Total, and SIS-ADL. The baseline assessment scores were selected to encompass all three domains under the ICF framework: body func- tion, activities, and participation. Wolf Motor Function Test-Time (WMFT-Time), Wolf Motor Function Test-Quality (WMFT-Quality), Chedoke Arm and Hand Activity Inventory (CAHAI), MAL-AOU, MAL-QOM, NEADL, FIM, SIS-Total, and SIS-ADL. The baseline assessment scores were selected to encompass all three domains under the ICF framework: body func- tion, activities, and participation. Study design and participantsh This study is a retrospective analysis of data collected for previous studies conducted by our research team; avail- able results have been published elsewhere [21, 22]. The inclusion criteria of the original studies were (1) at least 3 months after the onset of a first-ever unilateral cer- ebral stroke; (2) a baseline FMA-UE between 16 and 56; (3) ability to follow instructions, with one study includ- ing only participants without Wernicke’s aphasia; (4) a spasticity score of ≤ 3 on the Modified Ashworth Scale; and (5) no other neurologic or orthopedic disorders. The exclusion criteria of the original studies were (1) serious vision disorders in one study [22] and (2) psychiatric and balance problems in the other study [21]. Intervention and assessmenth The participants received one of the following therapy programs: InMotion robotic-assisted therapy, Bi-Manu- Track robotic therapy [21], robotic-priming mirror ther- apy, robotic-priming bilateral upper limb training [22], or conventional occupational therapy. Dosages were simi- lar across the therapy programs; participants received 3 weeks of therapy, 3 to 4 days a week, and 60 min a day. Assessments were completed before and after the thera- pies, and for most participants, at a 3-month follow-up. In addition to models with all 18 features, in considera- tion of clinical parsimony, we also built predictive mod- els with the four, five, and six most important features, which we identified by feature selection procedure (see details in the next paragraph). Therefore, for each target variable, 16 models were built (four algorithms x four numbers of features). Outcome measures and potential predictors SD standard deviation, IQR interquartile range, NIHSS National Institutes of Health Stroke Scale, FMA-UE Upper Extremity subscale of the Fugl-Meyer Assessment, BBT Box and Block Test, CAHAI Chedoke Arm and Hand Activity Inventory, WMFT Wolf Motor Function Test, FIM Functional Independence Measure, MAL Motor Activity Log, AOU Amount of Use, QOM Quality of Movement, NEADL Nottingham Extended Activities of Daily Living, SIS Stroke Impact Scale, ADL Activities of Daily Living participants. Of the 124 participants, 79 achieved MCID for MAL-AOU, 79 for MAL-QOM, 43 for NEADL, and 36 for SIS-ADL. Outcome measures and potential predictors 1 Flowchart for the machine-learning data analysis. SMOTE synthetic minority oversampling technique, KNN k-nearest neighbors, SVM support vector machine Fig. 1 Flowchart for the machine-learning data analysis. SMOTE synthetic minority oversampling technique, KNN k-nearest neighbors, SVM support vector machine Table 1 Participant Characteristics SD standard deviation, IQR interquartile range, NIHSS National Institutes of Health Stroke Scale, FMA-UE Upper Extremity subscale of the Fugl-Meyer Assessment, BBT Box and Block Test, CAHAI Chedoke Arm and Hand Activity Inventory, WMFT Wolf Motor Function Test, FIM Functional Independence Measure, MAL Motor Activity Log, AOU Amount of Use, QOM Quality of Movement, NEADL Nottingham Extended Activities of Daily Living, SIS Stroke Impact Scale, ADL Activities of Daily Living Mean ± SD/ Median (IQR)/ Participants, no. (%) Demographics Age (years) 55.75 ± 11.22 Male sex 84 (68%) Years of education 12.00 (6.00) Stroke characteristics Right-sided hemiplegia 70 (57%) Time since stroke (months) 14.00 (22.00) Hemorrhagic stroke diagnosis 55 (44%) NIHSS score 4.00 (3.00) Baseline Assessment Scores FMA-UE 30.00 (14.00) BBT 1.00 (13.25) CAHAI 31.50 (20.25) WMFT-Time 12.11 (8.14) WMFT-Quality 2.41 ± 0.54 FIM 110.00 (12.25) MAL-AOU 0.91 (1.01) MAL-QOM 0.59 (0.99) NEADL 28.00 (25.25) SIS-Total 63.13 ± 12.04 SIS-ADL 37.50 (10.25) Table 1 Participant Characteristics standardized to avoid dominating effects of features on scales of larger numbers [29]. For model tuning, grid search was used to identify values for hyperparameters that obtained the highest classification accuracy with stratified tenfold cross validation. For LR, the search procedure identified the optimal c value and maximum iterations. For KNN, the search procedure identified the optimal number of neighbors and the distance weight. For SVM, the search procedure identified the optimal kernel and c value, which specifies the size of the hyperplane margin and therefore regularizes the model. For RF, the search procedure identified the optimal number of estimators and maximum depth. All other hyperparameters were set as the default. After the models were constructed, model performance was tested using the testing set. Model performance was primarily assessed by classification accuracy and the area under the receiver operating characteristic curve (AUC). We also calculated specificity, sensitivity, negative predic- tive value (NPV), and positive predictive value (PPV). Descriptive statistics and normality checks were per- formed with R 4.0.3 software [30]. The construction and validation of the ML models and the corresponding data preprocessing were conducted using Python 3.8.2 software [31], with the packages sklearn 1.0 [32] and imblearn 0.8 [33]. Outcome measures and potential predictors p p Participants’ level of ADL was measured by three assess- ment tools with four PROMs: the MAL-AOU and MAL-QOM, NEADL, and SIS-ADL. For each meas- ure, participants who achieved the minimal clinically important difference (MCID) from pretest to posttest were labeled as responders, and those who did not were labeled as non-responders. For MAL, we adopted an MCID of 0.5 of average change, corresponding to 10% of the rating scale [23–25]. The MCIDs for NEADL total changes and SIS-ADL total changes were 6.1 [18] and 5.9 [26], respectively. For an ML model, the status of response to therapy (i.e., responders versus non-respond- ers) on a given PROM served as the predicted variable, called the “target” in ML terminology. Figure 1 visualizes steps for the data analysis using ML. For each target variable, the data set was first randomized and split into a training set and a testing set, with the training set containing 80% of the samples. The training set was used to build models, and the testing set was used to test the performance of the models. To select the most important features to use in the parsimonious models, feature selection was performed using the standardized training set by calculating mutual information gain (MI; also known as information gain). The testing data set was never used for model construction or feature selection. This ensured that the data used to test model perfor- mance did not influence any decisions about the models and was truly unseen until performance testing. We included 18 potential predictors, called “features” in ML terminology, in the ML models. The potential predic- tors can be grouped into three categories: (1) participant demographics: age, sex, and years of education; (2) stroke characteristics: time since stroke, the National Institute of Health Stroke Scale (NIHSS) score, side of hemiplegia, and diagnosis (hemorrhagic or ischemic); and (3) baseline assessment scores: FMA-UE, Box and Block Test (BBT), During model construction, the Synthetic Minor- ity Over-sampling Technique (SMOTE) [28] was used to minimize the effect of class imbalance, where models may favor the majority class, creating biases and potential false optimistic classification accuracy. Except for models built with RF, the data were also Chen et al. Journal of NeuroEngineering and Rehabilitation (2023) 20:25 Page 4 of 12 Fig. 1 Flowchart for the machine-learning data analysis. SMOTE synthetic minority oversampling technique, KNN k-nearest neighbors, SVM support vector machine Fig. Model performance Figure 2 visualizes the confusion matrices for the mod- els. Table 3 summaries the performance metrics as well as training scores and medians and interquartile ranges of the validation scores. Good model performance was achieved across the outcome measures. For all out- come measures, similar or slightly decreased prediction accuracies could be achieved with a reduced number of features. Among the MAL-AOU models with 18 fea- tures, LR yielded the best performance (accuracy = 0.72, AUC = 0.74). For MAL-AOU models with fewer fea- tures, RF with 6 features performed the best (accu- racy = 0.72, AUC = 0.80). For MAL-QOM models with 18 features, SVM and RF yielded the best performance (accuracy = 0.76, AUC = 0.83), and LR achieved similar performance (accuracy = 0.76, AUC = 0.81). Among the MAL-QOM models with fewer features, KNN with 5 fea- tures performed the best (accuracy = 0.76, AUC = 0.75). For NEADL models with 18 features, RF yielded the best performance (accuracy = 0.76, AUC = 0.81). For NEADL models with fewer features, the best performance occurred with RF fitted with 4 features (accuracy = 0.76, AUC = 0.87). For SIS-ADL predicted with 18 features, This current study extended from the existing literature by investigating the most important predictors for MCID achievements on multiple PROMs for activities and par- ticipation in chronic stroke using ML. We identified dif- ferent sets of the most important predictors for the target variables, reflecting the distinct, albeit related, aspects of ADL assessed in the four PROMs. We also obtained good model performances for the target variables, demonstrat- ing the feasibility of ML for predicting postintervention improvement on PROMs of activities and participa- tion in chronic stroke. Participant characteristics A total of 128 participants were located in our data base; four participants dropped out before the postinterven- tion assessment, resulting in missing data, and were excluded from the study. The study included 124 par- ticipants. Table 1 summarizes the demographics, stroke characteristics, and baseline assessment scores of the participants. Of the 124 participants, 79 achieved MCID for MAL-AOU, 79 for MAL-QOM, 43 for NEADL, and 36 for SIS-ADL. Chen et al. Journal of NeuroEngineering and Rehabilitation (2023) 20:25 Page 5 of 12 Chen et al. Journal of NeuroEngineering and Rehabilitation (2023) 20:25 Discussion ML is increasingly used in the prediction of postint- ervention prognosis in stroke. Previous studies have investigated prognostic predictors as well as the per- formance of predictive models. However, most studies were on acute to subacute stroke, and few studies exist on chronic stroke. Further, studies on postintervention improvements in subacute stroke included measures of motor function and measures of activities and participa- tion, whereas few studies on chronic stroke investigated activities and participation. In addition, most studies have included only one predicted outcome measure and focused on therapist-rated measures. The use of PROMs is attracting more attention in recent years as health care shifts toward patient-centered care, but few studies have investigated postintervention improvements measured by PROMs in chronic stroke. Most important predictors SVM yielded the best performance (accuracy = 0.96, AUC = 0.96). For SIS-ADL models fitted with fewer fea- tures, SVM with 5 features yielded the best performance (accuracy = 0.84, AUC = 0.92). Table 2 presents the MI gains for the predictors with gains higher than zero. Notably, across all target vari- ables, their corresponding baseline scores had non-zero MI gains for the achievement of MCID. Further, baseline UL motor function (FMA-UE and BBT) and baseline SIS- Total scores were important for all target variables. MAL scores were also at the top five important predictors for all target variables. ADL Activities of Daily Living, AOU Amount of Use, BBT Box and Block Test, CAHAI Chedoke Arm and Hand Activity Inventory, FMA-UE Upper Extremity subscale of the Fugl-Meyer Assessment, MAL Motor Activity Log, NEADL Nottingham Extended Activities of Daily Living, NIHSS National Institutes of Health Stroke Scale, QOM Quality of Movement, SIS Stroke Impact Scale, WMFT Wolf Motor Function Test Model performance In addition, we were able to build Table 2 Mutual information gains for the predictors sorted in descending order for each target variable ADL Activities of Daily Living, AOU Amount of Use, BBT Box and Block Test, CAHAI Chedoke Arm and Hand Activity Inventory, FMA-UE Upper Extremity subscale of the Fugl-Meyer Assessment, MAL Motor Activity Log, NEADL Nottingham Extended Activities of Daily Living, NIHSS National Institutes of Health Stroke Scale, QOM Quality of Movement, SIS Stroke Impact Scale, WMFT Wolf Motor Function Test MAL-AOU MAL-QOM NEADL SIS-ADL Predictor Gain Predictor Gain Predictor Gain Predictor Gain Time since stroke 0.14 SIS-Total 0.12 SIS-Total 0.07 FMA-UE 0.10 WMFT-Quality 0.13 WMFT-Quality 0.06 SIS-ADL 0.06 SIS-ADL 0.09 SIS-Total 0.10 MAL-QOM 0.06 NEADL 0.05 MAL-AOU 0.05 FMA-UE 0.06 FMA-UE 0.03 MAL-AOU 0.03 CAHAI 0.05 MAL-QOM 0.06 MAL-AOU 0.03 BBT 0.02 Diagnosis 0.06 CAHAI 0.04 FIM 0.03 NIHSS 0.02 SIS-Total 0.02 FIM 0.03 Side of hemiplegia 0.02 FIM 0.01 Time since stroke 0.01 WMFT-Time 0.03 NEADL 0.02 Other predictors 0 NIHSS 0.01 MAL-AOU 0.01 Sex 0.01 Other predictors 0 Other predictors 0 Years of education 0.01 Other predictors 0 Chen et al. Journal of NeuroEngineering and Rehabilitation (2023) 20:25 Page 6 of 12 Fig. 2 Confusion matrices for the predictive models. MAL Motor Activity Log, AOU Amount of Use, QOM Quality of Movement, NEADL Nottingham Extended Activities of Daily Living, SIS-ADL Stroke Impact Scale Activities of Daily Living domain, LR logistic regression, KNN k-nearest neighbors, SVM support vector machine, RF random forest Fig. 2 Confusion matrices for the predictive models. MAL Motor Activity Log, AOU Amount of Use, QOM Quality of Movement, NEADL Nottingham Extended Activities of Daily Living, SIS-ADL Stroke Impact Scale Activities of Daily Living domain, LR logistic regression, KNN k-nearest neighbors, SVM support vector machine, RF random forest using techniques to lower the effects of dimensionality and class imbalance.h parsimonious models with smaller sets of predictors that performed similar or just slightly worse than the full models, which could benefit clinical practice in the selec- tion of prioritized assessments. The practice of feature selection contributed to clini- cal parsimony. Clinically, it is more efficient if accurate prediction of prognosis can be obtained by assessment results from fewer tools. In our results, at least one of the models with fewer features for each target variable was able to achieve similar performance compared with 18 features. Model performance The results provided support for the clinical application of ML by finding that highly accurate pre- dictions of postintervention outcomes in stroke can be achieved with only a few clinical assessments and patient information. ML for predicting postintervention outcomes in stroke ML for predicting postintervention outcomes in stroke Emerging research has reported the feasibility of ML for the prediction of postintervention outcome in stroke. However, in the field of health care research, achieving the sample size of big data analysis is often difficult. This is because of a variety of limitations, such as patient pri- vacy policies, the heterogeneity of disease manifestation, the variability in care plans, and cost and time for inter- vention and data collection, to name just a few. Findings of this current study, however, indicates the feasibility of using ML for the prediction of postintervention outcome with a limited sample size. Despite the relatively smaller sample size, we were able to obtain high classification accuracies and acceptable to outstanding [34] AUCs Another issue working with our data set was class imbalance, where one of the classes (responders versus non-responders) outnumbered the other. In a data set with imbalanced classes, the learning machine may focus on finding patterns in the majority class when striving to increase classification accuracy. This usually results in a Chen et al. ML for predicting postintervention outcomes in stroke Journal of NeuroEngineering and Rehabilitation (2023) 20:25 Page 8 of 12 Table 3 (continued) Model Accuracy AUC Specificity Sensitivity NPV PPV Train score Validation, median (IQR) 6 features LR 0.52 0.57 0.63 0.33 0.63 0.33 0.60 0.65 (0.20) KNN 0.56 0.48 0.63 0.44 0.67 0.40 0.95 0.60 (0.20) SVM 0.64 0.62 0.50 0.89 0.89 0.50 0.55 0.70 (0.18) RF 0.72 0.85 0.75 0.67 0.80 0.60 0.80 0.70 (0.16) 5 features LR 0.64 0.72 0.75 0.44 0.71 0.50 0.62 0.65 (0.20) KNN 0.64 0.63 0.69 0.56 0.73 0.50 0.94 0.60 (0.10) SVM 0.64 0.76 1.00 0.00 0.64 0.00 0.70 0.60 (0.09) RF 0.68 0.82 0.75 0.56 0.75 0.56 0.86 0.68 (0.18) 4 features LR 0.64 0.72 0.75 0.44 0.71 0.50 0.65 0.60 (0.20) KNN 0.68 0.71 0.75 0.56 0.75 0.56 0.93 0.68 (0.20) SVM 0.60 0.70 0.63 0.56 0.71 0.45 0.62 0.60 (0.28) RF 0.76 0.87 0.75 0.78 0.86 0.64 0.80 0.70 (0.18) SIS-ADL 18 features LR 0.92 0.98 0.94 0.86 0.94 0.86 0.98 0.90 (0.08) KNN 0.80 0.75 0.94 0.43 0.81 0.75 0.96 0.68 (0.10) SVM 0.96 0.96 1.00 0.86 0.95 1.00 0.95 0.90 (0.15) RF 0.68 0.76 0.83 0.29 0.75 0.40 1.00 0.70 (0.09) 6 features LR 0.72 0.80 0.83 0.43 0.79 0.50 0.77 0.75 (0.27) KNN 0.72 0.77 0.72 0.71 0.87 0.50 0.78 0.70 (0.06) SVM 0.76 0.82 0.83 0.57 0.83 0.57 0.77 0.70 (0.18) RF 0.68 0.72 0.72 0.57 0.81 0.44 0.87 0.65 (0.19) 5 features LR 0.80 0.81 0.89 0.57 0.84 0.67 0.75 0.65 (0.19) KNN 0.76 0.76 0.83 0.57 0.83 0.57 0.73 0.65 (0.10) SVM 0.84 0.92 0.83 0.86 0.94 0.67 0.72 0.70 (0.13) RF 0.68 0.74 0.78 0.43 0.78 0.43 0.80 0.65 (0.16) 4 features LR 0.76 0.87 0.83 0.57 0.83 0.57 0.75 0.70 (0.09) KNN 0.68 0.69 0.78 0.43 0.78 0.43 0.82 0.70 (0.08) SVM 0.72 0.88 0.67 0.86 0.92 0.50 0.72 0.74 (0.20) RF 0.64 0.72 0.72 0.43 0.76 0.38 0.90 0.70 (0.19) IQR interquartile range, MAL Motor Activity Log, AOU Amount of Use, QOM Quality of Movement, NEADL Nottingham Extended Activities of Daily Living, SIS-ADL Stroke Impact Scale Activities of Daily Living domain, LR logistic regression, KNN k-nearest neighbors, SVM support vector machine, RF random forest, AUC area under the receiver operating characteristic curve, NPV negative predictive value, PPV positive predictive value IQR interquartile range, MAL Motor Activity Log, AOU Amount of Use, QOM Quality of Movement, NEADL Nottingham Extended Activities of Daily Living, SIS-ADL Stroke Impact Scale Activities of Daily Living domain, LR logistic regression, KNN k-nearest neighbors, SVM support vector machine, RF random forest, AUC area under the receiver operating characteristic curve, NPV negative predictive value, PPV positive predictive value used SMOTE to deal with class imbalance; of their 102 participants, 43 achieved MCID on FMA-UE and 59 did not. ML for predicting postintervention outcomes in stroke bias toward the majority class [29]. Consider an extreme example, where there are 90 cases in the positive class and 10 in the negative class, the classifier could conveni- ently classify all cases as positive and obtain a high train- ing accuracy of 0.90. However, the specificity and NPV would be zero. Among the techniques to work with class imbalance, we chose to use SMOTE because of our rela- tively smaller sample size (N = 124). Tozlu et al. [14] also ML for predicting postintervention outcomes in stroke Journal of NeuroEngineering and Rehabilitation (2023) 20:25 Page 7 of 12 Table 3 Model performance metrics and training and validation scores for the predictive models Model Accuracy AUC Specificity Sensitivity NPV PPV Train score Validation, median (IQR) MAL-AOU 18 features LR 0.72 0.74 0.56 0.81 0.63 0.76 0.66 0.60 (0.19) KNN 0.56 0.48 0.33 0.69 0.38 0.65 0.75 0.60 (0.17) SVM 0.60 0.56 0.33 0.75 0.43 0.67 0.86 0.63 (0.10) RF 0.68 0.76 0.44 0.88 0.67 0.74 1.00 0.65 (0.18) 6 features LR 0.68 0.74 0.78 0.63 0.54 0.83 0.59 0.47 (0.28) KNN 0.52 0.66 0.44 0.56 0.36 0.64 1.00 0.65 (0.18) SVM 0.60 0.69 0.56 0.63 0.45 0.71 0.89 0.65 (0.25) RF 0.72 0.80 0.67 0.75 0.60 0.80 1.00 0.70 (0.20) 5 features LR 0.60 0.69 0.78 0.50 0.47 0.80 0.57 0.58 (0.25) KNN 0.60 0.56 0.44 0.69 0.44 0.69 1.00 0.60 (0.16) SVM 0.52 0.77 0.78 0.38 0.41 0.75 0.57 0.50 (0.18) RF 0.64 0.69 0.33 0.81 0.50 0.68 1.00 0.70 (0.09) 4 features LR 0.60 0.70 0.67 0.56 0.46 0.75 0.58 0.53 (0.28) KNN 0.56 0.60 0.33 0.69 0.38 0.65 1.00 0.65 (0.19) SVM 0.64 0.74 0.67 0.63 0.50 0.77 0.61 0.60 (0.15) RF 0.64 0.64 0.44 0.75 0.50 0.71 1.00 0.74 (0.18) MAL-QOM 18 features LR 0.76 0.81 0.67 0.81 0.67 0.81 0.75 0.68 (0.10) KNN 0.72 0.78 0.56 0.81 0.63 0.76 0.78 0.50 (0.19) SVM 0.76 0.83 0.78 0.75 0.64 0.86 0.77 0.68 (0.10) RF 0.76 0.83 0.67 0.81 0.67 0.81 1.00 0.50 (0.19) 6 features LR 0.60 0.71 0.67 0.56 0.46 0.75 0.63 0.60 (0.26) KNN 0.52 0.57 0.56 0.50 0.38 0.67 0.73 0.65 (0.10) SVM 0.64 0.49 0.33 0.81 0.50 0.68 0.97 0.60 (0.10) RF 0.52 0.67 0.67 0.50 0.43 0.73 0.81 0.60 (0.19) 5 features LR 0.60 0.72 0.67 0.56 0.46 0.75 0.61 0.60 (0.15) KNN 0.76 0.75 0.56 0.88 0.71 0.78 1.00 0.60 (0.26) SVM 0.52 0.62 0.67 0.44 0.40 0.70 0.81 0.60 (0.26) RF 0.60 0.71 0.56 0.63 0.45 0.71 0.85 0.70 (0.16) 4 features LR 0.56 0.72 0.56 0.56 0.42 0.69 0.67 0.60 (0.19) KNN 0.60 0.62 0.33 0.75 0.43 0.67 1.00 0.70 (0.23) SVM 0.60 0.71 0.67 0.50 0.43 0.73 0.77 0.70 (0.10) RF 0.72 0.75 0.67 0.75 0.60 0.80 0.99 0.70 (0.21) NEADL 18 features LR 0.56 0.57 0.69 0.33 0.65 0.38 0.62 0.50 (0.26) KNN 0.52 0.41 0.63 0.33 0.63 0.33 0.97 0.60 (0.25) SVM 0.60 0.65 0.94 0.00 0.63 0.00 0.67 0.65 (0.10) RF 0.76 0.81 0.75 0.78 0.86 0.64 0.81 0.70 (0.16) Table 3 Model performance metrics and training and validation scores for the predictive models Chen et al. The most important predictors Stroke severity was previously reported to predict long-term post-stroke functional outcomes in acute and subacute stroke [46–48]; our results showed that stroke severity, as measured by NIHSS, can also predict postintervention improvements in NEADL in chronic stroke. The finding should be cautiously interpreted, however, because there may be an underrepresentation of severe cases in our study. Our participants had NIHSS scores ranging from 0 to 13, which correspond to no stroke symptoms, minor stroke, and moderate stroke. Therefore, this finding should not be generalized to patients with severe stroke in the chronic stage. In summary, our results that demo- graphic and stroke characteristics were among the most important predictors were largely consistent with previ- ous findings, and we recommend future studies include these characteristics in the potential predictors when performing feature selection. Lin et al. [8] analyzed data from a nation-wide disease registry and built predictive models for 90-day post- stroke scores on the modified Rankin Scale. They found that the 30-day modified Rankin Scale scores was the most important predictor for both ischemic and hem- orrhagic stroke. Our findings and previous findings together suggest that it is important to include the base- line score of an assessment as a potential predictor in future studies on postintervention outcome prediction. Baseline UL motor function, namely, the BBT and the FMA-UE, were found to be important predictors for achieving the MCID on all target variables. The finding was consistent with existing literature. In chronic stroke, baseline FMA-UE was found to predict postintervention UL motor function in two studies using ML [13, 14]. For studies using traditional statistical analysis, baseline BBT was found to predict postintervention outcomes of activ- ities and participation [37, 38], and FMA-UE was found to predict both UL motor function and activities and participation [25, 36, 39]. Our findings further supported the predictive value of UL motor function for postinter- vention achievement of MCID in the PROMs of activi- ties and participation in chronic stroke. Similar findings were also reported for studies using acute and subacute parameters to predict discharge assessment scores or long-term outcomes [5, 6, 40–43]. The similar findings across disease stages suggested that preintervention UL motor function is an important predictor for postinter- vention outcomes for all stages in stroke and should be included as a potential predictor if available in future pre- dictive studies. The most important predictors Our results showed that the baseline scores of a given PROM were among the important features for classifying responders versus non-responders on that measure. This Chen et al. Journal of NeuroEngineering and Rehabilitation (2023) 20:25 Page 9 of 12 postintervention UL motor function [14] and UL activity [39]. In acute to subacute stroke, age was found to pre- dict the possibility of home discharge after rehabilitation stay [11] and functional outcomes at discharge [6, 44, 45], at 3 months post-stroke [9], and at 6 months post- stroke [10]. Sex has also been previously reported as an important predictor for long-term post-stroke functional outcome [10] and postintervention UL activity [37]. Our results identified only sex and years of education in the lists of predictors with non-zero gains for MAL-QOM. Although the gains were negligibly small at 0.01, indi- cating their minimal relationship with postintervention achievement of MCID in MAL-QOM, the findings were partially in line with previous studies. was similar to findings of predictive studies of UL motor function in chronic stroke using ML [13, 14] and tradi- tional statistical methods [35]. In studies for the acute and subacute stages, similar findings have been reported with measures for motor function and ADL. Iwamoto et al. [36] found that FMA-UE scores at the initiation of inpatient rehabilitation were the most important predictor for identifying participants that would achieve an MCID on the FMA-UE 30 days after treatment. Harari et al. [4] built ML models to predict discharge scores of FIM, Ten-Meter Walk Test, Six-Min- ute Walk Test, and the Berg Balance Scale after inpatient rehabilitation stay; they found that the most impor- tant predictors for these scores were their own scores at admission. In two other studies with patients admitted to inpatient rehabilitation facilities, the discharge Barthel Index scores and improvements were both predicted by the admission Barthel Index scores [5, 6]. p y p Stroke characteristics, i.e., time since stroke, side of hemiplegia, NIHSS scores, and diagnosis (i.e., hemor- rhagic or ischemic) were identified as important predic- tors for one or two target variables. Previous studies also reported that time since stroke predicted functional out- comes in the subacute stage [4, 44] and postintervention UL motor function for the chronic stage [13, 14]. The most important predictors Note that, methodologically, feature selection could be conducted before model construction from the cohort or after model construction for specific models. This study identified the most important predictors a priori from the cohort, instead of post hoc from specific models. This decision took in considerations of the steps adopted by previous studies in stroke rehabilitation [10, 13, 14], clini- cal applications to identify a set of assessments to prior- itize regardless of chosen algorithms, and the reduction of overall complexity of this study. Predictive models and predictors across the four PROMs Demographic and stroke characteristics were fre- quently included as potential predictors in rehabili- tation outcome prediction. For example, in chronic stroke, age was previously reported as a predictor for Despite some overlapping predictors for the four target variables, the four sets of predictors were different. We chose these assessments because they include items for Chen et al. Journal of NeuroEngineering and Rehabilitation (2023) 20:25 Page 10 of 12 Page 10 of 12 different aspects of activities and participation. There is also a hierarchy among these assessments. The MAL assesses the more basic ADL. The NEADL considers mobility and community living activities. The SIS-ADL considers the daily activities of higher complexities, and some of the activities may require the collaboration of other body parts and/or use of instruments. The impor- tant predictors for each target variable likely reflected what each particular assessment tool captures. The find- ings highlight the importance of using different sets of predictors for these ADL assessments and support the use of feature selection to screen for the most relevant and meaningful predictors in future studies. rehabilitation. This study, as a secondary data analysis, did not collect data on all potential predictors, making it impossible to address these predictors. Future studies may investigate the predictive power of a wider range of predictors when investigating postintervention ADL in the stroke population. Finally, ML is characterized by its data-driven nature, and therefore the results of this study, as well as many other studies using ML, may not be readily generalized to data from other facilities or other patient characteristics. However, this study and previous studies have repeatedly confirmed the feasibility of ML in predicting postinter- vention outcomes in the stroke population. Further, some predictors were repeatedly reported and may be impor- tant to consider in future studies, such as UL motor func- tion, selected demographic and stroke characteristics, and baseline scores of assessments used to quantify the outcomes. We recommend that health care facilities develop their own models by taking findings of this and previous studies as references. Among the four PROMs, postintervention achieve- ment of MCID in SIS-ADL appeared to be predicted well across algorithms and numbers of features used. On the contrary, MCID achievement in NEADL required the more complex method, RF, to achieve good prediction performance. Predictive models and predictors across the four PROMs The NEADL concerns mobility and com- munity living activities in an extended context, and may involve aspects not as well captured by the predicting variables we used. Regardless, good prediction perfor- mance was achievable with the combination of a more complex prediction method and predictors that cover a wider range of aspects, such as general stroke sever- ity (NIHSS) and overall impact of the stroke (SIS). These measures include items for cognition, mobility, and emo- tion, among others, that may contribute to the extended aspects of activities and participation. Conclusion In this study, we obtained high accuracies and AUCs using ML to predict postintervention PROMs for activi- ties and participation in chronic stroke, demonstrating the feasibility of ML methods for this research task. We also identified the most important predictors for achiev- ing MCID on these PROMs. Consistent with existing literature, UL motor function, selected demographic and stroke characteristics, and the baseline scores of the PROMs were important predictors across the four PROMs. Individual predictors identified for the PROMs also reflected the characteristics and contexts of the ADL that these assessments capture. The study findings may contribute to precision rehabilitation by providing insights into the identification of patients that are likely to benefit from stroke rehabilitation. Study limitations Th l The major limitation of this study is the limited sam- ple size; however, we have made an effort to minimize model bias and variance that could result from it by using SMOTE, reducing dimensionality through fea- ture selection, and ensuring that the data used to test model performances did not affect model construction. Through these efforts, we were able to construct at least one model with acceptable to excellent metrics for each target variable. In fact, low specificity and/or sensitivity are commonly seen in the literature using ML to predict stroke rehabilitation outcomes with relatively small sam- ple sizes. Abbreviations ADL Activities of daily living AUC Area under the receiver operating characteristic curve BBT Box and Block Test CAHAI Chedoke Arm and Hand Activity Inventory FIM Functional Independence Measure FMA-UE Upper Extremity subscale of the Fugl-Meyer Assessment ICF International Classification of Functioning, Disability and Health KNN K-nearest neighbors LR Logistic regression MAL-AOU Motor Activity Log Amount of Use MAL-QOM Motor Activity Log Quality of Movement MCID Minimal clinically important difference MI Mutual information ML Machine learning NEADL Nottingham Extended Activities of Daily Living NIHSS National Institute of Health Stroke Scale NPV Negative predictive value PPV Positive predictive value Although we would recommend future studies use larger sample sizes, achieving the size of big data in health care is often difficult and/or costly. Future stud- ies may use more advanced techniques to minimize the effects of small sample sizes. f Further, the accurate prediction of postintervention ADL outcomes may be more complex and involve pre- dictors that were not included in this study. For example, nutritional status [49], aphasia [50, 51], and cognition [52] were reported to predict ADL outcomes after stroke Chen et al. Journal of NeuroEngineering and Rehabilitation (2023) 20:25 Page 11 of 12 Page 11 of 12 PROM Patient-reported outcome measure RF Random forest SIS Stroke Impact Scale SIS-ADL Activities of Daily Living domain of the Stroke Impact Scale SMOTE Synthetic Minority Over-sampling Technique SVM Support vector machine UL Upper limb WMFT Wolf Motor Function Test 8. Lin CH, Hsu KC, Johnson KR, Fann YC, Tsai CH, Sun Y, et al. Evaluation of machine learning methods to stroke outcome prediction using a nation‑ wide disease registry. Comput Methods Programs Biomed. 2020;190: 105381. 9. 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J Neuroeng Rehabil. 2020;17:131. g This research was funded in part by the Ministry of Science and Technology (grant numbers MOST-104-2314-B-002-019-MY3, MOST-107-2314-B-002-052, MOST-108-2314-B-002-165-MY3, and MOST-111-2314-B-002-168-MY3) and by the National Health Research Institutes (Grant Numbers NHRI-EX104-10403PI, NHRI-EX105-10403PI, NHRI-EX106-10403PI, NHRI-EX107-10403PI, NHRI-EX109- 10929PI, NHRI-EX110-10929PI, and NHRI-EX111-10929PI). 14. Tozlu C, Edwards D, Boes A, Labar D, Tsagaris KZ, Silverstein J, et al. Machine learning methods predict individual upper-limb motor impair‑ ment following therapy in chronic stroke. Neurorehabil Neural Repair. 2020;34:428–39. Author contributions YWC conceptualized the study, analyzed and interpreted the data, and wrote the manuscript. KcL conceptualized the study, interpreted the data, and supervised the project. YcL collected and validated the data. CJL man‑ aged data collection and project administration. All authors have read and approved the manuscript. 12. Rana S, Luo W, Tran T, Venkatesh S, Talman P, Phan T, et al. Application of machine learning techniques to identify data reliability and factors affect‑ ing outcome after stroke using electronic administrative records. Front Neurol. 2021;12: 670379. Received: 2 June 2022 Accepted: 14 February 2023 Received: 2 June 2022 Accepted: 14 February 2023 20. Duncan PW, Bode RK, Min Lai S, Perera S, Glycine Antagonist in Neu‑ roprotection Americans Investigators. Rasch analysis of a new stroke- specific outcome scale: the Stroke Impact Scale. Arch Phys Med Rehabil. 2003;84:950–63. 21. Hung CS, Lin KC, Chang WY, Huang WC, Chang YJ, Chen CL, et al. Unilateral vs bilateral hybrid approaches for upper limb rehabilitation in chronic stroke: a randomized controlled trial. Arch Phys Med Rehabil. 2019;100:2225–32. Study limitations Th l Machine learning- based three-month outcome prediction in acute ischemic stroke: a single Cerebrovascular-Specialty Hospital Study in South Korea. Diagn Basel Switz. 2021;11:1909. 10. Wang HL, Hsu WY, Lee MH, Weng HH, Chang SW, Yang JT, et al. Automatic machine-learning-based outcome prediction in patients with primary intracerebral hemorrhage. Front Neurol. 2019;10:910. 11. Imura T, Iwamoto Y, Inagawa T, Imada N, Tanaka R, Toda H, et al. Decision tree algorithm identifies stroke patients likely discharge home after rehabilitation using functional and environmental predictors. J Stroke Cerebrovasc Dis. 2021;30: 105636. Ethics approval and consent to participate All participants gave their informed consent before their participation. The study was approved by the Institutional Review Board and Ethics Committee of National Taiwan University and all participating clinical settings. 17. Uswatte G, Taub E, Morris D, Light K, Thompson PA. The Motor Activity Log-28: assessing daily use of the hemiparetic arm after stroke. Neurol‑ ogy. 2006;67:1189–94. 18. Wu CY, Chuang LL, Lin KC, Lee SD, Hong WH. Responsiveness, minimal detectable change, and minimal clinically important difference of the Nottingham Extended Activities of Daily Living Scale in patients with improved performance after stroke rehabilitation. Arch Phys Med Rehabil. 2011;92:1281–7. Competing interests The authors declare that they have no competing interests. 19. Hsueh IP, Huang SL, Chen MH, Jush SD, Hsieh CL. Evaluation of stroke patients with the extended activities of daily living scale in Taiwan. Disabil Rehabil. 2000;22:495–500. Received: 2 June 2022 Accepted: 14 February 2023 Publisher’s Note S i N i 44. Inouye M, Kishi K, Ikeda Y, Takada M, Katoh J, Iwahashi M, et al. Prediction of functional outcome after stroke rehabilitation. Am J Phys Med Rehabil. 2000;79:513–8. 45. Ishiwatari M, Honaga K, Tanuma A, Takakura T, Hatori K, Kurosu A, et al. Trunk impairment as a predictor of activities of daily living in acute stroke. Front Neurol. 2021;12: 665592. • 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: • 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: 46. Bertolin M, Van Patten R, Greif T, Fucetola R. Predicting cognitive function‑ ing, activities of daily living, and participation 6 months after mild to moderate stroke. Arch Clin Neuropsychol. 2018;33:562–76. 46. Bertolin M, Van Patten R, Greif T, Fucetola R. Predicting cognitive function‑ ing, activities of daily living, and participation 6 months after mild to moderate stroke. Arch Clin Neuropsychol. 2018;33:562–76. y 47. Lai SM, Duncan PW, Keighley J. References Lin KC, Fu T, Wu CY, Wang YH, Liu JS, Hsieh CJ, et al. Minimal detectable change and clinically important difference of the Stroke Impact Scale in stroke patients. Neurorehabil Neural Repair. 2010;24:486–92. 7. Heo J, Yoon JG, Park H, Kim YD, Nam HS, Heo JH. Machine learning-based model for prediction of outcomes in acute stroke. Stroke. 2019;50:1263–5. Chen et al. Journal of NeuroEngineering and Rehabilitation (2023) 20:25 Page 12 of 12 27. Imura T, Toda H, Iwamoto Y, Inagawa T, Imada N, Tanaka R, et al. Compari‑ son of supervised machine learning algorithms for classifying home dis‑ charge possibility in convalescent stroke patients: a secondary analysis. J Stroke Cerebrovasc Dis. 2021;30: 106011. 50. Lazar RM, Boehme AK. Aphasia as a predictor of stroke outcome. Curr Neurol Neurosci Rep. 2017;17:83. 51. Gialanella B, Prometti P, Vanoglio F, Comini L, Santoro R. Aphasia and activities of daily living in stroke patients. Eur J Phys Rehabil Med. 2016;52:782–90. 28. Chawla NV, Bowyer KW, Hall LO, Kegelmeyer WP. SMOTE: synthetic minor‑ ity over-sampling technique. J Artif Intell Res. 2002;16:321–57. 52. Gialanella B, Santoro R, Ferlucci C. Predicting outcome after stroke: the role of basic activities of daily living predicting outcome after stroke. Eur J Phys Rehabil Med. 2013;49:629–37. 29. Raschka S, Mirjalili V. Python Machine Learning: Machine Learning and Deep Learning with Python, scikit-learn, and TensorFlow 2. 3rd ed. Birmingham: Packt Publishing Ltd; 2019. 30. R Core Team. R: A language and environment for statistical computing [Internet]. R Foundation for Statistical Computing, Vienna, Austria. https:// www.R-project.org/ Publisher’s Note S i N i Springer Nature remains neutral with regard to jurisdictional claims in pub‑ lished maps and institutional affiliations. 31. Van Rossum G, Drake F. Python 3 Reference Manual. Scotts Valley: Create Space; 2009. 32. Pedregosa F, Varoquaux G, Gramfort A, et al. Scikit-learn: machine learning in Python. J Mach Learn Res. 2011;12:2825–30. 33. Lemaître G, Nogueira F, Aridas CK. Imbalanced-learn: A Python toolbox to tackle the curse of imbalanced datasets in machine learning. J Mach Learn Res. 2017;18:1–5. 34. Hosmer DW, Lemeshow S, Sturdivant RX. Assessing the Fit of the Model. Applied logistic regression. Hoboken: Wiley; 2013. p. 153–225. 35. Lee YY, Hsieh YW, Wu CY, Lin KC, Chen CK. Proximal Fugl-Meyer Assess‑ ment scores predict clinically important upper limb improvement after 3 stroke rehabilitative interventions. Arch Phys Med Rehabil. 2015;96:2137–44. 36. Iwamoto Y, Imura T, Tanaka R, Mitsutake T, Jung H, Suzukawa T, et al. Clinical prediction rule for identifying the stroke patients who will obtain clinically important improvement of upper limb motor function by robot- assisted upper limb. J Stroke Cerebrovasc Dis. 2022;31: 106517. 37. Hsieh YW, Lin KC, Wu CY, Lien HY, Chen JL, Chen CC, et al. Predicting clini‑ cally significant changes in motor and functional outcomes after robot- assisted stroke rehabilitation. Arch Phys Med Rehabil. 2014;95:316–21. 38. Huang PC, Hsieh YW, Wang CM, Wu CY, Huang SC, Lin KC. Predictors of motor, daily function, and quality-of-life improvements after upper- extremity robot-assisted rehabilitation in stroke. Am J Occup Ther. 2014;68:325–33. 39. Park SW, Wolf SL, Blanton S, Winstein C, Nichols-Larsen DS. The EXCITE Trial: Predicting a clinically meaningful motor activity log outcome. Neurorehabil Neural Repair. 2008;22:486–93. 40. Gebruers N, Truijen S, Engelborghs S, Dedeyn PP. Prediction of upper limb recovery, general disability, and rehabilitation status by activity measure‑ ments assessed by accelerometers or the Fugl-Meyer score in acute stroke. Am J Phys Med Rehabil. 2014;93:245–52. 41. Shelton FD, Volpe BT, Reding M. Motor impairment as a predictor of functional recovery and guide to rehabilitation treatment after stroke. Neurorehabil Neural Repair. 2001;15:229–37. 42. Chen CM, Tsai CC, Chung CY, Chen CL, Wu KP, Chen HC. Potential predictors for health-related quality of life in stroke patients undergoing inpatient rehabilitation. Health Qual Life Outcomes. 2015;13:118.f 43. Franceschini M, Goffredo M, Pournajaf S, Paravati S, Agosti M, De Pisi F, et al. Predictors of activities of daily living outcomes after upper limb robot-assisted therapy in subacute stroke patients. PLoS ONE. 2018;13: e0193235. • 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 S i N i Prediction of functional outcome after stroke: comparison of the Orpington Prognostic Scale and the NIH Stroke Scale. Stroke. 1998;29:1838–42. 48. Saxena SK, Ng T, Yong D, Fong N, Koh G. Functional outcomes in inpatient rehabilitative care of stroke patients: predictive factors and the effect of therapy intensity. Qual Prim Care. 2006;14:0–0. 48. Saxena SK, Ng T, Yong D, Fong N, Koh G. Functional outcomes in inpatient rehabilitative care of stroke patients: predictive factors and the effect of therapy intensity. Qual Prim Care. 2006;14:0–0. 49. Lee YC, Chiu EC. Nutritional status as a predictor of comprehensive activities of daily living function and quality of life in patients with stroke. NeuroRehabilitation. 2021;48:337–43. 49. Lee YC, Chiu EC. Nutritional status as a predictor of comprehensive activities of daily living function and quality of life in patients with stroke. NeuroRehabilitation. 2021;48:337–43.
https://openalex.org/W3000094516	https://nottingham-repository.worktribe.com/preview/3656159/BAME_MAS_authors_finalversion.pdf	English	null	Ethnic disparity in access to the memory assessment service between South Asian and white British older adults in the United Kingdom: A cohort study	International journal of geriatric psychiatry	2,020	cc-by	9,269	Ethnic disparity in access to the Memory Assessment Service between South Asian and White British older adults in the UK: a cohort study Giulia Ogliaria,b, Zoë Turnerb, Javid Khaliquec, Adam L. Gordona,d,e,f, John R.F. Gladmand,e,f, Neil H. Chadbornd,e a. University Hospitals of Derby and Burton NHS Foundation Trust, Derby, Derbyshire, United Kingdom b. Nottinghamshire Healthcare NHS Foundation Trust, Nottingham, Nottinghamshire, United Kingdom c. Independent community engagement consultant d. University of Nottingham, School of Medicine, Nottingham, United Kingdom e. Collaboration for Leadership in Applied Health Research and Care (CLAHRC) East Midlands, United Kingdom f. NIHR Nottingham Biomedical Research Centre, Nottingham, Nottinghamshire, United Kingdom Corresponding Author: Dr Neil H. Chadborn; Institute of Mental Health, Triumph Road, University of Nottingham, Nottingham, UK, NG7 2TU. Neil.Chadborn@nottingham.ac.uk g c. Independent community engagement consultant d. University of Nottingham, School of Medicine, Nottingham, United Kingdom e. Collaboration for Leadership in Applied Health Research and Care (CLAHRC) East Midlands, United Kingdom f. NIHR Nottingham Biomedical Research Centre, Nottingham, Nottinghamshire, United Kingdom Corresponding Author: Dr Neil H. Chadborn; Institute of Mental Health, Triumph Road, University of Nottingham, Nottingham, UK, NG7 2TU. Neil.Chadborn@nottingham.ac.uk Acknowledgements Jeff Day, Julie Aicha, Karen Hampson, Lyn Williams, members of the community at ICCA, Pakistani Centre Nottingham. This study is funded by the National Institute for Health Research (NIHR) Collaboration for Leadership in Applied Health Research and Care East Midlands (CLAHRC EM). The authors declare no conflicts of interest. The data that support the findings of this study are not publicly available due to privacy or ethical restrictions. Evidence Search: South Asians and dementia (LS011). Lindsay Snell (2019). Derby, UK: Evidence Search: South Asians and dementia (LS011). Lindsay Snell (2019). Derby, UK: University Hospitals of Derby and Burton NHS Foundation Trust Library and Knowledge Service. Ethnic disparity in access to the Memory Assessment Service between South Asian and White British older adults in the UK: a cohort study Background: Equality of access to memory assessment services by older adults from ethnic minorities is both an ethical imperative and a public health priority. Objective: To investigate whether timeliness of access to memory assessment service differs between older people of White British and South Asian ethnicity. Design: Longitudinal cohort. Setting: Nottingham Memory Study; outpatient secondary mental healthcare. ubjects: Our cohort comprised 3,654 White British and 32 South Asian older outpatient Methods: The criterion for timely access to memory assessment service was set at 90 days from referral. Relationships between ethnicity and likelihood of timely access to memory assessment service were analysed using binary logistic regression. Analyses were adjusted for socio-demographic factors, deprivation and previous access to rapid response mental health services. Results: Among White British outpatients, 2,272 people (62.2%) achieved timely access to memory assessment service. Among South Asian outpatients, fourteen people (43.8%) achieved timely access to memory assessment service. After full adjustment, South Asian outpatients had a 0.47-fold reduced likelihood of timely access, compared to White British outpatients (odds ratio 0.47, 95% confidence interval 0.23-0.95, p-value=0.035). The difference became non-significant when restricting analyses to outpatients reporting British nationality or English as first language. Older age, lower index of deprivation and previous access to rapid response mental health services were associated with reduced likelihood of timely access, while gender was not. Conclusions: In a UK mental healthcare service, older South Asian outpatients are less likely to access dementia diagnostic services in a timely way, compared to White British outpatients. Keywords: memory assessment services, dementia, healthcare disparities, barriers to mental healthcare, South Asian ethnicity, gender differences, aged, outpatients, cohort study. Keypoints: yp Older South Asian outpatients are less likely to achieve timely access to dementia services, once referred, compared to those White British in the UK. Older South Asian outpatients are less likely to achieve timely access to dementia services, once referred, compared to those White British in the UK. No ethnic gap in timely access to dementia services is found between White British and South Asian with British nationality or English as first language. Older age, lower deprivation and previous access to rapid response mental health services are associated with reduced likelihood of timely access to dementia services. The estimated referral rate to dementia services is lowest among South Asian older women. No ethnic gap in timely access to dementia services is found between White British and South Asian with British nationality or English as first language. y g g g Older age, lower deprivation and previous access to rapid response mental health services are associated with reduced likelihood of timely access to dementia services. Older age, lower deprivation and previous access to rapid response mental health services are associated with reduced likelihood of timely access to dementia services. y The estimated referral rate to dementia services is lowest among South Asian older women. The estimated referral rate to dementia services is lowest among South Asian older wom Introduction In many Western countries, the prevalence of dementia in Black, Asian and Minority Ethnic (BAME) groups is expected to increase as a result of increasing cultural diversity and population ageing1-5. In the UK, the South Asian minority – the largest BAME minority - accounts for about 7% of the total population and is steadily expanding and ageing3. As a result, the number of South Asian older adults with dementia is predicted to substantially and rapidly increase. Yet, South Asian older adults may face disparities in accessing dementia diagnostic services, compared to those White British leading to late or missed diagnosis of dementia6, 7. Equal access to services and timely recognition of dementia in South Asian older adults are policy goals8, 9. Timely diagnosis is essential for getting support, optimising safety and making choices about future care while the person with dementia retains the ability to participate in decision-making10, 11. Whereas higher deprivation is a barrier to dementia services and treatment in other Western countries12-13, data from the UK are conflicting14-16 and not detailing the South Asian minority. Qualitative literature has shown that barriers to timely diagnosis of dementia in older South Asian adults may include language, health beliefs, carers’ attitudes, stigma and lack of cross-cultural adaptation of services17-20. In particular, South Asian carers tend to delay help-seeking until a crisis occurs, due to attributing the symptoms of dementia to normal ageing or other physical or psychological causes, negative beliefs about psychiatry and sense of familial responsibility17. Knowledge about dementia may facilitate help-seeking but few campaigns target ethnic minorities21, 22. Furthermore, South Asians may seek help in primary care17 but further barriers may then arise in accessing secondary care. Quantitative studies exploring the disparities that South Asians may face after referral to memory assessment services are lacking. Our quantitative study aims to investigate whether older adults of South Asian ethnicity are less likely to achieve timely access to the memory assessment service, once referred, compared to older adults of White British ethnicity in the Nottingham Memory Study. A secondary aim is to explore whether likelihood of timely access may be modulated by age, gender, deprivation and previous access to rapid response mental health services. Nottinghamshire In Nottinghamshire, most of the resident population reports White British ethnicity (92.6%), while 2.9% reports Irish, Gypsy or other White ethnicity, 1.4% Mixed, 2.2% Asian / Asian British, 0.6% Black / Black British and 0.3% are from Other ethnic groups23, 24. Among the Asians, two thirds are South Asians (Indian, Pakistani or Bangladeshi)23. South Asians include both first-generation immigrants and UK-born people. About 2.8% of the population of Nottinghamshire was born in non- UK, non-EU countries23, 24. In Nottingham, 5.3% of older adults aged 65 years and over do not have English as first language; in particular, half of these are non-proficient in English24. Nottingham has been amongst the most deprived cities in England, over the last decade25, 26. Study design and participants Nottinghamshire Healthcare NHS Foundation Trust is a large secondary mental healthcare provider serving the catchment area of Nottinghamshire, UK, with around 800,000 residents. Older adults with suspected dementia are referred to its memory assessment service as outpatients, mainly by general practitioners. Access to memory assessment service is covered by the NHS and free of charge at the point of delivery. An electronic record is routinely created for each outpatient who is referred to the service. In this record, information on socio-demographic data is automatically entered at referral; data on age, gender, self-reported ethnicity, first language, nationality and domicile are recorded by the administrators, in specific mandatory fields, based on standardized codes. The record is then updated at every contact of the outpatient with the service, by recording all booked, attended, missed or cancelled visits. The dates of contacts as well as access to the other services of the Trust are recorded. We designed the Nottingham Memory Study, an outpatient longitudinal cohort study, with the primary aim to explore disparities in access for South Asian outpatients to the memory assessment service. We analysed all anonymised electronic records of outpatients who were referred as new consecutive outpatients to the memory assessment service of Nottinghamshire Healthcare NHS Foundation Trust, between 3rd March 2014 and 17th August 2018 (n = 3,819). We excluded those younger than 65 years at referral (n = 7) or of unknown gender (n = 1). Nottinghamshire Therefore, the Nottingham Memory Study is an outpatient longitudinal cohort study of 3,811 men and women aged 65 years and older who were referred as new consecutive outpatients to the memory assessment service of Nottinghamshire Healthcare NHS Foundation Trust between 3rd March 2014 and 17th August 2018. The Nottingham Memory Study is a one-wave cohort, with complete data on age and gender. In the total cohort, ethnicity was recorded in 3,781 (99.2%) outpatients, at referral, based on 2011 Census categories23. In the total cohort, most outpatients were White British (n = 3,654, 95.9%), while Irish and other White ethnicities accounted for 1.3%, Mixed ethnicity for 0.2%, all Asian / Asian British for 1%, all Black / Black British for 0.4% and Other ethnic group for 0.2%. Among the Asians, we grouped outpatients of Indian, Pakistani and Bangladeshi ethnicity as South Asians3. The current study includes 3,654 and 32 older adults of White British and South Asian ethnicity, respectively. Figure 1 shows the flow-chart of study inclusion criteria. Demographic and clinical characteristics Demographic and clinical characteristics We retrieved data on age, gender, marital status, nationality and first language, as recorded in the electronic records, at referral. We categorized nationality as British, non-British or unknown; we categorized first language as English, non-English or unknown. We also retrieved data on referral to Rapid Response Liaison Psychiatry, prior to referral to memory assessment service, for each outpatient. We estimated deprivation by the Index of Multiple Deprivation decile, which we extrapolated from postcodes25. The Index of Multiple Deprivation is the official measure of relative deprivation for small areas in England. It ranks every small area in England from 1 (most deprived area) to 32,844 (least deprived area)25. Deciles are calculated by ranking all small areas from most deprived to least deprived and dividing them into ten equal groups25. Therefore, the deciles range from the first, which includes the most deprived 10 percent of small areas nationally, to the tenth, which includes the least deprived 10 percent of small areas nationally25. Index of Multiple Deprivation is an area-level index, where higher values indicate lower deprivation25. Timely access On 21st December 2018, we retrospectively collected data on access to the memory assessment service, for each outpatient, from referral. By collecting data on 21st December 2018, we had a minimum follow-up of 120 days from referral for each outpatient. We calculated the time to access the memory assessment service as the time from referral to first attended visit. We defined as “timely access” the access within 90 days from referral. Ninety days is a timeframe used by the NHS in the context of long-term complex health needs27. Statistical analysis Statistical analysis We reported the characteristics of the study outpatients as number (percentages) for categorical variables and as mean (standard deviation, SD) for age, which we used as a continuous variable. Age was normally distributed in our study population. We plotted and visually inspected the distribution of the Index of Multiple Deprivation deciles in the study population, in White British and South Asian outpatients, respectively (Supplementary Figure 1); as these were not normally distributed, we reported the median (interquartile range, IQR) of the Index of Multiple Deprivation deciles. Differences in characteristics at referral between White British and South Asian outpatients were assessed using chi-square test for categorical variables, student’s T-test for mean age and Mann- Whitney U test for median Index of Multiple Deprivation. Moreover, we tested for gender-differences among White British and South Asian outpatients, respectively, in characteristics at referral. Moreover, we tested for gender-differences among White British and South Asian outpatients, respectively, in characteristics at referral. We estimated the annual rate of referral to memory assessment service for men and women aged 65 years and older, of South Asian and White British ethnicity, based on the 2011 Census23. We calculated the time to access the memory assessment service for each outpatient who had an attended visit during follow-up. We dichotomized the outpatients into 1) those who achieved access to the memory assessment service within 90 days and 2) those who either accessed the memory assessment service later than 90 days from referral or never accessed it. Binary logistic regression models were used to assess the association between ethnicity and likelihood of being assessed by memory assessment service within 90 days from referral (described in this study as “timely access”). The independent variable was ethnicity and the reference category was the White British ethnicity. We performed our analyses in three steps. Timely access In the first step, crude analyses were performed (Model 0). In the second step, we adjusted for age and gender (Model 1). Finally, analyses were further adjusted for Index of Multiple Deprivation decile and previous referral to the Rapid Response Liaison Psychiatry (Model 2). To explore potential barriers, we performed sensitivity analyses by including only 1) participants who reported British nationality and 2) participants who reported English as their first language, respectively. In these analyses, we excluded 1) participants with either non-British or unknown nationality and 2) participants with a first language other than English or unknown, respectively. Furthermore, to explore gender-differences, we repeated all analyses after stratifying by gender. All analyses were performed using SPSS software (version 20.0.0). Findings Characteristics of study population The total study population included 3,654 White British and 32 South Asian older outpatients (Figure 1). Table 1 shows the characteristics of the study population at referral to the memory assessment service. In the total population, mean age was 80.1 (SD 6.9) years and 57.4% of participants were women. South Asian outpatients were younger, more likely to be men and live in a less deprived area compared to those who were White British (Table 1 and Supplementary Figure 1). South Asian outpatients were less likely to report having British nationality and English as their first language compared to those who were White British (Table 1). No South Asian outpatient was known to Rapid Response Liaison Psychiatry while 10.3% of those White British were (Table 1). Gender-differences among South Asian and White British outpatients are detailed in Table 2. Among the South Asian outpatients, men were more likely to be married or in a civil partnership, to report British nationality and English as their first language, compared to women. Estimates of referral rates The estimated rate of referral to memory assessment service was 4.6 and 2.3 referrals per year for every 1,000 men and women, respectively, aged 65 years and older, of South Asian ethnicity. It was 4.6 and 5 referrals per year for every 1,000 men and women, respectively, aged 65 years and older, of White British ethnicity. Timely access and ethnicity Timely access and ethnicity Timely access and ethnicity In the total population, 2,286 (62.0%) outpatients were seen in memory assessment service within 90 days from referral. Among all those who were seen within 90 days, mean age was 79.7 (SD 6.8) and median Index of Multiple Deprivation decile 6 (IQR 4; 8); among all those who were not seen within 90 days, mean age was 80.9 (SD 7.0) and median Index of Multiple Deprivation decile 7 (IQR 4; 9). For White British, 2,272 (62.2%) were seen within 90 days, whereas for South Asian 14 (43.8%) were seen within 90 days. South Asian ethnicity was associated with a reduced likelihood of timely access to memory assessment service (Table 3). South Asian outpatients had a reduced likelihood to access memory assessment service within 90 days from referral compared to those White British (odds ratio (OR) 0.47, 95% Confidence Interval (CI) 0.24-0.95, p-value = 0.037, crude model). This association remained consistent after full adjustment (OR 0.47, 95% CI 0.23-0.95, p-value=0.035, Model 2). Timely access and age, deprivation and previous referral to the rapid response services Timely access and age, deprivation and previous referral to the rapid response services In the fully adjusted model, older age, lower deprivation and previous referral to the Rapid Response Liaison Psychiatry were associated with reduced likelihood of timely access to memory assessment service (all p-values <0.001), while gender was not (data not shown). Supplementary Figure 2 illustrates that the proportion of outpatients achieving timely access gradually declined with increasing age (p-value < 0.001). Supplementary Figure 3 and 4 show that likelihood of timely access to the memory assessment service was lowest in the outpatients in the ninth and tenth Index of Multiple Deprivation deciles – those from the least deprived areas. In particular, 55.1% and 44.5% of the outpatients in the ninth and tenth decile – those from the least deprived areas – achieved timely access versus 63.2% to 72.4% of the outpatients in the deciles from first to eight – those from the most deprived areas (p-value < 0.001). Among the outpatients not previously referred to the Rapid Response Liaison Psychiatry, 2,093 (63.3%) achieved timely access to the memory assessment service; among those previously referred to the Rapid Response Liaison Psychiatry, 193 (51.1%) achieved timely access (Supplementary Figure 5). Timely access and ethnicity The binary logistic regression analyses showed that the outpatients with previous referral to the Rapid Response Liaison Psychiatry had a 0.59-fold reduced likelihood of timely access, compared to those outpatients with no previous referral, after full adjustment (OR 0.59, 95% CI 0.48 – 0.74, p-value < 0.001). compared to those outpatients with no previous referral, after full adjustment (OR 0.59, 95% CI 0.48 – 0.74, p-value < 0.001). Similar findings were observed for the outcome of accessing memory assessment service within 120 days (Supplementary Table 1). Similar findings were observed for the outcome of accessing memory assessment service within 120 days (Supplementary Table 1). Nationality, language and gender No difference in timely access to memory assessment service by ethnicity was found when restricting the analyses to participants of British nationality (n=3,057) (Table 4), or to those with English as their first language (n=2,589), (Table 5).In gender-stratified analyses, the ethnic gap in likelihood of timely access to memory assessment service was not significant in women (Supplementary Table 2), while the ethnic gap tended to be significant in men (Supplementary Table 3). Discussion In our cohort study in a secondary mental healthcare setting, about two thirds of all outpatients achieved timely access to memory assessment service (within 90 days from referral). South Asian outpatients were less likely to achieve timely access to memory assessment service compared to White British, even after adjustment for covariates. Furthermore, factors associated with reduced likelihood of timely access to memory assessment service were older age, being known to rapid response mental health services and lower index of deprivation. Our findings of ethnic disparities in timely access to dementia diagnostic services, after referral, is complementary to other studies suggesting that older adults from ethnic minorities underuse dementia services, present to dementia services later in the illness and are globally less likely to receive diagnosis and treatment for dementia in Western countries6, 28. Previous literature has highlighted under-recognition of dementia in South Asian older adults in both the community and acute hospital settings in the UK and US29-32. In the THIN database, a nationally representative UK database of primary care electronic health records, Asian women were 18% less likely and Asian men 12% less likely to have a new dementia diagnosis, compared with White adults29. Likewise, in a large Californian insurance database, Asian-American had lower age-adjusted dementia incidence rates compared to White and African-American adults31; among Asian-Americans, South Asians had the lowest rate32. The true prevalence of dementia in South Asians living in Western countries remains unknown. We cannot infer whether lower rates of dementia diagnosis reflect a truly lower prevalence of dementia or a higher proportion of undiagnosed dementia. Yet, South Asian older adults have a high burden of cardiovascular and psychosocial risks factors for dementia. South Asians adults have a greater burden of diabetes33-35 and coronary heart disease36-37 than White adults. Lifetime social adversity, which contributes to cognitive decline, may disproportionately affect ethnic minorities and migrants38. All these may suggest a high risk of dementia in South Asians and raise concerns on undiagnosed dementia. In our study, South Asians appeared under-referred. Likewise, older adults from non-English- speaking backgrounds were under-referred to a memory clinic in Melbourne, Australia, in the 1990s39. In contrast, non-White older adults were equally referred to a memory clinic, compared to White adults, in London, in the early 2000s40. Moreover, African-Caribbean outpatients were well represented in a London memory service, but possibly presented at late stages of dementia41. Discussion However, London is the most ethnically diverse area across England3, 23 and may be more inclusive of ethnic minorities. The novel finding of our study is that South Asian older adults face a disparity in timely access to the memory assessment service, after referral, compared to those White British. This disparity is independent of age, gender, deprivation and previous referral to rapid response services. The dementia diagnostic pathway comprises multiple stages, settings and barriers. Previous literature has focused on awareness and recognition of dementia in South Asians in the community17, 21-22, primary care29 and acute care settings30. We explored the ethnic gap in that tract of the dementia diagnostic pathway from referral to first access to outpatient secondary mental healthcare services. We speculate that many outpatients were identified as having memory issues by their primary care or other medical practitioners, during a consultation for physical or mental health symptoms other than dementia or not attributed by the outpatients to dementia – outpatients were not seeking help for dementia. Although a referral to dementia services requires the consent, the outpatients and their caregivers may not fully understand its relevance or face barriers to attendance. These barriers could include intercurrent acute physical or mental illness, hospitalisation and death; these equally affect White British and South Asian outpatients. Further barriers specific to access to secondary care, compared to primary care, may be: unfamiliarity with the service; a complex booking system based on letters written in English; the need to be accompanied by a caregiver; or the stigma associated with mental healthcare hospitals. The latter may disproportionately affect ethnic minorities, as suggested by an Australian study on Asian caregivers42. Interestingly, in our study, outpatients with a previous referral to the rapid response psychiatric services had subsequent reduced likelihood of timely access to the memory assessment services. The referral to the rapid response services may have been triggered by delirium, which is associated with physical and mental co-morbidities and increased risk of hospitalisation, institutionalisation and death43. Therefore, those outpatients with a previous referral to the rapid response services may have been admitted to hospital or institutions, died, or were too frail to attend the services. Likewise, older age was associated with reduced likelihood of access. South Asian older adults in the UK are a heterogeneous population, with diverse immigration histories, cultural and linguistic backgrounds and socioeconomic status. Discussion Of note, the ethnic gap became null when restricting to outpatients with British nationality. Unfortunately, the number of South Asian outpatients with British nationality or English as first language was very small and our sensitivity analyses were likely underpowered. We hypothesised that British nationality and English as first language may reflect a longer residence in the UK, a British education and a higher level of integration into British society. Previous studies have highlighted language barriers44-49. Strengths and limitations To our knowledge, our study is the first quantitative study to show that South Asian older outpatients experience delay in access to dementia diagnostic services, compared to White British, after referral to secondary services. Strengths of our study are the large total cohort sample size, the longitudinal cohort design and the use of real-life, routinely collected data, which could facilitate its replication in other secondary mental healthcare settings in the UK. In our cohort, data on ethnicity were systematically collected. All ethnic minorities were represented in small numbers and in lower proportions compared to those in the general population. This supports the hypothesis of under- referral of older adults from ethnic minorities, in line with previous literature6, 7, 17. Our study was sufficiently powered for our main analyses on ethnic-related disparities in timely access. Yet, the small number of South Asian older adults is a major limitation, and the sensitivity analyses in the sample with British nationality or English language are likely underpowered. In particular, the under-recording of the first language limits us in drawing conclusions on the role of the language barrier. Access within ninety days from referral may be deemed not timely; yet, our data showed that about one third of all patients did not achieve this outcome. A further limitation is that the true prevalence of diagnosed and undiagnosed dementia by ethnicity in the community remains unknown. Nevertheless, our study provides important new insights about referral and access to secondary mental healthcare under universal health coverage. Implications References 1. Prince M, Wimo A, Guerchet M, at al. World Alzheimer Report 2015. The global impact of dementia: An analysis of prevalence, incidence, cost & trends. London: Alzheimer’s disease International: London; 2015. 2. US Census Bureau. 2017 National Population Projections Datasets. Projections for the United States: 2017 to 2060. Available at: https://www.census.gov/data/datasets/2017/demo/popproj/2017-popproj.html (accessed: 25th August 2019). 3. Office for National Statistics. Ethnicity and National Identity in England and Wales: 2011. Available at: https://www.ons.gov.uk/peoplepopulationandcommunity (accessed: 25th August 2019) 4. Parliament of Australia. Joanne Simon-Davies. Population and migration statistics in Australia. Research Paper Series. 7 December 2018. Available at: https://parlinfo.aph.gov.au (accessed: 25th August 2019). 5. House of Commons. All-Party Parliamentary Group on Dementia. Dementia does not discriminate. The experiences of black, Asian and minority ethnic communities. London, UK 2013. 6. Cooper C, Tandy AR, Balamurali TB, Livingston G. A systematic review and meta-analysis of ethnic differences in use of dementia treatment, care, and research. Am J Geriatr Psychiatry. 2010;18(3):193-203. 7. Mukadam N, Lewis G, Mueller C, et al. Ethnic differences in cognition and age in people diagnosed with dementia: A study of electronic health records in two large mental healthcare providers. Int J Geriatr Psychiatry. 2019;34(3):504-510. 8. National Collaborating Centre for Mental Health. 2018. “The Dementia Care Pathway. Full implementation guidance.” pp. 1-42. Royal College of Psychiatrists, London, UK. 9. NHS England, Older People’s Mental Health and Dementia. 2017. Implementation guide and resource pack for dementia care. pp. 1-14. NHS England, Leeds, UK. 10. Livingston G, Sommerlad A, Orgeta V, et al. Dementia prevention, intervention, and care. Lancet. 2017;390:2673–734. 11. Livingston G, Leavey G, Manela M, et al. Making decisions for people with dementia who lack capacity: qualitative study of family carers in UK. BMJ. 2010;341:c4184. 12. Qian W, Schweizer TA, Fischer CE. Impact of socioeconomic status on initial clinical presentation to a memory disorders clinic. Int Psychogeriatr. 2014;26(4):597-603. 13. Zilkens RR, Duke J, Horner B, Semmens JB, Bruce DG. Australian population trends and disparities in cholinesterase inhibitor use, 2003 to 2010. Alzheimers Dement. 2014;10(3):310-8. 14. Cooper C, Blanchard M, Selwood A, Livingston G. Antidementia drugs: prescription by level of cognitive impairment or by socio-economic group? Aging Ment Health. 2010;14(1):85-9. 15. Cooper C, Lodwick R, Walters K, Raine R, Manthorpe J, Iliffe S, Petersen I. Inequalities in receipt of mental and physical healthcare in people with dementia in the UK. Age Ageing. 2017 May;46(3):393-400. 16. Implications Future research should elucidate the causes of delayed or missed access to memory assessment services, after referral. Qualitative interviews with outpatients who did not achieve timely access (or their caregivers) may provide insight. Quality improvement projects should target women from ethnic minorities. In conclusion, our study shows that South Asian older outpatients are less likely to achieve timely access to secondary healthcare dementia diagnostic services, after referral, compared to those White British in the UK. Our findings may be relevant to other Western countries where South Asian communities are growing and ageing. References Cooper C, Lodwick R, Walters K, Raine R, Manthorpe J, Iliffe S, Petersen I. Observational cohort study: deprivation and access to anti-dementia drugs in the UK. Age Ageing. 2016 Jan;45(1):148- 54. 17. Mukadam N, Cooper C, Basit B, Livingston G. Why do ethnic elders present later to UK dementia services? A qualitative study. Int Psychogeriatr. 2011;23(7):1070-7. 18. Lawrence V, Murray J, Samsi K, Banerjee S. Attitudes and support needs of Black Caribbean, south Asian and White British carers of people with dementia in the UK. Br J Psychiatry. 2008;193(3):240-6. 19. Bhattacharyya S, Benbow SM. Mental health services for black and minority ethnic elders in the United Kingdom: a systematic review of innovative practice with service provision and policy implications. Int Psychogeriatr. 2013;25(3):359-73. 20. Lawrence V, Samsi K, Banerjee S, Morgan C, Murray J. Threat to valued elements of life: the experience of dementia across three ethnic groups. Gerontologist. 2011;51(1):39-50. 21. Mukadam N, Waugh A, Cooper C, Livingston G. What would encourage help-seeking for memory problems among UK-based South Asians? A qualitative study. BMJ Open. 2015;5(9):e007990. 22. Mukadam N, Cooper C, Livingston G. Improving access to dementia services for people from minority ethnic groups. Curr Opin Psychiatry. 2013;26(4):409-14. 23. Office for National Statistics. 2011 Census – Population and Household Estimates for England and Wales. Newport: Office for National Statistics, 2012. 24. Nomis. Official labour market statistics. https://www.nomisweb.co.uk/census/2011 (accessed: 17th November 2019). 25. Department for Communities and Local Government. English Indices of Deprivation 2015. Available at: http://www.gov.uk/government/statistics/english-indices-of-deprivation-2015 (accessed: 25th August 2019). 26. Department for Communities and Local Government. English Indices of Deprivation 2019. Available at: https://www.gov.uk/government/statistics/english-indices-of-deprivation-2019 (accessed: 17th November 2019). 27. https://www.nhs.uk/conditions/social-care-and-support-guide/money-work-and-benefits/nhs- continuing-healthcare/ (accessed: 3rd December 2019). 28. Nielsen TR, Vogel A, Phung TK, Gade A, Waldemar G. Over- and under-diagnosis of dementia in ethnic minorities: a nationwide register-based study. Int J Geriatr Psychiatry. 2011;26(11):1128-35. 29. Pham TM, Petersen I, Walters K, et al. Trends in dementia diagnosis rates in UK ethnic groups: analysis of UK primary care data. Clin Epidemiol. 2018;10:949-960. 30. Sommerlad A, Perera G, Singh-Manoux A, et al. Accuracy of general hospital dementia diagnoses in England: Sensitivity, specificity, and predictors of diagnostic accuracy 2008-2016. Alzheimers Dement. 2018;14(7):933-943. 31. Mayeda ER, Glymour MM, Quesenberry CP, Whitmer RA. Inequalities in dementia incidence between six racial and ethnic groups over 14 years. Alzheimers Dement. 2016 Mar;12(3):216-24. 32. Mayeda ER, Glymour MM, Quesenberry CP Jr, Whitmer RA. References Heterogeneity in 14-year Dementia Incidence Between Asian American Subgroups. Alzheimer Dis Assoc Disord. 2017 Jul- Sep;31(3):181-186. 33. Anand SS, Yusuf S, Vuksan V, et al. Differences in risk factors, atherosclerosis, and cardiovascular disease between ethnic groups in Canada: the Study of Health Assessment and Risk in Ethnic groups (SHARE). Lancet. 2000 July;356(9226):279-84. 34. Kanaya AM, Herrington D, Vittinghoff E, et al. Understanding the high prevalence of diabetes in U.S. south Asians compared with four racial/ethnic groups: the MASALA and MESA studies. Diabetes Care. 2014 Jun;37(6):1621-8. 35. Tillin T, Hughes AD, Godsland IF, et al. Insulin resistance and truncal obesity as important determinants of the greater incidence of diabetes in Indian Asians and African Caribbeans compared with Europeans: the Southall And Brent REvisited (SABRE) cohort. Diabetes Care. 2013 Feb;36(2):383-93. 36. Palaniappan L, Wang Y, Fortmann SP. Coronary heart disease mortality for six ethnic groups in California, 1990-2000. Ann Epidemiol. 2004 Aug;14(7):499-506. 37. Palaniappan LP, Araneta MR, Assimes TL, et al. Call to action: cardiovascular disease in Asian Americans: a science advisory from the American Heart Association. Circulation. 2010 Sep;122(12):1242-52. 38. Glymour MM, Manly JJ. Lifecourse social conditions and racial and ethnic patterns of cognitive aging. Neuropsychol Rev. 2008 Sep;18(3):223-54. 39. LoGiudice D, Hassett A, Cook R, Flicker L, Ames D. Equity of access to a memory clinic in Melbourne? Non-English speaking background attenders are more severely demented and have increased rates of psychiatric disorders. Int J Geriatr Psychiatry. 2001;16(3):327-34. 40. Banerjee S, Willis R, Matthews D, et al. Improving the quality of care for mild to moderate dementia: an evaluation of the Croydon Memory Service Model. Int J Geriatr Psychiatry. 2007;22(8):782-8. 41. Tuerk R, Sauer J. Dementia in a Black and minority ethnic population: characteristics of presentation to an inner London memory service. BJPsych Bull. 2015 Aug;39(4):162-6. 42. Haralambous B, Dow B, Tinney J, et al. Help seeking in older Asian people with dementia in Melbourne: using the Cultural Exchange Model to explore barriers and enablers. J Cross Cult Gerontol. 2014 Mar;29(1):69-86. 43. Witlox J, Eurelings LS, de Jonghe JF, et al. Delirium in elderly patients and the risk of postdischarge mortality, institutionalization, and dementia: a meta-analysis. JAMA. 2010 Jul;304(4):443-51. 44. Ayalon L, Areán PA. Knowledge of Alzheimer's disease in four ethnic groups of older adults. Int J Geriatr Psychiatry. 2004;19(1):51-7. 45. Giebel CM, Zubair M, Jolley D, et al. References South Asian older adults with memory impairment: improving assessment and access to dementia care. Int J Geriatr Psychiatry. 2015;30(4):345-56. 46. Nielsen TR, Vogel A, Riepe MW, et al. Assessment of dementia in ethnic minority patients in Europe: a European Alzheimer's Disease Consortium survey. Int Psychogeriatr. 2011;23(1):86- 95. 47. Parveen S, Peltier C, Oyebode JR. Perceptions of dementia and use of services in minority ethnic communities: a scoping exercise. Health Soc Care Community. 2017;25(2):734-742. 48. Goeman D, King J, Koch S. Development of a model of dementia support and pathway for culturally and linguistically diverse communities using co-creation and participatory action research. BMJ Open. 2016;6(12). Figure 1. Flow-chart of study inclusion criteria Abbreviations: n: number, MAS: Memory Assessment Service, M/F: men / women. Figure 1. Flow-chart of study inclusion criteria Figure 1. Flow-chart of study inclusion criteria Abbreviations: n: number, MAS: Memory Assessment Service, M/F: men / women. Table 1. Characteristics of the study population at referral to MAS, by ethnicity Total n=3686 White British n=3654 South Asian n=32 p-value Women, n (%) 2114 (57.4) 2103 (57.6) 11 (34.4) 0.008 Age, mean (±SD) 80.1 (6.9) 80.2 (6.9) 76.1 (4.9) 0.001 Marital status, n (%) • Married / civil partnership • Widowed / single / divorced / separated • No data / not disclosed / not known 1863 (50.5) 1577 (42.8) 246 (6.7) 1841 (50.4) 1567 (42.9) 246 (6.7) 22 (68.8) 10 (31.3) 0 (0) 0.072 Nationality, n (%) • British • Non-British • unknown 3057 (82.9) 18 (0.5) 611 (16.6) 3048 (83.4) 6 (0.2) 600 (16.4) 9 (28.1) 12 (37.5) 11 (34.4) <0.001 First language, n (%) • English • Non-English • unknown 2589 (70.2) 11 (0.3) 1086 (29.5) 2580 (70.6) 2 (0.1) 1072 (29.3) 9 (28.1) 9 (28.1) 14 (43.8) <0.001 IMD, median (IQR) 6 (4; 8) 6 (4; 8) 8 (5; 10) 0.002 Known to RRLP, n (%) 378 (10.3) 378 (10.3) 0 (0) 0.055 Abbreviations: MAS: Memory Assessment Service, SD: standard deviation, IQR: interquartile range, IMD: Index of Multiple Deprivation, RRLP: Rapid Response Liaison Psychiatry. P-values are calculated by chi-square test for categorical variables. The p-value for difference in mean age is calculated by student’s T-test for means. The p-value for difference in median IMD is calculated by non-parametric Mann-Whitney U test. of the study population at referral to MAS, by ethnicity Table 1. Abbreviations: MAS: Memory Assessment Service, SD: standard deviation, IMD: Index of Multiple Deprivation, IQR: interquartile range, RRLP: Rapid Response Liaison Psychiatry. P-value* indicates gender-differences among the White British participants; p-value indicates gender- differences among South Asian participants. P-values are calculated by chi-square test for categorical variables. The P-value for difference in mean age is calculated by student’s T-test for means. The p-value for differences in median IMD is calculated by non-parametric Mann-Whitney U test. References Characteristics of the study population at referral to MAS, by ethnicity Abbreviations: MAS: Memory Assessment Service, SD: standard deviation, IQR: interquartile range, IMD: Index of Multiple Deprivation, RRLP: Rapid Response Liaison Psychiatry. P-values are calculated by chi-square test for categorical variables. The p-value for difference in mean age is calculated by student’s T-test for means. The p-value for difference in median IMD is calculated by non-parametric Mann-Whitney U test. IMD: Index of Multiple Deprivation, RRLP: Rapid Response Liaison Psychiatry. P-values are calculated by chi-square test for categorical variables. The p-value for difference in mean age is calculated by student’s T-test for means. The p-value for difference in median IMD is calculated by non-parametric Mann-Whitney U test. Table 2. Gender-differences among White British and South Asian participants, at referral to MAS White British South Asian Women n=2103 Men n=1551 p-value Women n=11 Men n=21 p- value** Age, mean (±SD) 80.8 (6.9) 79.4 (6.8) <0.001 74.8 (6.8) 76.8 (3.7) 0.297 Marital status, n (%) • Married / civil partnership • Widowed / single / divorced / separated • No data / not disclosed / not known 814 (38.7) 1128 (53.6) 161 (7.7) 1027 (66.2) 439 (28.3) 85 (5.5) <0.001 5 (45.5) 6 (54.5) 0 (0) 17 (81.0) 4 (19.0) 0(0) 0.040 Nationality, n (%) • British • Non-British • unknown 1750 (83.2) 4 (0.2) 349 (16.6) 1298 (83.7) 2 (0.1) 251 (16.2) 0.852 1 (9.1) 3 (27.3) 7 (63.6) 8 (38.1) 9 (42.9) 4 (19.0) 0.033 First language, n (%) • English • Non-English • unknown 1480 (70.4) 2 (0.1) 621 (29.5) 1100 (70.9) 0 (0) 451 (29.1) 0.455 1 (9.1) 4 (36.4) 6 (54.5) 8 (38.1) 5 (23.8) 8 (38.1) 0.222 IMD, median (IQR) 6 (4; 8) 6 (4; 8) 0.020 7 (6;10) 9 (5; 10) 0.935 Known to RRLP, n (%) 225 (10.7) 153 (9.9) 0.413 0 (0) 0 (0) NA erences among White British and South Asian participants, at referral to MAS South Asian Abbreviations: MAS: Memory Assessment Service, SD: standard deviation, IMD: Index of Multiple Deprivation, IQR: interquartile range, RRLP: Rapid Response Liaison Psychiatry. P-value* indicates gender-differences among the White British participants; p-value** indicates gender- differences among South Asian participants. P-values are calculated by chi-square test for categorical variables. The P-value for difference in mean age is calculated by student’s T-test for means. The p-value for differences in median IMD is calculated by non-parametric Mann-Whitney U test. References Table 3. Association between South Asian ethnicity and outcome of accessing MAS within 90 days from referral, compared to White British ethnicity Model OR [95% CI] p-value 0 0.47 [0.24 – 0.95] 0.037 1 0.42 [0.21 – 0.86] 0.017 2 0.47 [0.23 – 0.95] 0.035 Binary logistic regression analyses. Abbreviations: MAS: Memory Assessment Service, OR: odds ratio, CI: confidence interval. Model 0 is adjusted by ethnicity only. Model 1 is adjusted by ethnicity, age and gender. Model 2 is adjusted by ethnicity, age, gender, previous referral to the RRLP and IMD decile. The analyses in Model 0 and 1 are performed in the total population of 3,686 outpatients. The analyses in Model 2 are performed in 3,683 outpatients, due to missing data on Index of Multiple Deprivation decile for three White British outpatients. able 3. Association between South Asian ethnicity and outcome of accessing MAS ithin 90 days from referral, compared to White British ethnicity Binary logistic regression analyses. Abbreviations: MAS: Memory Assessment Service, OR: odds ratio, CI: confidence interval. Model 0 is adjusted by ethnicity only. Model 1 is adjusted by ethnicity, age and gender. Model 2 is adjusted by ethnicity, age, gender, previous referral to the RRLP and IMD decile. The analyses in Model 0 and 1 are performed in the total population of 3,686 outpatients. The analyses in Model 2 are performed in 3,683 outpatients, due to missing data on Index of Multiple Deprivation decile for three White British outpatients. Binary logistic regression analyses. Abbreviations: MAS: Memory Assessment Service, OR: odds ratio, CI: confidence interval. Model 0 is adjusted by ethnicity only. Model 1 is adjusted by ethnicity, age and gender. Model 2 is adjusted by ethnicity, age, gender, previous referral to the RRLP and IMD decile. The analyses in Model 0 and 1 are performed in the total population of 3,686 outpatients. The analyses in Model 2 are performed in 3,683 outpatients, due to missing data on Index of Multiple Deprivation decile for three White British outpatients. Binary logistic regression analyses. Abbreviations: MAS: Memory Assessment Servic Table 4. Association between South Asian ethnicity and outcome of accessing MAS within 90 days from referral, compared to White British ethnicity, in subpopulation who reported British nationality (n = 3,057) Model OR [95% CI] p-value 0 0.99 [0.25 – 3.97] 0.990 1 0.90 [0.22 – 3.63] 0.884 2 1.04 [0.25 – 4.25] 0.961 Binary logistic regression analyses. References Abbreviations: MAS: Memory Assessment Service, OR: odds ratio, CI: confidence interval. Model 0 is adjusted by ethnicity only. Model 1 is adjusted by ethnicity, age and gender. Model 2 is adjusted by ethnicity, age, gender, previous referral to the Rapid Response Liaison Psychiatry and Index of Multiple Deprivation decile. The analyses in Model 0 and 1 are performed in 3,057 outpatients. The analyses in Model 2 are performed in 3,054 outpatients, due to missing data on Index of Multiple Deprivation for three White British outpatients. Table 4. Association between South Asian ethnicity and outcome of accessing MAS within 90 days from referral, compared to White British ethnicity, in subpopulation who reported British nationality (n = 3,057) Model OR [95% CI] p-value 0 0.99 [0.25 – 3.97] 0.990 1 0.90 [0.22 – 3.63] 0.884 2 1.04 [0.25 – 4.25] 0.961 able 4. Association between South Asian ethnicity and outcome of accessing MAS ithin 90 days from referral, compared to White British ethnicity, in subpopulation Binary logistic regression analyses. Abbreviations: MAS: Memory Assessment Servic Binary logistic regression analyses. Abbreviations: MAS: Memory Assessment Service, OR: odds ratio, CI: confidence interval. Model 0 is adjusted by ethnicity only. Model 1 is adjusted by ethnicity, age and gender. Model 2 is adjusted by ethnicity, age, gender, previous referral to the Rapid Response Liaison Psychiatry and Index of Multiple Deprivation decile. The analyses in Model 0 and 1 are performed in 3,057 outpatients. The analyses in Model 2 are performed in 3,054 outpatients, due to missing data on Index of Multiple Deprivation for three White British outpatients. Binary logistic regression analyses. Abbreviations: MAS: Memory Assessment Service, OR: odds ratio, CI: confidence interval. Model 0 is adjusted by ethnicity only. Model 1 is adjusted by ethnicity, age and gender. Model 2 is adjusted by ethnicity, age, gender, previous referral to the Rapid Response Liaison Psychiatry and Index of Multiple Deprivation decile. The analyses in Model 0 and 1 are performed in 3,057 outpatients. The analyses in Model 2 are performed in 3,054 outpatients, due to missing data on Index of Multiple Deprivation for three White British outpatients. Table 5. References Association between South Asian ethnicity and outcome of accessing MAS within 90 days from referral, compared to White British ethnicity, in subpopulation who reported English as their first language (n = 2,589) Model OR [95% CI] p-value 0 0.63 [0.17 – 2.34] 0.486 1 0.59 [0.16 – 2.22] 0.438 2 0.75 [0.20 – 2.88] 0.677 Binary logistic regression analyses. Abbreviations: MAS: Memory Assessment Service, OR: odds ratio, CI: confidence interval. Model 0 is adjusted by ethnicity only. Model 1 is adjusted by ethnicity, age and gender. Model 2 is adjusted by ethnicity, age, gender, previous referral to the Rapid Response Liaison Psychiatry and Index of Multiple Deprivation decile. The analyses in Model 0 and 1 are performed in 2,589 outpatients. The analyses in Model 2 are performed in 2,587 outpatients, due to missing data on Index of Multiple Deprivation for two White British outpatients. Binary logistic regression analyses. Abbreviations: MAS: Memory Assessment Servic Binary logistic regression analyses. Abbreviations: MAS: Memory Assessment Service, OR: odds ratio, CI: confidence interval. Model 0 is adjusted by ethnicity only. Model 1 is adjusted by ethnicity, age and gender. Model 2 is adjusted by ethnicity, age, gender, previous referral to the Rapid Response Liaison Psychiatry and Index of Multiple Deprivation decile. The analyses in Model 0 and 1 are performed in 2,589 outpatients. The analyses in Model 2 are performed in 2,587 outpatients, due to missing data on Index of Multiple Deprivation for two White British outpatients. Binary logistic regression analyses. Abbreviations: MAS: Memory Assessment Service, OR: odds ratio, CI: confidence interval. Model 0 is adjusted by ethnicity only. Model 1 is adjusted by ethnicity, age and gender. Model 2 is adjusted by ethnicity, age, gender, previous referral to the Rapid Response Liaison Psychiatry and Index of Multiple Deprivation decile. The analyses in Model 0 and 1 are performed in 2,589 outpatients. The analyses in Model 2 are performed in 2,587 outpatients, due to missing data on Index of Multiple Deprivation for two White British outpatients. Supplementary Table 1. Association between South Asian ethnicity and outcome of accessing MAS within 120 days from referral, compared to White British ethnicity Model OR [95% CI] p-value 0 0.58 [0.29 – 1.17] 0.128 1 0.51 [0.25 – 1.02] 0.058 2 0.53 [0.26 – 1.07] 0.077 Binary logistic regression analyses. Abbreviations: MAS: Memory Assessment Service, OR: odds ratio, CI: confidence interval. Model 0 is adjusted by ethnicity only. References Model 1 is adjusted by ethnicity, age and gender. Model 2 is adjusted by ethnicity, age, gender, previous referral to the Rapid Response Liaison Psychiatry and Index of Multiple Deprivation decile. The analyses in Model 0 and 1 are performed in the total population of 3,686 outpatients. The analyses in Model 2 are performed in 3,683 outpatients, due to missing data on IMD for three White British outpatients. Supplementary Table 1. Association between South Asian ethnicity and outcome o accessing MAS within 120 days from referral, compared to White British ethnicit Binary logistic regression analyses. Abbreviations: MAS: Memory Assessment Service, OR: odds ratio, CI: confidence interval. Model 0 is adjusted by ethnicity only. Model 1 is adjusted by ethnicity, age and gender. Model 2 is adjusted by ethnicity, age, gender, previous referral to the Rapid Response Liaison Psychiatry and Index of Multiple Deprivation decile. The analyses in Model 0 and 1 are performed in the total population of 3,686 outpatients. The analyses in Model 2 are performed in 3,683 outpatients, due to missing data on IMD for three White British outpatients. Binary logistic regression analyses. Abbreviations: MAS: Memory Assessment Service, OR: odds ratio, CI: confidence interval. Model 0 is adjusted by ethnicity only. Model 1 is adjusted by ethnicity, age and gender. Model 2 is adjusted by ethnicity, age, gender, previous referral to the Rapid Response Liaison Psychiatry and Index of Multiple Deprivation decile. The analyses in Model 0 and 1 are performed in the total population of 3,686 outpatients. The analyses in Model 2 are performed in 3,683 outpatients, due to missing data on IMD for three White British outpatients. Binary logistic regression analyses. Abbreviations: MAS: Memory Assessment Servic Supplementary Table 2. Association between South Asian ethnicity and outcome of accessing MAS within 90 days from referral, compared to White British ethnicity, in women (n = 2,114) Model OR [95% CI] p-value 0 0.52 [0.16 – 1.70] 0.277 1 0.44 [0.13 – 1.46] 0.181 2 0.49 [0.15 – 1.62] 0.242 Binary logistic regression analyses. Abbreviations: MAS: Memory Assessment Service, OR: odds ratio, CI: confidence interval. Model 0 is adjusted by ethnicity only. Model 1 is adjusted by ethnicity and age. Model 2 is adjusted by ethnicity, age, previous referral to the Rapid Response Liaison Psychiatry and Index of Multiple Deprivation decile. All the analyses are performed in the total sample of 2,114 women. Binary logistic regression analyses. References Abbreviations: MAS: Memory Assessment Servic Binary logistic regression analyses. Abbreviations: MAS: Memory Assessment Service, OR: odds ratio, CI: confidence interval. Model 0 is adjusted by ethnicity only. Model 1 is adjusted by ethnicity and age. Model 2 is adjusted by ethnicity, age, previous referral to the Rapid Response Liaison Psychiatry and Index of Multiple Deprivation decile. All the analyses are performed in the total sample of 2,114 women. Binary logistic regression analyses. Abbreviations: MAS: Memory Assessment Service, OR: odds ratio, CI: confidence interval. Model 0 is adjusted by ethnicity only. Model 1 is adjusted by ethnicity and age. Model 2 is adjusted by ethnicity, age, previous referral to the Rapid Response Liaison Psychiatry and Index of Multiple Deprivation decile. All the analyses are performed in the total sample of 2,114 women. Supplementary Table 3. Association between South Asian ethnicity and outcome of accessing MAS within 90 days from referral, compared to White British ethnicity, in men (n = 1,572) Model OR [95% CI] p-value 0 0.44 [0.19 – 1.06] 0.068 1 0.41 [0.17 – 0.99] 0.047 2 0.46 [0.19 – 1.12] 0.086 Binary logistic regression analyses. Abbreviations: MAS: Memory Assessment Service, OR: odds ratio, CI: confidence interval. Model 0 is adjusted by ethnicity only. Model 1 is adjusted by ethnicity and age. Model 2 is adjusted by ethnicity, age, previous referral to the Rapid Response Liaison Psychiatry and Index of Multiple Deprivation decile. The analyses in Model 0 and 1 are performed in the total sample of 1,572 men. The analyses in Model 2 are performed in 1,569 men, due to missing data on Index of Multiple Deprivation for three White British men. Binary logistic regression analyses. Abbreviations: MAS: Memory Assessment Servic Binary logistic regression analyses. Abbreviations: MAS: Memory Assessment Service, OR: odds ratio, CI: confidence interval. Model 0 is adjusted by ethnicity only. Model 1 is adjusted by ethnicity and age. Model 2 is adjusted by ethnicity, age, previous referral to the Rapid Response Liaison Psychiatry and Index of Multiple Deprivation decile. The analyses in Model 0 and 1 are performed in the total sample of 1,572 men. The analyses in Model 2 are performed in 1,569 men, due to missing data on Index of Multiple Deprivation for three White British men. Binary logistic regression analyses. Abbreviations: MAS: Memory Assessment Service, OR: odds ratio, CI: confidence interval. Model 0 is adjusted by ethnicity only. References Model 1 is adjusted by ethnicity and age. Model 2 is adjusted by ethnicity, age, previous referral to the Rapid Response Liaison Psychiatry and Index of Multiple Deprivation decile. The analyses in Model 0 and 1 are performed in the total sample of 1,572 men. The analyses in Model 2 are performed in 1,569 men, due to missing data on Index of Multiple Deprivation for three White British men. Supplementary Figure 1. Index of Multiple Deprivation decile distribution at referral, by ethnicity Abbreviations: IMD: Index of Multiple Deprivation. The graph on the left shows data on 3,651 White British outpatients (three White British outpatients had missing data on IMD and were excluded); the graph on the right shows data on 32 South Asian outpatients. Abbreviations: IMD: Index of Multiple Deprivation. The graph on the left shows data on 3,651 White British outpatients (three White British outpatients had missing data on IMD and were excluded); the graph on the right shows data on 32 South Asian outpatients. Supplementary Figure 2. Timely access to MAS by age category, in the total study population. Supplementary Figure 2. Timely access to MAS by age category, in the total study population. Abbreviations: MAS: memory assessment service. Number of outpatients in each age category: 65 to 69 years, n = 274; 70 to 74 years, n = 559; 75 to 79 years, n = 819; 80 to 84 years, n = 980; 85 to 89 years, n = 746; 90plus, n = 308. Abbreviations: MAS: memory assessment service. Number of outpatients in each age category: 65 to 69 years, n = 274; 70 to 74 years, n = 559; 75 to 79 years, n = 819; 80 to 84 years, n = 980; 85 to 89 years, n = 746; 90plus, n = 308. Supplementary Figure 3. Timely access to MAS by Index of Multiple Deprivation decile, in the total study population Supplementary Figure 3. Timely access to MAS by Index of Multiple Deprivation decile, in the total study population Supplementary Figure 3. Timely access to MAS by Index of Multiple Deprivation decile, in the total study population Abbreviations: n: number, IMD: Index of Multiple Deprivation, MAS: memory assessment service. This graph shows data on a total of n = 3,683 outpatients. Three outpatients had missing data on IMD and were excluded. Abbreviations: n: number, IMD: Index of Multiple Deprivation, MAS: memory assessment service. References This graph shows data on a total of n = 3,683 outpatients. Three Abbreviations: n: number, IMD: Index of Multiple Deprivation, MAS: memor assessment service. This graph shows data on a total of n = 3,683 outpatients. Thre outpatients had missing data on IMD and were excluded. Abbreviations: n: number, IMD: Index of Multiple Deprivation, MAS: memory assessment service. This graph shows data on a total of n = 3,683 outpatients. Three outpatients had missing data on IMD and were excluded. Supplementary Figure 4. Timely access to MAS by Index of Multiple Deprivation decile, in the total population (shown as percentage of IMD decile category). Abbreviations: MAS: memory assessment service, IMD: Index of Multiple Deprivation. Number of outpatients per IMD decile: first decile, n = 160; second decile, n = 323; third decile, 291; fourth decile, n = 399; fifth decile, n = 379; sixth decile, n = 447; seventh decile, n = 451; eight decile, n = 416; ninth decile, n = 345; tenth decile, n = 472. This graph shows data on a total of n = 3,683 outpatients. Three outpatients had missing data on IMD and were excluded. Supplementary Figure 4. Timely access to MAS by Index of Multiple Deprivation decile, in the total population (shown as percentage of IMD decile category). Abbreviations: MAS: memory assessment service, IMD: Index of Multiple Deprivation. Number of outpatients per IMD decile: first decile, n = 160; second decile, n = 323; third decile, 291; fourth decile, n = 399; fifth decile, n = 379; sixth decile, n = 447; seventh decile, n = 451; eight decile, n = 416; ninth decile, n = 345; tenth decile, n = 472. This graph shows data on a total of n = 3,683 outpatients. Three outpatients had missing data on IMD and were excluded. upplementary Figure 5. Timely access to MAS by previous referral to the Rapid esponse Liaison Psychiatry, in the total study population. upplementary Figure 5. Timely access to MAS by previous referral to the Rapid esponse Liaison Psychiatry, in the total study population. Supplementary Figure 5. Timely access to MAS by previous referral to the Rapid Response Liaison Psychiatry, in the total study population. Abbreviations: MAS: memory assessment service. Number of outpatients by referral category: not previously referred to Rapid Response Liaison Psychiatry, n = 3,308; previously referred to Rapid Response Liaison Psychiatry, n = 378. References Abbreviations: MAS: memory assessment service. Number of outpatients by referral category: not previously referred to Rapid Response Liaison Psychiatry, n = 3,308; previously referred to Rapid Response Liaison Psychiatry, n = 378. Abbreviations: MAS: memory assessment service. Number of outpatients by referral category: not previously referred to Rapid Response Liaison Psychiatry, n = 3,308; previously referred to Rapid Response Liaison Psychiatry, n = 378. For reviewers only Figure 1 for reviewers only. Age distribution at referral, by ethnicity. Abbreviation: n: number. Abbreviation: n: number.
W2558136607.txt	https://bmcendocrdisord.biomedcentral.com/track/pdf/10.1186/s12902-016-0149-z	en		Effect of sitagliptin on blood glucose control in patients with type 2 diabetes mellitus who are treatment naive or poorly responsive to existing antidiabetic drugs: the JAMP study	BMC endocrine disorders	2,016	cc-by	7,147	Sakura et al. BMC Endocrine Disorders (2016) 16:70 DOI 10.1186/s12902-016-0149-z RESEARCH ARTICLE Open Access Effect of sitagliptin on blood glucose control in patients with type 2 diabetes mellitus who are treatment naive or poorly responsive to existing antidiabetic drugs: the JAMP study Hiroshi Sakura1, Naotake Hashimoto2, Kazuo Sasamoto3, Hiroshi Ohashi4, Sumiko Hasumi5, Noriko Ujihara6, Tadasu Kasahara7, Osamu Tomonaga8, Hideo Nunome9, Masashi Honda10, Yasuhiko Iwamoto11 and for the JAMP Study Investigators Abstract Background: To investigate the ameliorating effect of sitagliptin, a dipeptidyl peptidase-4 inhibitor, on blood glucose control in patients with type 2 diabetes mellitus who were previously untreated with or who have a poor responsive to existing antidiabetic drugs. Methods: Sitagliptin (50 mg/day) was added on to the pre-existing therapy for type 2 diabetes and changes in the glycated hemoglobin (HbA1c) level after 3 months of treatment were compared with the baseline and performed exploratory analysis. Results: HbA1c levels were significantly decreased after 1 month of treatment compared to baseline, with a mean change in HbA1c level from baseline of −0.73% (range, −0.80 to −0.67) in the entire study population at 3 months. Patients who received a medium dose of glimepiride showed the least improvement in HbA1c levels. The percentage of patients who achieved an HbA1c level of <7.0% significantly increased after 1 month of treatment, reaching 53.1% at 3 months. The percentage of patients who achieved a fasting blood glucose level of <130 mg/dL significantly increased after 1 month of treatment, reaching 50.9% at 3 months. Conclusions: Sitagliptin improved the HbA1c level and rate of achieving the target control levels in patients with type 2 diabetes mellitus who were previously untreated with, or poorly responsive to, existing antidiabetic drugs. Thus, sitagliptin is expected to be useful in this patient group. However, the additional administration of sitagliptin in patients treated with medium-dose glimepiride only slightly improved blood glucose control when corrected for baseline HbA1c level. Keywords: Sitagliptin, Diabetes mellitus, DPP-4 inhibitor, HbA1c, Glimepiride Correspondence: sakuragm@dnh.twmu.ac.jp 1 Department of Medicine, Medical Center East, Tokyo Women’s Medical University School of Medicine , 2-1-10 Nishiogu, Arakawa-ku, Tokyo 116-8567, Japan Full list of author information is available at the end of the article © 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. Sakura et al. BMC Endocrine Disorders (2016) 16:70 Background The 2012 National Health and Nutrition Survey conducted by the Ministry of Health, Labour and Welfare of Japan (MHLW) estimated that approximately 20.5 million people were strongly suspected of having diabetes mellitus; these patients are considered the at-risk group of individuals in whom diabetes mellitus cannot be ruled out [1]. An increasing amount of patients strongly suspected of having diabetes mellitus are currently being treated with existing antidiabetic drugs (men, 65.9% and women, 64.3%) [1]. However, existing antidiabetic drugs have various drawbacks, including insufficient efficacy, short-lasting effect [2], body weight increase and inconvenient administration. Thus, new drugs that have a different mechanism of action and that show improved efficacy, safety and tolerability are required. Dipeptidyl peptidase-4 (DPP-4) inhibitors exhibit antidiabetic effects by stimulating insulin secretion through highly selective inhibition of DPP-4, an enzyme that inactivates incretins such as glucagon-like peptide 1 and gastric inhibitory polypeptide via a mechanism different from that of conventional hypoglycemic drugs. Many reports have demonstrated the superior efficacy and safety of DPP-4 inhibitors [3–5], among which sitagliptin was the first to gain approval in Japan in 2009. Many reports of sitagliptin had published, but we have examined the additional effect of sitagliptin in 7 pre-existing therapy groups. Treatment of these patients often includes other antidiabetic medications because they often have diverse complications. The objective of this study is to investigate the ameliorating effect of sitagliptin on blood glucose control in patients with type 2 diabetes mellitus, who were previously untreated with, or who had a poor responsive to, existing antidiabetic drugs. Methods Study design This open-label, central registration, multi-center, prospective observational study was conducted at the Tokyo Women’s Medical University Hospital and 69 collaborating institutions in Japan. Patients were enrolled from 1 January 2011 to 30 June 2013, and followed up until 30 June 2014. This study was conducted with the approval of the ethic committee of the Tokyo Women’s Medical University (UMIN000019154). Page 2 of 11 receiving diet/exercise therapy or/and oral antidiabetic drug therapy. The participants were treated with diet and exercise therapy, low-dose glimepiride (0.5–1 mg), medium-dose glimepiride (1.5–2 mg), biguanides, thiazolidines, αglucosidase inhibitors or two or more of these drugs in combination during the observation period. And patients treated with more than 2 mg of glimepiride or other SUs were excluded from the study. The all patients provided written informed consent before participation. Patients who met any of the following criteria were excluded from the study: (i) history of severe ketosis, diabetic coma or pre-coma within the past 6 months; (ii) severe infection before or after surgical treatment, or serious external injury; (iii) pregnancy, possible pregnancy or lactation; (iv) moderate renal impairment (serum creatinine level ≥1.5 mg/dL in men and ≥1.3 mg/ dL in women); (v) patients on insulin therapy; (vi) patients on treatment with rapid-acting insulin secretagogues; (vii) history of allergy to the ingredients of the study drug; and (viii) a medical reason that makes the patient unsuitable for participation in the study as judged by the investigator. Treatment with (v) and/or (vi) were not covered by national health insurance at the time of setting the study protocol. Treatments The pre-existing therapy for type 2 diabetes were not changed during the observation period and entire study period (for the first 3 months after add-on sitagliptin). Thereafter, sitagliptin (50 mg) was administered once daily as a first-line treatment (single-drug therapy) or as an additional treatment (combination therapy; Fig. 1). During the 3-month period from the initiation of sitagliptin treatment (baseline), administration of sitagliptin was continued without the addition of any other drugs or dose increases. At 3 months, the sitagliptin dose was increased from 50 to 100 mg/day and other antidiabetic drugs were added, changed or discontinued at the investigator’s discretion. No restrictions were imposed on the use of drugs for treating concurrent diseases, but dose changes or the addition of new drugs was avoided whenever possible during the study period. Evaluation Study subjects The study subjects were male or female, 20 years of age or older, and outpatients with type 2 diabetes mellitus and inadequately controlled blood glucose levels (a glycated hemoglobin (HbA1c) level of ≥6.9% (52 mmol/ mol) or a fasting blood glucose level of ≥ 130 mg/dL [6] during the observation period) after at least 1 month of All end points were defined in the study protocol prior to implementation of the study. The primary end point was the change in HbA1c level at 3 months from baseline. The secondary end points were as follows: the percentage of patients who achieved a normal HbA1c level (<7.0%, 53 mmol/mol [6]) and the percentage of patients who achieved a normal fasting blood glucose level Sakura et al. BMC Endocrine Disorders (2016) 16:70 Page 3 of 11 Fig. 1 Study design. 1 Criteria for inadequate blood glucose control: HbA1c level ≥6.9% (52 mmol/mol) or fasting blood glucose level ≥130 mg/ dL. 2 Specific investigational tests (optional): GA, 1.5AG, C-peptide, proinsulin-to-insulin ratio (<130 mg/dL) at 1, 3 and 12 months after the start of treatment; changes in HbA1c from baseline at 1 and 12 months after starting treatment; and fasting blood glucose level, homeostatic model of assessment-β index and blood lipid levels (low-density lipoprotein cholesterol [LDL-C], triglycerides [TG], and high-density lipoprotein cholesterol [HDL-C]) at 3 and 12 months after starting treatment. In addition, the HbA1c level at 3 months of treatment and the change in HbA1c level from baseline were calculated for each patient group classified according to the concomitant drug used. The approximate linear correlation between the baseline HbA1c level and the change in HbA1c level after 3 months of treatment was examined statistically using an exploratory approach. Multiple linear regression analysis was performed using the deviation from the correlation line as the dependent variable. Safety assessments included the incidence of adverse drug reactions and hypoglycemia during the study period. At the start of the study in 2011, HbA1c values were expressed in The Japan Diabetes Society levels, the standard system in Japan, but were changed to National Glycohemoglobin Standardization Program (NGSP) system values at the end of the study in accordance with the “Report of the Committee on the Classification and Diagnostic Criteria of Diabetes Mellitus (Revision for International Harmonization),” issued by The Japan Diabetes Society [7]. Pursuant to the above change, the lower limit of inadequate blood glucose control was also changed. Therefore, patients with an HbA1c level of ≥6.9% (52 mmol/mol) were enrolled at the start of the study, but at the data analysis stage, the percentage of patients who achieved the target HbA1c level of ≥7.0% (53 mmol/mol) or <7.0% (53 mmol/mol) was calculated. Statistical analysis Data analysis was performed using IBM SPSS Statistics 22.0 for Windows (IBM Japan, Ltd.). Observed values were analyzed using a paired t test, while a betweengroup comparison of changes was performed using analysis of variance. The chi-square test was used to analyze the percentage of patients who achieved the HbA1c target level, and correlation was evaluated using Pearson’s test. The factors that affect the fasting blood glucoselowering effect were evaluated using simple and multiple regression analyses, with the significance level set at 5% (two-sided). Demographic characteristics are presented as the mean ± standard deviation (SD), and the observed values are presented as the mean ± standard error (SE). Changes are presented as the mean (95% confidence interval [CI]). Results Figure 2 shows the flow diagram of the patient enrollment in the study. Of the 779 patients with T2DM enrolled, 651 were included in the Efficacy analysis (Diet/ exercise therapy, 189; Low-dose glimepiride, 72; Mediumdose glimepiride, 50; Biguanide, 99; Thiazolidine, 38; α-GI, 18; Combination therapy, 185). Table 1 shows the baseline Sakura et al. BMC Endocrine Disorders (2016) 16:70 Page 4 of 11 Fig. 2 Patient enrollment flow diagram demographics of the 651 patients who were classified according to the concomitant drug used and evaluated to determine treatment efficacy. Past or concurrent illnesses in the entire study population are also presented in Table 1. There were 22 (3.4%) participants at 3 months and 37 (5.7%) participants at 12 months reported as poor adherence of sitagliptin. The HbA1c level significantly decreased after one month of treatment compared to baseline (p < 0.05), and this reduction was maintained throughout 12 months of treatment (Additional flile 1: Figure S1). The change (95% CI) in HbA1c level from baseline in the entire patient population was −0.73% (−0.80 to −0.67) at 3 months of treatment. There was no significant difference in the change in HbA1c level between the patient groups treated with different concomitant drugs (Fig. 3 and Additional file 3). The HbA1c level normalization rate, expressed as the percentage of patients who achieved an HbA1c level of <7.0% (53 mmol/mol), significantly increased at 1 month compared to baseline (p < 0.05), reaching an increase in 53.1% at 3 months (Additional flile 2: Figure S2). The fasting blood glucose normalization rate, calculated as the percentage of patients who achieved a fasting blood glucose level of <130 mg/dL, significantly increased at 1 month compared to baseline (p < 0.05), reaching an increase of 50.9% at 3 months (S2). We enrolled patients who meet the criteria of a glycated hemoglobin (HbA1c) level of ≥6.9% (52 mmol/mol) or a fasting blood glucose level of ≥ 130 mg/dL [6] during the observation period. Consequently, this study include patients who were achieved the HbA1c goal/or the fasting blood glucose goal at baseline. Table 2 shows the changes (95% CI) in most laboratory parameters at 3 and 12 months after the start of treatment compared with baseline. Improvement was observed in many of the diabetes mellitus-related parameters. The factors that affect the blood glucose-lowering effect of sitagliptin were also investigated. A negative correlation (R = −0.528, p < 0.05) was observed between the baseline HbA1c level and the change in HbA1c level from baseline to 3 months (S3). Related factors other than baseline HbA1c level were also investigated. For this purpose, the approximate linear correlation between the baseline HbA1c level and that at 3 months of treatment was calculated. A single regression analysis was performed using the deviation from the correlation line as the dependent variable. The results indicate that age, body mass index, mean blood pressure and concomitant administration of medium-dose glimepiride significantly affected sitagliptin efficacy (Table 3). Using the factors that showed a significant effect, multiple regression analysis was performed on the deviation from the approximate linear correlation. Only medium-dose glimepiride significantly affected sitagliptin efficacy (p = 0.017; Table 3). Table 4 shows the changes in anti-diabetic medication during the study and 86 patients were increased the dose of sitagliptin after the 3 months of follow-up visits. Adverse events observed in the 711 safety-evaluable patients included clinical symptoms in 37 patients and laboratory abnormalities in 18 patients (Table 5). 19 out of 55 patients discontinued administration of sitagliptin because of the adverse event. Four of these events, anemia (sitagliptin single-drug group), hypoglycemia (multipledrug co-administration group, low-dose glimepiride Group Parameter (Sitagliptin only) +Sitagliptin (combination therapy) Overall Diet/exercise therapy Low-dose glimepiride (0.5–1.0 mg) Medium-dose glimepiride (1.5–2.0 mg) Biguanide Thiazolidine α-GI Coadministration of two or more drugs n 651 189 72 50 99 38 18 185 Age (y) 63.8 ± 11.8 64.6 ± 12.7 68.1 ± 11.1 65.3 ± 11.2 59.3 ± 11.4 64.3 ± 12.2 63.9 ± 11.7 63.2 ± 10.8 Sex (male %) 434 (66.7) 121 (64.0) 44 (61.1) 37 (74.0) 72 (72.7) 27 (71.1) 13 (72.2) 120 (64.9) BMI (kg/m2) 25.2 ± 4.2 24.5 ± 4.1 24.3 ± 4.0 24.5 ± 3.5 25.8 ± 3.6 26.4 ± 7.1 25.9 ± 3.9 25.7 ± 4.1 Abdominal circumference (cm) 88.3 ± 11.1 87.3 ± 10.7 88.7 ± 10.7 85.1 ± 8.2 88.9 ± 9.0 92.9 ± 21.4 92.4 ± 7.2 87.9 ± 10.1 Disease duration (y) 8.8 ± 6.7 6.2 ± 5.7 8.2 ± 6.4 10.2 ± 8.1 9.4 ± 6.8 10.5 ± 7.1 8.7 ± 5.7 10.5 ± 6.5 Smoking habit (%) 143 (22.7) 32 (17.4) 14 (20.3) 16 (34.0) 26 (27.1) Drinking habit (%) 301 (48.1) 79 (42.5) 31 (47.0) 33 (68.8) 51 (53.1) HbA1c (%) 7.86 ± 1.07 7.67 ± 1.09 7.74 ± 0.86 8.23 ± 1.31 8.05 ± 1.10 7.64 ± 0.74 7.26 ± 0.76 8.00 ± 1.04 Converted mean HbA1c (mmol/mol) 62 60 61 66 64 60 56 64 Fasting blood glucose (mg/dL) 159.2 ± 41.5 152.7 ± 43.5 156.6 ± 35.4 166.5 ± 38.7 173.5 ± 48.1 154.1 ± 38.3 146.0 ± 43.3 158.9 ± 36.5 HOMA-IR 2.83 ± 1.80 2.36 ± 1.45 2.70 ± 1.69 2.97 ± 1.92 3.34 ± 1.72 2.68 ± 2.03 2.69 ± 1.75 3.01 ± 2.03 HOMA-β (%) 32.1 ± 27.1 32.6 ± 33.9 29.4 ± 20.1 33.4 ± 25.7 31.5 ± 22.7 29.1 ± 21.3 36.0 ± 35.2 33.0 ± 25.3 2.05 ± 0.96 5 (13.5) 19 (52.8) 4 (22.2) 46 (25.6) 10 (55.6) 78 (44.3) C-Peptide (ng/mL) 2.10 ± 0.89 2.10 ± 0.90 2.36 ± 1.64 2.08 ± 0.58 2.14 ± 0.74 1.97 ± 0.78 2.00 ± 0.74 SBP (mmHg) 130.9 ± 14.9 128.9 ± 16.1 134.0 ± 13.4 131.8 ± 11.4 131.0 ± 16.5 131.6 ± 16.3 130.5 ± 13.4 131.4 ± 13.8 DBP (mmHg) 76.5 ± 10.5 75.7 ± 9.7 76.5 ± 9.5 77.3 ± 10.3 79.6 ± 11.9 76.8 ± 11.4 79.7 ± 12.0 75.1 ± 10.2 Hypertension 393 (60.4) 107 (56.6) 46 (63.9) 29 (58) 60 (60.6) 24 (63.2) 13 (72.2) 114 (61.6) Dyslipidemia 417 (64.1) 102 (54) 41 (56.9) 34 (68) 72 (72.7) 25 (65.8) 12 (66.7) 131 (70.8) Hyperuricemia 65 (10) 17 (9) 4 (5.6) 2 (4) 13 (13.1) 5 (13.2) 7 (38.9) 17 (9.2) Retinopathy 48 (7.4) 7 (3.7) 3 (4.2) 2 (4) 18 (18.2) 1 (2.6) 3 (16.7) 14 (7.6) Arteriosclerosis obliterans 55 (8.4) 8 (4.2) 2 (2.8) 2 (4) 25 (25.3) 2 (5.3) 2 (11.1) 14 (7.6) Atrial fibrillation 16 (2.5) 5 (2.6) 2 (2.8) 2 (4) 2 (2) 1 (2.6) 0 (0) 4 (2.2) Renal disease 49 (7.5) 5 (2.6) 2 (2.8) 2 (4) 16 (16.2) 3 (7.9) 4 (22.2) 17 (9.2) Hepatic disease 56 (8.6) 15 (7.9) 3 (4.2) 4 (8) 14 (14.1) 4 (10.5) 3 (16.7) 13 (7) 18 (2.8) 5 (2.6) 2 (2.8) 1 (2) 3 (3) 0 (0) 2 (11.1) 5 (2.7) 45 (6.9) 8 (4.2) 4 (5.6) 3 (6) 6 (6.1) 3 (7.9) 4 (22.2) 17 (9.2) Angina pectoris 27 (4.1) 9 (4.8) 2 (2.8) 3 (6) 6 (6.1) 1 (2.6) 1 (5.6) 5 (2.7) Cardiac failure 11 (1.7) 3 (1.6) 3 (4.2) 1 (2) 1 (1) 0 (0) 0 (0) 3 (1.6) Data presented as n (%) or mean ± SD Page 5 of 11 Myocardial infarction Cerebral stroke Sakura et al. BMC Endocrine Disorders (2016) 16:70 Table 1 Patient demographic characteristics Sakura et al. BMC Endocrine Disorders (2016) 16:70 Page 6 of 11 Fig. 3 ΔHbA1c level according to concomitant drug type (3 months) co-administration group), urticaria (multiple-drug coadministration group), were judged by the investigator to be causally related to the study drug. The incidence of an adverse drug reaction was 0.56%. Hypoglycemia occurred in three patients (0.42%), but all the cases were mild. They were judged to be causally related to the study drug in one patient each in the multiple drug co-administration and low-dose glimepiride groups, and as possibly causally related to the study drug in one patient in the sitagliptin single-drug group. Discussion In the present study, the overall sitagliptin-induced decrease (95% CI) in HbA1c level from baseline was −0.73% (range, −0.80 to −0.67) at 3 months. In many studies, sitagliptin reportedly improved HbA1c and fasting blood glucose levels both as a single-drug therapy [3, 8–10] and as a combination therapy with other antidiabetic drugs [4, 5, 11]. In our study, we excluded patients undergoing insulin use, also some clinical trials [12, 13] reported about the combination therapy about sitagliptin and insulin. The patients in the present study had chronic diabetes mellitus, and many had already been inadequately controlled with antidiabetic drugs and additionally received sitagliptin. This suggests that the conditions under which this study was conducted were closer to routine clinical practice than those of another Japanese dose-finding study for sitagliptin [9]. Our study showed favorable, significant results for patients taking sitagliptin, which were similar to those obtained in other studies, despite the differences in study conditions. In the present study, no significant difference in the change in HbA1c level was observed between the groups of patients who received different concomitant drugs. However, the extent of the improvement was greater in the patients with a higher baseline HbA1c level [S3]. This suggests the need to correct for the effect of the baseline HbA1c level when interpreting data obtained in studies with widely scattered baseline HbA1c values. Sitagliptin-induced improvement in the HbA1c level was more difficult to achieve in the medium-dose glimepiride group than in the other groups, based on the exploratory study on the factors that affect the blood glucose-lowering effect of sitagliptin, the multiple regression analysis of the correlation between the baseline HbA1c level and the change in HbA1c level from baseline based on the deviation from the approximate linear correlation (Table 3). The baseline HbA1c level in the medium-dose glimepiride group was high (8.24%), but the sitagliptin-induced improvement in HbA1c level failed to meet the expectation that the higher the baseline HbA1c level, the greater the improvement [14, 15]. Although there is no report stating that Glimepiride is likely to cause the secondary failure, in comparison with Sulfonylurea, Biguanide and Thiazolidinedione, it is reported that Sulfonylurea causes the secondary failure [2]. Although Glimepiride doesn’t beget the secondary failure easily in Sulfonylurea [16], compared to other drug groups in this study, it is also presumed that in Glimepiride middle-dose group, the pancreas may become exhausted. We can’t identify the cause of it because there were some patients who didn’t laboratory test. Sakura et al. BMC Endocrine Disorders (2016) 16:70 Page 7 of 11 Table 2 Laboratory test results Parameter n 0 month (mean ± SE) n 3 months (mean ± SE) Δ3 months, mean (95% CI) p n 12 months (mean ± SE) Δ12 months, mean (95% CI) p HbA1c 649 7.86 ± 0.04 635 634 7.12 ± 0.04 −0.73 (−0.80, −0.67) 0.001> 577 576 7.11 ± 0.04 −0.69 (−0.76, −0.62) 0.001> Fasting blood glucose 493 159.2 ± 1.9 446 413 135.1 ± 1.6 −26.3 (−29.7, −22.9) 0.001> 394 361 135.9 ± 1.7 −23.2 (−27.3, −19.2) 0.001> HOMA-R 384 2.83 ± 0.09 367 318 2.71 ± 0.14 −0.36 (−0.51, −0.22) 0.001> 331 275 2.66 ± 0.11 −0.29 (−0.47, −0.10) 0.002 HOMA-β 382 32.1 ± 1.4 364 315 47 ± 3.2 11.5 (8.4, 14.6) 0.001> 324 270 43.9 ± 2 11.1 (7.6, 14.6) 0.001> Fasting insulin 384 7.22 ± 0.21 367 318 8.09 ± 0.43 0.21 (−0.15, 0.57) 0.254 331 275 7.77 ± 0.29 0.29 (−0.10, 0.67) 0.147 1.5AG 254 6.4 ± 0.3 248 240 11.3 ± 0.4 4.8 (4.3, 5.2) 0.001> 213 207 10.2 ± 0.4 3.8 (3.2, 4.3) 0.001> Glycoalbumin 254 21.1 ± 0.2 248 240 18.2 ± 0.2 −2.7 (−3.1, −2.4) 0.001> 213 207 18.5 ± 0.2 −2.5 (−2.9, −2.2) 0.001> C-Peptide 200 2.1 ± 0.06 197 177 2.07 ± 0.06 0 (−0.11, 0.1) 0.925 152 137 2.17 ± 0.08 0.05 (−0.06, 0.15) 0.380 Proinsulin-to-insulin ratio 193 0.51 ± 0.02 187 169 0.45 ± 0.02 −0.05 (−0.08, −0.02) 0.033 150 135 0.49 ± 0.02 −0.01 (−0.05, 0.03) 0.857 CPI 200 1.38 ± 0.05 197 177 1.56 ± 0.05 0.23 (0.15, 0.32) 0.001> 152 137 1.66 ± 0.07 0.28 (0.18, 0.38) 0.001> SBP 646 130.9 ± 0.6 635 634 129.3 ± 0.6 −1.7 (−2.9, −0.6) 0.004 576 575 130.2 ± 0.6 −0.3 (−1.6, 0.9) 0.587 DBP 646 76.5 ± 0.4 635 634 75.3 ± 0.4 −1.2 (−1.9, −0.4) 0.003 576 575 75.6 ± 0.4 −0.9 (−1.7, −0.1) 0.022 BMI 630 25.19 ± 0.17 601 599 25.24 ± 0.17 0 (−0.10, 0.05) 0.987 552 549 25.1 ± 0.18 −0.08 (−0.20, 0) 0.064 Triglycerides 589 149.3 ± 4.1 569 551 146.7 ± 5.1 −6.1 (−13.0, 0.7) 0.081 537 517 142.5 ± 4.2 −8.9 (−16.0, −1.3) 0.021 HDL-C 574 55 ± 0.6 555 537 53.8 ± 0.6 −1.1 (−1.8, −0.5) 0.001 527 506 53.6 ± 0.6 −1.6 (−2.3, −0.9) 0.001> LDL-C 589 113.9 ± 1.2 563 545 110 ± 1.2 −3.2 (−4.9, −1.5) 0.001> 533 512 110.3 ± 1.2 −3.4 (−5.6, −1.2) 0.003 Converted mean HbA1c(mmol/mol) 62 54 54 Upper row: Observed value (mean ± SE) Lower row: Change (mean [95% CI]) p-value: versus 0 month, paired t-test However the duration of Glimepiride middle-dose group was 10.2 years, it was longer than overall. Consequently, the effect of sitagliptin might not be obtained easily. By contrast, in a study where glimepiride was administered at a low or high dose and sitagliptin was added after dose reduction of glimepiride [17], sufficient efficacy was achieved in both groups. Patients in actual clinical settings have diverse treatment histories, which requires future studies on sitagliptin co-administration with medium- or high-dose glimepiride. Although, smoking and drinking rate are both high in the medium-dose glimepiride group. But we judged it is not significant elements in the multiple regression analysis. To prevent complications, the target HbA1c level is <7.0% (53 mmol/mol) and the target fasting blood glucose level is 130 mg/dL for blood glucose control [6]. In the present study, the rate of achieving the target control level improved for both HbA1c and fasting blood glucose levels. Laboratory test results showed that the homeostatic model of assessment (β index) significantly increased at 3 month after starting treatment compared with its baseline value, suggesting that sitagliptin enhances insulin secretion. Patients with type 2 diabetes mellitus have reduced numbers of pancreatic β cells [18, 19]. In an animal experiment, sitagliptin reportedly had pancreatic β cell-protecting and growth-promoting effects [20], in our present study, the Cpeptide reactivity index (CPI) significantly increased at 3 months after starting treatment. It was more likely because of improved beta-cell function rather than increased beta-cell mass because it was just 3 months after the start Sakura et al. BMC Endocrine Disorders (2016) 16:70 Page 8 of 11 Table 3 Single and multiple regression analyses of baseline HbA1c and ΔHbA1c levels (at 3 months) using the deviation from the approximate linear correlation coefficient as the dependent variable Single regression analysis Independent variable Age Regression coefficient Multiple regression analysis Lower Upper Limit Limit P-value 0.006 0.002 0.011 0.008‡ Partial regression coefficient Lower Upper Limit Limit P-value 0.004 −0.001 0.009 0.149 −0.022 −0.140 0.096 0.715 Smoking habit 0.015 −0.121 0.151 0.83 Drinking habit −0.037 −0.152 0.078 0.523 Duration of diabetes mellitus −0.004 −0.012 0.005 0.377 Body mass index −0.015 −0.028 −0.001 0.029‡ −0.009 −0.023 0.005 0.207 Mean blood pressure −0.008 −0.013 −0.002 0.005‡ −0.004 −0.008 0.001 0.095 Diet/exercise group 0.097 −0.027 0.22 0.126 Low-dose glimepiride group 0.054 −0.124 0.232 0.555 Medium-dose glimepiride group −0.264 −0.472 −0.056 0.013‡ −0.249 −0.452 −0.045 0.017§ Biguanide group −0.073 −0.228 0.081 0.351 Thiazolidine group 0.082 −0.159 0.323 0.504 αGI group 0.028 −0.308 0.364 0.871 −0.008 −0.131 0.115 0.9 Sex (male) Multi-drug coadministration group ‡ p < 0.05, single regression analysis § p < 0.05, multiple regression analysis DPP-4 inhibitors do not induce body weight increases [8, 24–26]. In the present study, sitagliptin did not cause any increase in the body mass index. Sitagliptin also decreases postprandial triglyceride level [27] and lowers blood pressure, blood lipid level [28] and alkaline phosphatase level [29]. Our present study also showed decreased systolic and diastolic blood pressures, decreased LDL-C level and decreased triglyceride levels, which is similar to results from previously published studies. Incretin is secreted from the gastrointestinal tract by the stimulation of molecules such as glucose and fats that are produced after food is taken into the digestive organs. Thus, incretin enhances insulin secretion from the pancreatic β cells when the blood glucose level increases after a meal. By contrast, sitagliptin decreased both postprandial and fasting blood glucose levels [26]. In the present study, sitagliptin improved fasting blood glucose levels and the rate of achieving the target control level. Talk about adherence, Walker et al. reported 22% of DM patients are defined as poor adherence of of sitagliptin, and considering this was an observation in human subjects. However, Nishimura et al. reported that CPI increased from baseline to 3, 6, 12, 18, and 24 months after the start of sitagliptin administration [21]. This indicated the pancreatic β cell-protective effect of sitagliptin in a clinical setting. Nishimura et al.also reported that greater CPI increase after sitagliptin administration were associated with the response to sitagliptin [21]. In our study, the number of patients who test CPI was limited. So we didn’t analysis CPI as the elements of logistic analysis. But CPI may be benchmark of the efficacy of Sitagliptin. However, the insulin secretion-enhancing effect of glucagon-like peptide 1 is reportedly dependent on the blood glucose level [22]. A meta-analysis on the effect of DPP-4 inhibitors revealed that the hypoglycemic risk from DPP-4 inhibitors is similar to that from placebo or thiazolidines but lower than that from sulfonylurea drugs [23]. Sitagliptin did not induce excessive hypoglycemia [10], and in the present study, mild hypoglycemia occurred in only three patients. Table 4 Changes in antidiabetic medication during the study (After 3 months of follow-up) n = 585 Agent Sulfonylurea Biguanide Thiazolidinedione α-Glucosidase inhibitors Sitagliptin Patients who received additional antidiabetic agent(s) 26 18 9 2 0 Patients who increased dosage 8 26 0 0 86 Patients who decreased dosage 7 4 0 0 2 Patients who stopped receiving agent(s) 8 3 20 0 0 Sakura et al. BMC Endocrine Disorders (2016) 16:70 Page 9 of 11 Table 5 Summary of adverse events Hypoglycemia 3 Hyperglycemia 1 Hematopoietic disorders 3 Cardiovascular disease 8 Respiratory disorders 2 Skin eruption 2 Gastrointestinal disorders 5 Hepatobiliary disorders 7 Renal and urinary disorders 2 Neuropsychiatric disorders 2 Others 20 Total 55 medicine [30], but our study showed only 5.7% of poor adherence of sitagliptin. We designed this study period because Iwamoto et al. reported that clinical treatment with sitagliptin for 12 weeks provided significant and clinically meaningful reductions in HbA1c. Although, if study drug doesn’t work enough, to fix medication for a long time is not good for patients. On the other hand, Nishimura et al. reported that a patient’s HbA1c change at 3 months may be a predictor of their HbA1c change at 24 months [11]. And our study also showed the HbA1c level of 12 month was similar to 3 month. According to the result of this study, we assume that 3 months of observation period is not too short to evaluate the clinical effects of sitagliptin. Study limitations This was an open-label observational study (patients were not allocated to the groups), the number of patients were different in the each groups because we categorized by the type of pre-existing therapy for type 2 diabetes. In this study, we excluded participants undergoing insulin use and rapid-acting insulin secretagogues. Dose increases of sitagliptin, and additional administration, dose changes or discontinuation of other antidiabetic drugs were allowed starting after 3 months of treatment. This short duration is a limitations to assessing the long-term usefulness of sitagliptin administration. However, this study is significant in that it evaluated sitagliptin efficacy in patients treated with different concomitant drugs in actual clinical settings. Conclusion Sitagliptin administration improved the HbA1c level and the rate of achieving the target control levels in patients with type 2 diabetes mellitus who were previously untreated with, or who were poorly responsive to, existing antidiabetic drugs. Thus, sitagliptin is expected to be effective for this patient group. Concomitant administration of sitagliptin to patients treated with medium-dose glimepiride only slightly improved blood glucose control after correction for the baseline HbA1c level. Additional files Additional file 1: Figure S1. HbA1c level and changes over time. (TIF 160 kb) Additional file 2: Figure S2. Rate of achieving the target fasting blood glucose level (HbA1c level). (TIF 268 kb) Additional file 3: Correlation between baseline HbA1c level and change in HbA1c level after 3 months of treatment. (Strongly effective: Below the correlation line; Weakly effective: Above the correlation line). (TIF 113 kb) Abbreviations 95% CI: 95% Confidence interval; BMI: Body mass index; CPI: C-peptide reactivity index; DBP: Diastolic blood pressure; DPP-4: Dipeptidyl peptidase-4; HbA1c: Glycated hemoglobin; HDL-C: High-density lipoprotein cholesterol; LDL-C: Low-density lipoprotein cholesterol; MHLW: Ministry of Health, Labour and Welfare of Japan; NGSP: National Glycohemoglobin Standardization Program; SBP: Systolic blood pressure; SD: Standard deviation; SE: Standard error; TG: Triglycerides; α-GI: α-glucosidase inhibitors Acknowledgments We would like to express our sincere gratitude to Mr. Shogo Shishikura (MSD K.K.,) (Teikyo University Graduate School of Public Health, MPH) for scientific advice including references for revising the manuscript. We also would like to thank and Nouvelle Place Inc. for conducting the data analyses. Funding This study was funded by Japan Diabetes Foundation. Availability of data and materials Data are available on request to the authors. Authors’ contributions Conceived and designed the experiments: HS NH KS HO SH NU TK OT HN MH YI; Enrolled patients: HS NH KS HO NU TK OT HN MH; Analyzed the data: HS YI; Wrote the first draft of the manuscript: HS YI; Contributed to the writing of the manuscript: HS NH KS HO SH NU TK OT HN MH YI; Agree with manuscript results and conclusions: HS NH KS HO SH NU TK OT HN MH YI. All authors read and approved the final manuscript. Competing interest Hiroshi Sakura received honoraria from Mitsubishi Tanabe Pharma Corporation and research grant from Ono Pharmaceutical Co., Ltd. Other authors declare that they have no conflict of interest. Consent for publication Not applicable. Ethics approval and consent to participate Written informed consent was obtained from all individual participants included in the study. This study was conducted with the approval of the ethic committee of the Tokyo Women’s Medical University (UMIN000019154) and all procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. JAMP Study Investigators The following investigators were involved in this study: Akiko Sato (Maruyama Internal Medicine Clinic); Akinori Yamashita (Yamashita Clinic); Akira Miyashita (Miyashita Surgery Clinic); Asako Kokubo (Kokubo Clinic); Atsuro Tsuchiya (Tsuchiya Clinic); Dai Hirohara (Hanazono Clinic); Daiji Kogure (Kogure Clinic); Daijo Kasahara (Kasahara Clinic); Hideki Tanaka (Internal Medicine, Seiwa Clinic); Hideki Tanaka (Internal Medicine, Nishiarai Hospital); Hideo Tezuka (Tezuka Clinic); Hiroyuki Kuroki (Internal Medicine, Johsai Sakura et al. BMC Endocrine Disorders (2016) 16:70 Hospital); Jun Ogino (Department of Diabetes, Endocrine and Metabolic Diseases, Tokyo Women’s Medical University Yachiyo Medical Center); Kanu Kin (Internal Medicine, Nishiarai Lifestyle-related diseases Clinic); Kanu Kin (Internal Medicine, Nishiarai Hospital); Kazuko Muto (Tokyo Women’s Medical University); Kazuo Suzuki (Kenkokan Suzuki Clinic of Internal Medicine); Keiko Iseki (Iseki Clinic); Keita Watanabe (Watanabe Clinic); Kenshi Higami (Higami Hospital); Kenzo Matsumura (Matsumura Gastroenterological Clinic); Kiyotaka Nakajima (Ebisu Clinic); Koki Shin (Shin Clinic); Kuniya Koizumi (Kuniya Clinic); Maki Saneshige (Mugishima Medical Clinic); Makio Sekine (Sekine Clinic); Makoto Yaida (Urban Heights Clinic); Mari Kiuchi (Physician, Kanauchi Medical Clinic); Mari Mugishima (Mugishima Medical Clinic); Mari Osawa (Department of Diabetes Mellitus, Institute of Geriatrics, Tokyo Women’s Medical University); Masae Banno (Banno Medical Clinic); Masahiro Yamamoto (Internal Medicine 1, Shimane University Faculty of Medicine); Masatake Hiratsuka (Higashishinagawa Clinic); Masumi Hosoya (Yasui Clinic); Michika Atsuta (Internal Medicine, Nishiarai Lifestyle-related diseases Clinic); Mitsutoshi Kato (Kato Clinic of Internal Medicine); Miwa Morita (Internal Medicine 1, Shimane University Faculty of Medicine); Munehiro Miyamae (Johsai Hospital); Mutsumi Iijima (Abe Hospital); Naomi Okuyama (Shinjuku Mitsui Building Clinic); Nobuo Hisano (Mejiro Medical Clinic); Norihiro Tsuchiya (Omotesando Naika Ganka); Rie Wada (Kanauchi Medical Clinic); Rie Wada (Nerimasakuradai Clinic); Ryuji Momose (Momo Medical Clinic); Sachiko Otake (Tokyo Women’s Medical University); Satoko Maruyama (Shinjuku Mitsui Building Clinic); Satoru Takada (Internal Medicine, Social welfare corporation Shineikai Takinogawa Hospital); Shigeki Dan (Ube Internal Medicine and Pediatrics Hospital); Shigeki Nishizawa (Nishizawa Medical Clinic); Shigeo Yamashita (Department of Diabetes and Endocrinology, JR Tokyo General Hospital); Shingo Saneshige (Internal Medicine, Kamiochiai Shin Clinic); Shinichi Teno (Teno Clinic); Shinji Tsuruta (Diabetic Medicine, Itabashi Chuo Medical Center); Shinobu Kumakura (Kumakura Medical Clinic); Sumiko Kijima (Abe Hospital); Takashi Kondo (Kondo Clinic); Takeo Onishi (Internal Medicine, Onishi Clinic); Taku Kudo (Internal Medicine, Social welfare corporation Shineikai Takinogawa Hospital); Tatsushi Sugiura (Internal Medicine, Seiwa Clinic); Toshihiko Ishiguro (Kaname Clinic); Yasue Suzuki (Suzuki Medical Clinic); Yasuhiro Tomita (Nakanobu Clinic); Yasuko Takano (Department of Diabetes, Shiseikai Daini Hospital); Yoshihisa Akimoto (Akimoto Yoshi Medical Clinic); Yoshiko Odanaka (Ito Internal Medicine Pediatrics Clinic); Yoshimasa Tasaka (Tokyo Women’s Medical University); Yoshitaka Aiso (Internal Medicine, Diabetes, Aiso Clinic); Yukiko Inoue (Inoue Medical Clinic); Yukinobu Kobayashi (Kobayashi Clinic). Author details 1 Department of Medicine, Medical Center East, Tokyo Women’s Medical University School of Medicine , 2-1-10 Nishiogu, Arakawa-ku, Tokyo 116-8567, Japan. 2Department of Diabetes, Endocrine and Metabolic Diseases, Tokyo Women’s Medical University Yachiyo Medical Center, 477-96, Owada-shinden, Yachiyo-shi, Chiba 276-8524, Japan. 3Internal Medicine, Suzuki Clinic, 2-10-14, Koyasu-machi, Hachioji-shi, Tokyo 192-0904, Japan. 4Internal Medicine, Oyama East Clinic, 1-32-1, Ekihigashi-dori, Oyama-shi, Tochigi 323-0022, Japan. 5Internal Medicine, Nishiyamado-Keiwa Hospital, 3247-1, Kounosu, Naka-shi, Ibaraki 311-0133, Japan. 6Department of Medicine, Diabetes Center, Institute of Geriatrics, Tokyo Women’s Medical University, Shibuya Cross Tower 22F, 2-15-1, Shibuya, Shibuya-ku, Tokyo 150-0002, Japan. 7Josai Hospital, 2-42-11, Kamiogi, Suginami-ku, Tokyo 167-0043, Japan. 8Diabetes and Lifestyle Center, Tomonaga Clinic, Shinyon curumu Building 9F, 4-2-23, Shinjuku, Shinjuku-ku, Tokyo 160-0022, Japan. 9Diabetes Center, Edogawa Hospital, Medical Plaza Shinozaki, SK Building, 7-15-12, Shinozaki-machi, Edogawa-ku, Tokyo 133-0057, Japan. 10Nishikawa Clinic, 2-16-3, Towa, Adachi-ku, Tokyo 120-0003, Japan. 11The Institute for Adult Diseases, Asahi Life Foundation, Asahiseimeisunaga building, 2-2-6, Nihonbashi Bakuro-cho, Chuo-ku, Tokyo 103-0002, Japan. 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https://openalex.org/W2906644679	https://europepmc.org/articles/pmc6306497?pdf=render	English	null	Waterlogging of Winter Crops at Early and Late Stages: Impacts on Leaf Physiology, Growth and Yield	Frontiers in plant science	2,018	cc-by	14,858	Waterlogging of Winter Crops at Early and Late Stages: Impacts on Leaf Physiology, Growth and Yield Rocío Antonella Ploschuk1, Daniel Julio Miralles1, Timothy David Colmer2, Edmundo Leonardo Ploschuk3 and Gustavo Gabriel Striker1,2* 1 IFEVA, Facultad de Agronomía, Universidad de Buenos Aires, CONICET, Buenos Aires, Argentina, 2 Faculty of Science, School of Agriculture and Environment, The University of Western Australia, Crawley, WA, Australia, 3 Facultad de Agronomía, Cátedra de Cultivos Industriales, Universidad de Buenos Aires, Buenos Aires, Argentina Waterlogging is expected to increase as a consequence of global climate change, constraining crop production in various parts of the world. This study assessed tolerance to 14-days of early- or late-stage waterlogging of the major winter crops wheat, barley, rapeseed and ﬁeld pea. Aerenchyma formation in adventitious roots, leaf physiological parameters (net photosynthesis, stomatal and mesophyll conductances, chlorophyll ﬂuorescence), shoot and root growth during and after waterlogging, and seed production were evaluated. Wheat produced adventitious roots with 20– 22% of aerenchyma, photosynthesis was maintained during waterlogging, and seed production was 86 and 71% of controls for early- and late-waterlogging events. In barley and rapeseed, plants were less affected by early- than by late-waterlogging. Barley adventitious roots contained 19% aerenchyma, whereas rapeseed did not form aerenchyma. In barley, photosynthesis was reduced during early-waterlogging mainly by stomatal limitations, and by non-stomatal constraints (lower mesophyll conductance and damage to photosynthetic apparatus as revealed by chlorophyll ﬂuorescence) during late-waterlogging. In rapeseed, photosynthesis was mostly reduced by non- stomatal limitations during early- and late-waterlogging, which also impacted shoot and root growth. Early-waterlogged plants of both barley and rapeseed were able to recover in growth upon drainage, and seed production reached ca. 79–85% of the controls, while late-waterlogged plants only attained 26–32% in seed production. Field pea showed no ability to develop root aerenchyma when waterlogged, and its photosynthesis (and stomatal and mesophyll conductances) was rapidly decreased by the stress. Consequently, waterlogging drastically reduced ﬁeld pea seed production to 6% of controls both at early- and late-stages with plants being unable to resume growth upon drainage. In conclusion, wheat generates a set of adaptive responses to withstand 14 days of waterlogging, barley and rapeseed can still produce signiﬁcant yield if transiently waterlogged during early plant stages but are more adversely impacted at the late stage, and ﬁeld pea is not suitable for areas prone to waterlogging events of 14 days at either growth stage. Keywords: waterlogging, crops, aerenchyma, photosynthesis, yield ORIGINAL RESEARCH published: 20 December 2018 doi: 10.3389/fpls.2018.01863 Edited by: y Johannes Kromdijk, University of Cambridge, United Kingdom Reviewed by: Angelika Mustroph, University of Bayreuth, Germany Nobuhito Sekiya, Mie University, Japan Sunita A. Ramesh, The University of Adelaide, Australia *Correspondence: Gustavo Gabriel Striker striker@agro.uba.ar Specialty section: This article was submitted to Crop and Product Physiology, a section of the journal Frontiers in Plant Science INTRODUCTION conductivity is severely reduced (Tan et al., 2018). Some species have the capacity to grow adventitious roots with aerenchyma, facilitating the diﬀusion of oxygen from the shoot into and along the roots, which allows cells to continue respiration and water and nutrient uptake (Armstrong, 1979; Justin and Armstrong, 1987; Colmer, 2003; Striker et al., 2007; Colmer and Greenway, 2011). The species studied present variable ability to generate adventitious roots with aerenchyma and thus a high gas-ﬁlled volume (porosity); root porosity in wheat can reach values of 19– 30% (Huang et al., 1994) and in barley 7–23% (Pang et al., 2004), whereas roots of rapeseed (Voesenek et al., 1999) and of ﬁeld pea (Healy and Armstrong, 1972) did not develop aerenchyma when in an oxygen deﬁcient medium. Over the last decades, the number of waterlogging episodes on croplands has increased worldwide, mainly due to more intense and unpredictable rainfalls associated with climate change (Hirabayashi et al., 2013; Core Writing Team et al., 2014). In turn, soils with signiﬁcant content of clay or intensely compacted by the repeated use of agricultural machinery can experience poor drainage, also entailing an increase in waterlogging occurrence (Jackson, 2004). Waterlogging impacts around 16% of soils in United States, aﬀecting also irrigated areas of India, China, and Pakistan (Pang et al., 2004). Moreover, the occurrence of ﬂash ﬂoods is expected to increase in Europe due to an intensiﬁed hydrological cycle by global warming (Feyen et al., 2012). In the Argentine Pampas, a vast plain of around 50 Mha, about 31% of the area has suﬀered recurrently from waterlogging events since 2002 (Kuppel et al., 2015). To illustrate economic losses associated with waterlogging or ﬂoods, in the United States the lost crop production was around $360 million per year during 2010–2016, and was even a greater loss than caused by drought in three out of the 7 years (Pedersen et al., 2017; USDA, 2017). So, soil waterlogging is a major abiotic stress of increasing importance, and it causes signiﬁcant yield losses of various crops. Therefore, it is necessary to understand how crop plants respond to waterlogging to identify traits contributing to tolerance. The impaired root functioning under waterlogging impacts the physiological responses of the shoots, particularly the carbon ﬁxation. INTRODUCTION Such lack of oxygen is rapidly reﬂected as a drop in the soil redox potential (Ponnamperuma, 1972). Plants react to soil oxygen deprivation through a series of anatomical, morphological and physiological responses to mitigate the eﬀects of soil anoxia. Soon after waterlogging, root metabolism shifts from aerobic respiration to the less eﬃcient fermentation to produce energy, leading to a reduction in ATP production (Gibbs and Greenway, 2003). The energy deﬁcit at root level results in a lack of phosphorylation of aquaporins, a reaction necessary to allow these proteins to regulate cell water ﬂux, so root hydraulic Wheat and barley are the most substantial cool-season cereals in the world, each having an annual production, respectively, of up to 749 and 141 million tons FAOSTAT, 2016. Rapeseed is the second largest oil-seed crop after soybean, presenting a production of 68 million ton FAOSTAT, 2016, providing oil with a high level of omega-9 (oleic fatty acid), one of the healthiest oils (Kwon et al., 1991). Field pea is an important cool season grain legume, with about 14 million ton of dry peas produced each year FAOSTAT, 2016. Each of these four species can suﬀer yield losses resulting from waterlogging (Siddique et al., 1993; Zhou and Lin, 1995; Setter and Waters, 2003; Pang et al., 2004). Depending on the waterlogging duration, soil type and genotypes, reductions in yield range from 15 to 25% in wheat and barley (Setter and Waters, 2003; Herzog et al., 2016), 30–45% in rapeseed (Gutierrez Boem et al., 1996), and 50–90% in ﬁeld pea (Cannell et al., 1979). Low photosynthesis rates can be a constraint to shoot and root growth, and ultimately reduce seed production (Sinclair and Horie, 1989). Only some studies have analyzed the impact of waterlogging throughout the entire plant life-cycle, describing responses in vegetative growth and seed production. Citation: Citation: Ploschuk RA, Miralles DJ, Colmer TD, Ploschuk EL and Striker GG (2018) Waterlogging of Winter Crops at Early and Late Stages: Impacts on Leaf Physiology, Growth and Yield. Front. Plant Sci. 9:1863. doi: 10.3389/fpls.2018.01863 Citation: Ploschuk RA, Miralles DJ, Colmer TD, Ploschuk EL and Striker GG (2018) Waterlogging of Winter Crops at Early and Late Stages: Impacts on Leaf Physiology, Growth and Yield. Front. Plant Sci. 9:1863. doi: 10.3389/fpls.2018.01863 December 2018 \| Volume 9 \| Article 1863 1 Frontiers in Plant Science \| www.frontiersin.org Waterlogging of Winter Crops Ploschuk et al. Frontiers in Plant Science \| www.frontiersin.org INTRODUCTION Waterlogging-induced partial stomatal closure could constrain internal CO2 levels and limit carbon ﬁxation (Malik et al., 2001; Striker et al., 2005), but this variable has rarely been quantiﬁed (e.g., in wheat, Wu et al., 2014; in rapeseed, Leul and Zhou, 1998). Apart from stomatal limitations, photosynthesis rates can also be determined by non-stomatal factors, such as mesophyll conductance (Jones, 1985; Flexas and Medrano, 2002), damage to photosystem II (PSII) caused by reactive oxygen species (ROS) (Nishiyama et al., 2006; Ashraf, 2012), and leaf chlorosis related to chlorophyll degradation due to an accelerated leaf senescence (Hörtensteiner, 2006; Araki et al., 2012). Although there are reports regarding the impact of waterlogging on carbon ﬁxation of the winter crops in this study (e.g., Huang et al., 1994 for wheat; Pang et al., 2004 for barley; Leul and Zhou, 1998 for rapeseed, and Malik et al., 2015 for ﬁeld pea), comparisons between these species are scarce (see de San Celedonio et al., 2017 for wheat and barley). gg g y g Wheat and barley are the most substantial cool-season cereals in the world, each having an annual production, respectively, of up to 749 and 141 million tons FAOSTAT, 2016. Rapeseed is the second largest oil-seed crop after soybean, presenting a production of 68 million ton FAOSTAT, 2016, providing oil with a high level of omega-9 (oleic fatty acid), one of the healthiest oils (Kwon et al., 1991). Field pea is an important cool season grain legume, with about 14 million ton of dry peas produced each year FAOSTAT, 2016. Each of these four species can suﬀer yield losses resulting from waterlogging (Siddique et al., 1993; Zhou and Lin, 1995; Setter and Waters, 2003; Pang et al., 2004). Depending on the waterlogging duration, soil type and genotypes, reductions in yield range from 15 to 25% in wheat and barley (Setter and Waters, 2003; Herzog et al., 2016), 30–45% in rapeseed (Gutierrez Boem et al., 1996), and 50–90% in ﬁeld pea (Cannell et al., 1979). Soil waterlogging imposes a considerable slow-down of the oxygen exchange between soil and roots, as gas diﬀusion rates are 10,000 times slower in water than in air (Armstrong, 1979). Consequently, waterlogged soils can quickly become anoxic at depths greater than a few centimeters, as the oxygen demand by roots and soil microorganisms’ respiration largely exceeds the inﬂux from the atmosphere (Visser and Voesenek, 2005). INTRODUCTION found that seed production is more compromised in wheat and barley when waterlogged at late stages (e.g., around ﬂowering) as compared with earlier stages, which probably relates to the importance of photoassimilate supply during these stages. A rise in temperatures and air vapor pressure deﬁcit (VPDair) during the later stages (i.e., mid-late spring) as compared to earlier ones (i.e., late winter to early spring), could also exacerbate the eﬀects of waterlogging. (isopyrazam+azoxystrobin) for wheat and barley; Orquesta, BASF R⃝ (ﬂuxapyroxad+epoxiconazole+pyraclostrobin) for ﬁeld pea, and K Mamboretá R⃝(captan) for rapeseed, at 50 and 80 days after sowing (DAS)] and insecticides [D Mamboretá R⃝ (dimethoate) for wheat, barley and rapeseed at 55 and 104 DAS]. y Three treatments were applied to plants: (i) well-drained controls watered daily and allowed to drain freely, (ii) waterlogged at an ‘early’ stage and (iii) waterlogged at a ‘late’ stage. Early-waterlogging, which coincided with the vegetative stage, was imposed at 65 DAS. Containers with their valves closed were ﬁlled with tap water for 14 days to reach 1–2 cm of water above the soil level of the tubes. After the waterlogging period ended, valves were opened so that the water drained and from then onwards, plants were watered daily to ﬁeld capacity until the end of the experiment, to assess their recovery. Late- waterlogging, which occurred during plant reproductive stages (85 DAS for wheat, barley and rapeseed; 87 DAS for ﬁeld pea), also lasted 14 days, and again recovery post-waterlogging was also monitored. In both waterlogging treatments, the purpose of having all species waterlogged at the same time was to have reliable comparisons among them, as diﬀerences in the environmental conditions [e.g., temperature and atmospheric vapor pressure deﬁcit (VPDair)] if waterlogged at diﬀerent moments could diﬀerentially impact on plant responses to waterlogging (e.g., Grassini et al., 2007; de San Celedonio et al., 2014). This study evaluated the tolerances to waterlogging in four widely used winter crops: wheat, barley, rapeseed and ﬁeld pea. Plants of these species were exposed to a 14-day waterlogging period imposed at an ‘early’ or a ‘late’ plant stage. In addition, to shed light on the factors limiting growth, a suite of parameters that inﬂuence carbon ﬁxation, such as stomatal and mesophyll conductances, leaf greenness and damage to PSII, along with internal CO2 were examined. INTRODUCTION These parameters, as well as root aerenchyma and growth, were measured not only during the waterlogging periods but also after, so that plant recovery was also assessed to maturity, as well as the resulting seed production. Site and Plant Material The experiment was carried out at the School of Agriculture, University of Buenos Aires, Argentina (34◦35′ S, 58◦29′ W), under outdoor conditions during July to November of 2016 [VPDair, maximum (Tmax) and minimum (Tmin) air temperature and photoperiod in Supplementary Figure S1]. Seeds of wheat (Triticum aestivum, cv. AGP FAST from Buck breeder, Argentina), barley (Hordeum vulgare, cv. Andreia from Quilmes malting, Argentina), rapeseed (Brassica napus, cv. Hyola 575 CL, from Advanta Seeds, Argentina) and ﬁeld pea (Pisum sativum, cv. Viper from AFA-Federated Argentinean Farmers Society, Argentina) were sown on July 12th. The varieties chosen, widely grown in Argentina, have similar durations of life cycle (i.e., ﬂowering and maturity). Fertilizer (Nitrofull Emerger R⃝, Argentina: 12% N, 5% P, 15% K, 2% Mg, 8% S, 3% Ca, 0.02% Zn, 0.2% Fe, 0.02% Mn and 0.015% Bo; % are by weight) was applied to the substrate of all tubes, distributed in three doses of 0.7 g each (2.1 g total per tube). This total amount per tube was based on providing N at a level equivalent to 200 kg per ha (typical dose used under ﬁeld conditions). In all treatments, the ﬁrst dose was added 58 DAS (1 week before early-waterlogging), the second dose was applied at 80 DAS (to coincide with 1 day after early waterlogging period ended), and the third dose was applied 102 DAS (to coincide with the start of recovery period after the late waterlogging). The split- application was aimed at reducing potential eﬀects of any nutrient leaching from the tubes either by watering or by waterlogging and subsequent drainage. INTRODUCTION As examples: in wheat, 20 days of waterlogging on 3–4 leaf-stage plants resulted in a ﬁnal dry mass and yield representing 95 and 90% of controls, respectively (Collaku and Harrison, 2002; Pampana et al., 2016a); in barley, plants attained 85 and 90% of controls in dry mass and yield when waterlogged for 20 days at 3–4 leaf-stage (Masoni et al., 2016); in rapeseed, 21 days of soil hypoxia, applied to 5- leaf stage plants, constrained growth as the stressed individuals attained 77% in dry mass and 73% in yield compared to the control (Leul and Zhou, 1998); in ﬁeld pea, 5 days of waterlogging had a substantial impact on plant dry mass accumulation and seed production as, respectively, stressed plants attained 35–50% and 5–25% of controls (Jackson, 1979; Pampana et al., 2016b). Nevertheless, information on root and shoot growth rates during waterlogging and subsequent recovery is scarce, and studies to functionally link leaf physiological responses with growth (i.e., RGR) and seed production (but see Li et al., 2011 for wheat are few). In addition, waterlogging can aﬀect plants diﬀerently depending on the growth stage (e.g., de San Celedonio et al., 2014 for wheat and barley; Gutierrez Boem et al., 1996 for rapeseed; Belford et al., 1980 for ﬁeld pea). de San Celedonio et al. (2014) Soil waterlogging imposes a considerable slow-down of the oxygen exchange between soil and roots, as gas diﬀusion rates are 10,000 times slower in water than in air (Armstrong, 1979). Consequently, waterlogged soils can quickly become anoxic at depths greater than a few centimeters, as the oxygen demand by roots and soil microorganisms’ respiration largely exceeds the inﬂux from the atmosphere (Visser and Voesenek, 2005). Such lack of oxygen is rapidly reﬂected as a drop in the soil redox potential (Ponnamperuma, 1972). Plants react to soil oxygen deprivation through a series of anatomical, morphological and physiological responses to mitigate the eﬀects of soil anoxia. Soon after waterlogging, root metabolism shifts from aerobic respiration to the less eﬃcient fermentation to produce energy, leading to a reduction in ATP production (Gibbs and Greenway, 2003). The energy deﬁcit at root level results in a lack of phosphorylation of aquaporins, a reaction necessary to allow these proteins to regulate cell water ﬂux, so root hydraulic December 2018 \| Volume 9 \| Article 1863 2 Waterlogging of Winter Crops Ploschuk et al. Experimental Design Plants were cultivated in polyvinyl chloride (PVC) tubes of 66 cm length and 10 cm diameter, with a capacity of 5.2 L. The bottom end of each PVC tube was ﬁtted with a ﬁne mesh which retained the soil but allowed water drainage (or entry during waterlogging, see below). Tubes were placed in 1 m side plastic cubical containers, which had a valve located at the bottom (so waterlogging and drainage could be regulated). Each tube was ﬁlled with a mixture (3:1) of sand and silty clay loam soil (Typic Argiudoll). 60 tubes were used per species (240 tubes in total). Three seeds per tube were sown, and after 1 week, seedlings were thinned to one per tube. Before sowing, ﬁeld pea seeds were inoculated with Rhizobium leguminosarum biovar viceae (Signum, Rizobacter R⃝) and sprayed with fungicide [Maxim XL, Syngenta R⃝ (Mefenoxam+ﬂudioxonil)] as a common agronomic practice. Fertilizer applications are described below. Tubes were kept free of weeds by hand removal and any diseases and insect pests were controlled as required, by applying fungicides [Reﬂect Xtra, Syngenta R⃝ Frontiers in Plant Science \| www.frontiersin.org Leaf Physiological Measurements Net photosynthesis (Pn), stomatal conductance (gs) and internal CO2 (Ci) were measured on the topmost fully expanded leaves of control and waterlogged plants, from the beginning of the treatments until the end of the experiment. Measurements were taken using a portable infrared gas analyser ((IRGA) Li-Cor 6400, Li-Cor Inc., Lincoln, NE, United States) under saturating light of 1500 µmol m−2 s−1 PPFD provided by the 6400-40 leaf chamber ﬂuorometer using a mix of 90% red and 10% blue light. Air ﬂow (300 mmol s−1) and CO2 concentration (400 µmol mol−1) in the reference chamber and block temperature (24◦C) were automatically controlled. Mesophyll conductance (gm) was calculated using the equation from Bernacchi et al., 2002: Root Aerenchyma Formation Aerenchyma formation was assessed in adventitious roots (white colored, 3.5–4 cm minimum length) of all species taken from control and waterlogged plants at the end of early- and late- waterlogging (n = 4 per species and treatment combination). Root segments of 2.5–3 cm were preserved in 70% ethanol, and then segments of 1 cm length were taken 2 cm from the apex and dehydrated in a series of increasing ethanol concentrations and then embedded in paraﬃn wax. Cross sections of 7–8 µm thick were cut using a steel blade and rotary microtome (Leica RM 2235, Leica Microsystems, Germany), then stained with safranin and fast green and mounted on Canada balsam. Representative images of root sections were photographed using an optical microscope (Zeiss Axioplan; Zeiss, Oberkochen, Germany) connected to a digital camera. The percentage occupied by aerenchyma in each cross-section was determined by adding up the areas of all present lacunae, to then divide it by the total area of the cross-section (Striker et al., 2014). These areas were quantiﬁed by hand using the free software ImageJ version 1.47 (National Institutes of Health: Bethesda, MD, United States). The ﬂowering period of each species was described by dating the beginning and end of ﬂowering. The approximate beginning of seed ﬁlling was also recorded. These stages were described according to the status of at least 50% of the plants of each species (Supplementary Figure S3). Plant Growth, Dry Mass and Seed Production Responses gm = Pn Ci 0∗(J+8(Pn+Rd)) J−4(Pn−Rd) Plants were harvested: (i) at the beginning of early-waterlogging (65 DAS), (ii) at the end of early-waterlogging (79 DAS), (iii) when late-waterlogging started (85 DAS with the exception of ﬁeld pea, which was harvested at 87 DAS), (iv) at the end of late-waterlogging (99 DAS with exception of ﬁeld pea, which was harvested at 101 DAS) and (v) at maturity. Six replicates were sampled for each species and treatments at all harvests. Each plant was divided into roots, shoots and, in case of mature plants, also seeds. All harvested material was oven dried at 57◦C for 72 h and weighed. The relative growth rates (RGR) were calculated for shoots and roots following the procedure by Hunt (1982). where Pn, Ci, and J are net photosynthesis, leaf internal CO2 concentration and electron transport rate, respectively. Leaf Rd (day respiration) values were extracted from the literature (Barbour et al., 2010; Shrestha, 2017; Walker et al., 2017). 0∗is the speciﬁcity factor of Rubisco for CO2 and O2 and was estimated from the response of 0∗to temperature as described by Bernacchi et al. (2002) by using the leaf temperature measured by the thermocouple in the chamber of the Li-Cor 6400. where Pn, Ci, and J are net photosynthesis, leaf internal CO2 concentration and electron transport rate, respectively. Leaf Rd (day respiration) values were extracted from the literature (Barbour et al., 2010; Shrestha, 2017; Walker et al., 2017). 0∗is the speciﬁcity factor of Rubisco for CO2 and O2 and was estimated from the response of 0∗to temperature as described by Bernacchi et al. (2002) by using the leaf temperature measured by the thermocouple in the chamber of the Li-Cor 6400. Leaf greenness was measured in young (top-most fully expanded leaf) and adult-basal leaves (lower third of the plant) using a chlorophyll meter (SPAD-502, Konica Minolta Sensing Inc., Osaka, Japan). This parameter is useful to examine the eﬀects of waterlogging on leaf yellowing, associated with N remobilisation and senescence in relation to leaf age among the tested species. Frontiers in Plant Science \| www.frontiersin.org Environmental Growing Conditions Air temperature and relative humidity were monitored with an automatic meteorological station (Davis Vantage Pro2, CA, United States) at the site of the experiment. VPDair was estimated as the diﬀerence between the actual air vapor pressure (ea) and the saturated vapor pressure (es) using the Clausius–Clapeyron equation. VPDair values ranged between 0.49 and 0.89 kPa during early-waterlogging and between 0.69 and 1.07 kPa during late- waterlogging. Air mean temperatures ranged between 10.4 and 20.2◦C during early-waterlogging and between 15 and 21.6◦C during late-waterlogging (Supplementary Figure S1). The redox potential of the substrate in control and waterlogged conditions was measured with a redox electrode (Fiedler et al., 2007). The soil pH, measured at both conditions, December 2018 \| Volume 9 \| Article 1863 Frontiers in Plant Science \| www.frontiersin.org 3 Waterlogging of Winter Crops Ploschuk et al. was used to transform the readings to Eh at pH 7 (Eh7) assuming a slope of 59 mV per pH unit (Bohn, 1971). Values for Eh7 dropped from 400–440 mV in drained conditions to 110–124 mV after 1 week of waterlogging and remained between 109 and 121 mV by the end of the 2nd week of waterlogging. Five days after waterlogging ceased was enough to allow Eh7 to recover to similar values to those of the well-drained control tubes. The evolution of Eh7 (reduction and recovery) was similar for both early- and late-waterlogging events (Supplementary Figure S2). and four replicates were used. SPAD and Fv/Fm measurements were taken two times per week for the entire duration of the experiment, and ﬁve replicates were used for each of these measurements. Statistical Analyses Physiological variables (A, gs, gm, Ci, SPADadult leaf, SPADyoung leaf, and Fv/Fm) were analyzed by a three-way ANOVA with ‘species,’ ‘treatment’ and ‘time’ as main factors. Calculated RGR values were compared between controls and each waterlogging treatment for each species and period of analysis using Student’s t-test (degrees of freedom = 10). Maximum quantum eﬃciency of PSII (Fv/Fm) was measured on top-most fully expanded leaves after a dark-adaptation period of 30 min by using leaf-clips and the OS-30p portable ﬂuorometer (Opti-Sciences Inc., United States). This parameter indicates the proportion of functional PSII reaction centers, so that it can be used to quantify the degree of photoinhibition (Maxwell and Johnson, 2000). Root aerenchyma percentages and dry mass responses were analyzed by a two-way ANOVA with ‘species’ and ‘treatment’ as main factors. Additional contrasts using Fisher’s LSD test were performed to compare treatments within species for shoot, root, and seed dry masses. Normality and homogeneity of variances of the data were checked before ANOVAs. Statistical analyses were performed using Infostat software (Di Rienzo et al., 2011) and All IRGA measurements were taken two times per week during waterlogging, and once a week during the recovery period, December 2018 \| Volume 9 \| Article 1863 Frontiers in Plant Science \| www.frontiersin.org 4 Waterlogging of Winter Crops Ploschuk et al. FIGURE 1 \| Net photosynthesis (Pn: A–D), stomatal conductance (gs: E–H), mesophyll conductance (gm: I–L) and internal CO2 (Ci: M–P) of control, early-waterlogged (Early wl) and late-waterlogged (Late wl) plants of wheat (A,E,I,M), barley (B,F,J,N), rapeseed (C,G,K,O) and ﬁeld pea (D,H,L,P) on the top-most fully expanded leaves, over time (DAS is days after sowing). Note that the scale for gm of ﬁeld pea (L) differs to those of the other species (I–K). Measurements were taken under saturating light of 1500 mmol m−2 s−1 PPFD provided by the 6400-40 leaf chamber ﬂuorometer using a mix of 90% red and 10% blue light. Air ﬂow, CO2 concentration in the reference chamber and block temperature were automatically controlled by the equipment at 300 mmol s−1, 400 µmol mol−1 (ppm) and 24◦C, respectively. Gray and black bars on the x-axis represent the 14-day early- and late- waterlogging periods, respectively. The dashed horizontal line in (M–P) indicates external CO2. Pn LSDinteraction = 4.99 µmol m−2 s−1; gs LSDinteraction = 0.10 mmol m−2 s−1; gm LSDinteraction = 0.26 mmol m−2 s−1, Ci LSDinteraction = 111 ppm. Statistical Analyses Measurements were taken under saturating light of 1500 mmol m−2 s−1 PPFD provided by the 6400-40 leaf chamber ﬂuorometer using a mix of 90% red and 10% blue light. Air ﬂow, CO2 concentration in the reference chamber and block temperature were automatically controlled by the equipment at 300 mmol s−1, 400 µmol mol−1 (ppm) and 24◦C, respectively. Gray and black bars on the x-axis represent the 14-day early- and late- waterlogging periods, respectively. The dashed horizontal line in (M–P) indicates external CO2. Pn LSDinteraction = 4.99 µmol m−2 s−1; gs LSDinteraction = 0.10 mmol m−2 s−1; gm LSDinteraction = 0.26 mmol m−2 s−1, Ci LSDinteraction = 111 ppm. The bars represent the LSD (Fisher’s protected least signiﬁcant difference at P = 0.05). ANOVA results are presented in Table 2. Values are means ± standard errors of four replicates. Measurements for rapeseed at 100 and 107 DAS are missing because of complete leaf abscission, after which the newly sprouted leaves were big enough to measure. graphs were made with GraphPad Prism 5 for Windows PRISM, GraphPad. 5 (2009)1. experiment, depending on the species and the timing of the stress (Figures 1–4, signiﬁcant ‘species × treatment’, ‘treatment × time’ and ‘species × treatment × time’ interactions in Table 1). In wheat, the early-waterlogging did not aﬀect physiological performance. Values measured for stomatal conductance (gs), mesophyll conductance (gm), internal CO2 (Ci) (Figures 1E,I,M) and Fv/Fm (Figure 2A) showed similar patterns with time for both the early-waterlogged and control plants. In agreement, there were no diﬀerences between treatments in net photosynthesis (Pn) and relative growth rates (RGRs) for shoot and roots (Figures 1A, 4A,E). Only slightly lower leaf greenness values (SPAD) for adult leaves (85% of controls) were evident in waterlogged plants by the end of the stress (Figures 3A,E). Through the recovery period, plants were able to restore SPAD values 5–7 days after the waterlogging was Statistical Analyses The bars represent the LSD (Fisher’s protected least signiﬁcant difference at P = 0.05). ANOVA results are presented in Table 2. Values are means ± standard errors of four replicates. Measurements for rapeseed at 100 and 107 DAS are missing because of complete leaf abscission, after which the newly sprouted leaves were big enough to measure. A–D), stomatal conductance (gs: E–H), mesophyll conductance (gm: I–L) and internal CO2 (Ci: M–P) of control, FIGURE 1 \| Net photosynthesis (Pn: A–D), stomatal conductance (gs: E–H), mesophyll conductance (gm: I–L) and internal CO2 (Ci: M–P) of control, early-waterlogged (Early wl) and late-waterlogged (Late wl) plants of wheat (A,E,I,M), barley (B,F,J,N), rapeseed (C,G,K,O) and ﬁeld pea (D,H,L,P) on the top-most fully expanded leaves, over time (DAS is days after sowing). Note that the scale for gm of ﬁeld pea (L) differs to those of the other species (I–K). Measurements were taken under saturating light of 1500 mmol m−2 s−1 PPFD provided by the 6400-40 leaf chamber ﬂuorometer using a mix of 90% red and 10% blue light. Air ﬂow, CO2 concentration in the reference chamber and block temperature were automatically controlled by the equipment at 300 mmol s−1, 400 µmol mol−1 (ppm) and 24◦C, respectively. Gray and black bars on the x-axis represent the 14-day early- and late- waterlogging periods, respectively. The dashed horizontal line in (M–P) indicates external CO2. Pn LSDinteraction = 4.99 µmol m−2 s−1; gs LSDinteraction = 0.10 mmol m−2 s−1; gm LSDinteraction = 0.26 mmol m−2 s−1, Ci LSDinteraction = 111 ppm. The bars represent the LSD (Fisher’s protected least signiﬁcant difference at P = 0.05). ANOVA results are presented in Table 2. Values are means ± standard errors of four replicates. Measurements for rapeseed at 100 and 107 DAS are missing because of complete leaf abscission, after which the newly sprouted leaves were big enough to measure. phyll conductance (gm: I–L) and internal CO2 (Ci: M–P) of control, FIGURE 1 \| Net photosynthesis (Pn: A–D), stomatal conductance (gs: E–H), mesophyll conductance (gm: I–L) and internal CO2 (Ci: M–P) of control, early-waterlogged (Early wl) and late-waterlogged (Late wl) plants of wheat (A,E,I,M), barley (B,F,J,N), rapeseed (C,G,K,O) and ﬁeld pea (D,H,L,P) on the top-most fully expanded leaves, over time (DAS is days after sowing). Note that the scale for gm of ﬁeld pea (L) differs to those of the other species (I–K). 1www.graphpad.com Waterlogging Affected Species Differentially for Leaf Physiology and Growth During Early and Late Life-Cycle Stages Waterlogging aﬀected diﬀerently all leaf physiological parameters measured and, thereby shoot and root growth along the 1www.graphpad.com December 2018 \| Volume 9 \| Article 1863 Frontiers in Plant Science \| www.frontiersin.org Frontiers in Plant Science \| www.frontiersin.org 5 Waterlogging of Winter Crops Ploschuk et al. FIGURE 2 \| Continued the 14-day early- and late-waterlogging periods, respectively. Fv/Fm LSDinteraction = 0.06. The bars represent the LSD (Fisher’s protected least signiﬁcant difference at P = 0.05). ANOVA results are presented in Table 2. Values are means ± standard errors of ﬁve replicates. Measurements for rapeseed at 100, 103, and 107 DAS are missing because of complete leaf abscission, after which the newly sprouted leaves were big enough to measure. FIGURE 2 \| Chlorophyll ﬂuorescence (Fv/Fm) of control, early-waterlogged (Early wl) and late- waterlogged (Late wl) plants of wheat (A), barley (B), rapeseed (C), and ﬁeld pea (D) on the top-most fully expanded leaves, over time (DAS is days after sowing). Gray and black bars on the x-axis represent (Continued) removed (Figures 3A,E), coinciding with the maintenance of the rest of the parameters until maturity (Figures 1A,E,I,M, 2A). Interestingly, 73% higher Pn values compared to controls were observed in previously waterlogged plants 1 week before maturity (Figure 1A), which also showed a 42% higher shoot RGR compared to controls during the 99–130 DAS period (Figure 4A). Similarly to what occurred in early-waterlogging, during late- waterlogging of wheat no diﬀerences in any physiological variable were observed, as gs, gm, Ci, Fv/Fm, SPAD, and Pn remained similar to controls (Figures 1A,E,M, 2A, 3A,E). Along recovery, physiological parameters maintained similar to controls, except for SPAD in adult leaves which began to decline until near maturity, reaching 18% of controls 1 week after water subsided; denoting accelerated plant senescence (Figure 3A). Although root growth was 13% of controls during late-waterlogging, shoot RGR presented no diﬀerences compared to controls; and during recovery root and shoot RGR attained 63 and 59% of controls, respectively (Figures 4I,M). In barley, early-waterlogging induced changes in leaf physiology, such as gs reduced to 38% of controls from 1 week of waterlogging (Figure 1F), followed by Ci lessened to 73% of controls by the end of the stress (Figure 1N); however, there were no changes either in gm or Fv/Fm. Pn was reduced to 58% of controls 1 week after waterlogging was imposed, likely due to stomatal limitations (Figure 1B). Waterlogging Affected Species Differentially for Leaf Physiology and Growth During Early and Late Life-Cycle Stages Measurements for rapeseed at 100, 103, and 107 DAS are missing because of complete leaf abscission, after which the newly sprouted leaves were big enough to measure. FIGURE 3 \| Leaf greenness of adult leaves located on the basal third of the plant (SPADadultleaf: A–D) and from the top-most fully expanded leaf (SPADyoungleaf: E–H) of control, early- waterlogged (Early wl) and late-waterlogged (Late wl) plants of wheat (A,E), barley (B,F), rapeseed (C,G) and ﬁeld pea (D,H), over time (DAS is days after sowing). Gray and black bars on the x-axis represent the 14-day early- and late-waterlogging periods, respectively. SPADadultleaf LSDinteraction = 5.90, SPADyoungleaf LSDinteraction = 5.79. The bars represent the LSD (Fisher’s protected least signiﬁcant difference at P = 0.05). Values are means ± standard errors of ﬁve replicates. Measurements for rapeseed at 100, 103, and 107 DAS are missing because of complete leaf abscission, after which the newly sprouted leaves were big enough to measure. imposed (55–15% of controls) (Figure 1B). A progressive fall in SPAD of adult leaves occurred 1 week after waterlogging until reaching minimum values (3.7–6.3 SPAD units) at the end of the stress (Figure 3B). Such poor physiological performance during the stress was in turn related to a negative root RGR due to root mortality (Figure 4N). During the recovery, gs and gm of previously waterlogged plants could not reach control values (Figures 1F,J), although Ci was fully restored a few days after the stress (Figure 1N). Adult leaves continued with very low SPAD values (i.e., senescing leaves) (Figure 3B). Despite Fv/Fm was restored during recovery (Figure 2B), Pn values could not be re-established up to control levels until 1 week before maturity (Figure 1B). So, plants were not able to resume root growth (RGR was still negative) and shoot RGR was close to zero, both symptoms of an approaching end of the plant life-cycle (Figures 4J,N). root RGR was negative, caused mainly by tissue death during waterlogging (Figure 4G). However, during recovery, rapeseed showed great ability to sprout buds (i.e., branching) soon after the stress was removed, allowing re-establishing growth from new leaves. Two weeks after waterlogging removal, gs and gm of these new leaves exhibited a peak, reaching higher values than controls (125 and 212% of controls, respectively), to later remain similar to controls until maturity (Figures 1G,K). Waterlogging Affected Species Differentially for Leaf Physiology and Growth During Early and Late Life-Cycle Stages Waterlogged plants showed a trend toward low SPAD values in adult and young leaves during the stress (Figures 3B,F). Shoot growth was not aﬀected but root RGR was reduced to 33% of controls due to early-waterlogging. Afterwards, during the recovery, barley showed an important ability to restore its gs, Ci, and SPAD values, along with a full recovery of Pn (similar to controls) in only 1 week after removing the stress (Figures 1B,F,N, 3B,F). In line with this, a 5.7-fold higher root RGR during the 79–99 DAS recovery period was observed concerning controls, and subsequently, shoot RGR was 53% higher than controls during the 99–130 DAS period (Figures 4B,F). Late-waterlogging resulted in more adverse eﬀects on the physiology and growth of barley, compared to early- waterlogging. Firstly, a decrease in gs (values attained 27% of controls) occurred 5 days after waterlogging (Figure 1F), and then gm had a drastic reduction 1 week after waterlogging started, attaining 9% of controls (Figure 1J). In addition, Fv/Fm dropped by 77% of controls at the end of the stress, indicating damage to PSII (Figure 2B), which concurred with a 5.7-fold raise in Ci by the end of waterlogging (Figure 1N). In line with these responses, plants showed reductions in Pn 5 days after waterlogging was FIGURE 2 \| Chlorophyll ﬂuorescence (Fv/Fm) of control, early-waterlogged (Early wl) and late- waterlogged (Late wl) plants of wheat (A), barley (B), rapeseed (C), and ﬁeld pea (D) on the top-most fully expanded leaves, over time (DAS is days after sowing). Gray and black bars on the x-axis represent (Continued December 2018 \| Volume 9 \| Article 1863 Frontiers in Plant Science \| www.frontiersin.org 6 Waterlogging of Winter Crops Ploschuk et al. FIGURE 3 \| Leaf greenness of adult leaves located on the basal third of the plant (SPADadultleaf: A–D) and from the top-most fully expanded leaf (SPADyoungleaf: E–H) of control, early- waterlogged (Early wl) and late-waterlogged (Late wl) plants of wheat (A,E), barley (B,F), rapeseed (C,G) and ﬁeld pea (D,H), over time (DAS is days after sowing). Gray and black bars on the x-axis represent the 14-day early- and late-waterlogging periods, respectively. SPADadultleaf LSDinteraction = 5.90, SPADyoungleaf LSDinteraction = 5.79. The bars represent the LSD (Fisher’s protected least signiﬁcant difference at P = 0.05). Values are means ± standard errors of ﬁve replicates. Frontiers in Plant Science \| www.frontiersin.org Waterlogging Affected Species Differentially for Leaf Physiology and Growth During Early and Late Life-Cycle Stages Fv/Fm and Ci of these new leaves showed similar values than controls since waterlogging was removed (Figure 2C), while SPAD values were 43% higher than those of controls, a pattern that persisted until maturity (Figure 3C). This recovery allowed reaching Pn values 28% higher than controls 2 weeks after waterlogging, remaining similar to controls onwards (Figure 1C). During the 79–99 DAS recovery period, root RGR was 3.6-fold higher compared to controls (Figure 4C) while shoot RGR was 65% of controls. Later, during 99–130 DAS, shoot RGR in recovered plants was 79% higher than the controls (Figure 4G). Rapeseed showed important changes in leaf physiology due to early-waterlogging, as gs was drastically reduced during the stress period, reaching values close to zero from 1 week after waterlogging (Figure 1G). A reduction in gm occurred 3 days after waterlogging, to continue dropping until reaching minimum values 1 week after the beginning of the stress (Figure 1K). Simultaneously, early-waterlogged plants presented Fv/Fm values 63% of controls, indicating PSII damage (Figure 2C), followed by a rise of 2.1-fold higher than controls in Ci (Figure 1O). At the same time, Pn was progressively reduced during waterlogging until reaching values close to zero. Early- waterlogged plants showed a drastic reduction in SPAD values on adult leaves, indicating early leaf senescence (Figure 3C) and most of the leaves dropped oﬀby the end of the stress. Waterlogging had a drastic impact on root growth also, as As in early-waterlogging, rapeseed plants exposed to waterlogging at late-stage showed important diﬀerences in leaf physiological performance and growth during the stress. Reductions in gs and gm occurred 5 days after the stress was imposed, reaching values close to zero by 1 week of waterlogging (Figures 1G,K). Additionally, signiﬁcant diﬀerences for leaf greenness were observed, not only in adult leaves but also in younger ones (65 and 37% of controls, respectively) (Figures 3C,G). Anticipated leaf senescence was followed by abscission both in young and adult leaves (abscised leaves were included in the quantiﬁcation of shoot dry mass). In addition, Fv/Fm values stood around 65% of controls throughout late-waterlogging indicating damage to the PSII (Figure 2C), December 2018 \| Volume 9 \| Article 1863 7 Waterlogging of Winter Crops Ploschuk et al. FIGURE 4 \| Shoot and root relative growth rate (RGR) of control vs. early-waterlogged plants (Early wl) (A–D; E–H, respectively) and control vs. Waterlogging Affected Species Differentially for Leaf Physiology and Growth During Early and Late Life-Cycle Stages late-waterlogged plants (Late wl) (I–L; M–P, respectively) of wheat (A,E,I,M), barley (B,F,J,N), rapeseed (C,G,K,O), and ﬁeld pea (D,H,L,P), over time (DAS is days after sowing). RGRs comparisons from control vs. early-waterlogged plants considered three periods: early-waterlogging [65–79 days after sowing (DAS)], early recovery (79–99 DAS excepting for ﬁeld pea, which occurred at 79–101 DAS) and late recovery (99–130 DAS excepting for ﬁeld pea, which occurred 101–130 DAS). RGRs comparisons from controls vs. late-waterlogged plants considered two periods: late-waterlogging (85–99 DAS excepting for ﬁeld pea, which was during 87–101 DAS) and the recovery until the end of the experiment (99–130 DAS excepting for ﬁeld pea, which lasted from 101 to 130 DAS). Asterisks indicate signiﬁcant differences between treatments within species (∗P = 0.05, ∗∗P = 0.01, ∗∗∗P = 0.001). Values are means ± standard errors of 6 replicates. FIGURE 4 \| Shoot and root relative growth rate (RGR) of control vs. early-waterlogged plants (Early wl) (A–D; E–H, respectively) and control vs. late-waterlogged plants (Late wl) (I–L; M–P, respectively) of wheat (A,E,I,M), barley (B,F,J,N), rapeseed (C,G,K,O), and ﬁeld pea (D,H,L,P), over time (DAS is days after sowing). RGRs comparisons from control vs. early-waterlogged plants considered three periods: early-waterlogging [65–79 days after sowing (DAS)], early recovery (79–99 DAS excepting for ﬁeld pea, which occurred at 79–101 DAS) and late recovery (99–130 DAS excepting for ﬁeld pea, which occurred 101–130 DAS). RGRs comparisons from controls vs. late-waterlogged plants considered two periods: late-waterlogging (85–99 DAS excepting for ﬁeld pea, which was during 87–101 DAS) and the recovery until the end of the experiment (99–130 DAS excepting for ﬁeld pea, which lasted from 101 to 130 DAS). Asterisks indicate signiﬁcant differences between treatments within species (∗P = 0.05, ∗∗P = 0.01, ∗∗∗P = 0.001). Values are means ± standard errors of 6 replicates. FIGURE 4 \| Shoot and root relative growth rate (RGR) of control vs. early-waterlogged plants (Early wl) (A–D; E–H, respectively) plants (Late wl) (I–L; M–P, respectively) of wheat (A,E,I,M), barley (B,F,J,N), rapeseed (C,G,K,O), and ﬁeld pea (D,H,L,P), over TABLE 1 \| F-values of three-way ANOVA (factors: ‘species’, ‘treatment’, and ‘time’) for net photosynthesis (Pn), stomatal conductance (gs), mesophyll conductance (gm), internal CO2 (Ci), Fv/Fm, and SPAD values of adult and young leaves of control, early- and late- waterlogged plants of wheat, barley, rapeseed and ﬁeld peaA. Root Aerenchyma Formation Under Waterlogging Differed Among Species In rapeseed (Figures 5G–I) and ﬁeld pea (Figures 5J–L), both species of relatively low waterlogging tolerance, in spite of forming relatively short adventitious roots (3–4 cm length) in the root-shoot junction, no noticeable root aerenchyma formation was found after neither early- nor late-waterlogging in the examined cross-sections. Roots from control plants evaluated at the same times when each waterlogging period ended also did not contain aerenchyma. Field pea plants subjected to late-waterlogging behaved in a similar way to those exposed to this stress at the early-stage. Plants presented an abrupt fall in physiological performance soon after the stress was imposed, reaching values close to zero in gs and gm in only 3 days after the stress was imposed (Figures 1H,L). A drastic reduction in Fv/Fm occurred 1 week after waterlogging (Figure 2D), as well as a rise in Ci (Figure 1P) and negligible values of Pn (Figure 1D). Additionally, SPAD values from both adult and young leaves were progressively reduced (Figures 3D,H) indicating early leaf senescence in Root Aerenchyma Formation Under Waterlogging Differed Among Species Aerenchyma formation in adventitious roots in response to waterlogging depended on the species (‘species × treatment’ interaction in Table 2). In wheat, waterlogging at early- stage induced the development of aerenchyma-lacunae in the root cortex, with an average of 20.4 ± 4.4% of the root cross-sectional area occupied by lacunae (Figure 5B). Wheat roots subjected to the stress at late stage showed a similar capacity to develop aerenchyma, with an average of 22.2 ± 2.9% (Figure 5C). Wheat roots from controls either from the early- or the late-waterlogging treatment contained low proportions of aerenchyma (2% on average) (Figure 5A). g In ﬁeld pea, early-waterlogging severely aﬀected leaf physiology and growth. Values close to zero were observed in gs 1 week after waterlogging started, along with a similar pattern in gm (Figures 1H,L). A progressive decline in SPAD of adult leaves was observed 1 week after waterlogging was applied, joined by decreases in SPAD of young leaves at the end of the stress (Figures 3D,H). Fv/Fm declined to 49% of controls (i.e., damage to PSII), and a rise in Ci occurred at the same time (twofold higher compared to controls) (Figures 1P, 2D) matching the near to zero values for Pn (Figure 1D). Field pea had not only its root RGR aﬀected, but also its shoot RGR reduced during early-waterlogging (ca. 38% of controls) (Figures 4D,H). During the recovery, ﬁeld pea was not able to restore leaf functioning (Figures 1D,H,L,P, 2D, 3D,H). Root RGR continued showing negative values throughout the experiment, and shoot RGR was even more reduced with respect to controls during the recovery than along the waterlogging period (5% of controls in 79–101 DAS) (Figures 4D,H). In adventitious roots of barley, aerenchyma also formed in the cortex in response to waterlogging. Root samples from the early-waterlogging had lacunae occupying 18.9 ± 3.6% of the root cross-sectional area (Figure 5E). Late-waterlogging also triggered the formation of aerenchyma in the roots of barley, but the lacunae were 13.9 ± 3.3%, being slightly lower than for roots from the early-waterlogging treatment (Figure 5F) (Table 2). Contrastingly, roots of control plants for both, early- and late-waterlogging treatments, only presented 1.5% of aerenchyma on average (Figure 5D). Waterlogging Affected Species Differentially for Leaf Physiology and Growth During Early and Late Life-Cycle Stages \| y ( p , , ) p y ( n), (gs), p y (gm), internal CO2 (Ci), Fv/Fm, and SPAD values of adult and young leaves of control, early- and late- waterlogged plants of wheat, barley, rapeseed and ﬁeld peaA. Variables Species (Sp) Treatment (T) Time Sp × T Sp × time T × time Sp × T × time Net photosynthesis 255.30∗∗∗ 287.27∗∗∗ 51.06∗∗∗ 57.23∗∗∗ 6.41∗∗∗ 17.24∗∗∗ 5.43∗∗∗ Stomatal conductance 98.47∗∗∗ 132.55∗∗∗ 110.23∗∗∗ 31.85∗∗∗ 3.93∗∗∗ 13.30∗∗∗ 4.43∗∗∗ Mesophyll conductance 65.04∗∗∗ 31.62∗∗∗ 3.83∗∗∗ 9.88∗∗∗ 2.25∗∗∗ 3.42∗∗∗ 3.17∗∗∗ Internal CO2 55.51∗∗∗ 25.90∗∗∗ 23.01∗∗∗ 9.88∗∗∗ 3.06∗∗∗ 6.05∗∗∗ 2.73∗∗∗ Fv/Fm 196.81∗∗∗ 134.90∗∗∗ 15.88∗∗∗ 75.36∗∗∗ 8.98∗∗∗ 7.62∗∗∗ 6.93∗∗∗ SPADadultleaf 92.40∗∗∗ 480.42∗∗∗ 152.85∗∗∗ 68.18∗∗∗ 25.77∗∗∗ 21.56∗∗∗ 13.08∗∗∗ SPADyoungleaf 269.69∗∗∗ 120.42∗∗∗ 21.64∗∗∗ 58.92∗∗∗ 10.77∗∗∗ 7.77∗∗∗ 5.99∗∗∗ ∗∗∗P = 0.001. ADegrees of freedom for each source of variation were: 3 (‘species’), 2 (‘treatment’), 6 (‘species × treatment’). 12, 35, 18, 46, and 360 (‘time,’ ‘species × time,’ ‘treatment’ × ‘time,’ ‘species × treatment × time’ and ‘error’ for Pn, gs, gm, and Ci, respectively). 15, 44, 23, 61, and 536 (‘time,’ ‘species × time,’ ‘treatment’ × ‘time,’ ‘species × treatment × time’ and ‘error’ for Fv/Fm, SPAD of adult and young leaves, respectively). ∗∗∗P = 0.001. ADegrees of freedom for each source of variation were: 3 (‘species’), 2 (‘treatment’), 6 (‘species × treatment’). 12, 35, 18, 46, and 360 (‘time,’ ‘species × time,’ ‘treatment’ × ‘time,’ ‘species × treatment × time’ and ‘error’ for Pn, gs, gm, and Ci, respectively). 15, 44, 23, 61, and 536 (‘time,’ ‘species × time,’ ‘treatment’ × ‘time,’ ‘species × treatment × time’ and ‘error’ for Fv/Fm, SPAD of adult and young leaves, respectively). December 2018 \| Volume 9 \| Article 1863 Frontiers in Plant Science \| www.frontiersin.org 8 Waterlogging of Winter Crops Ploschuk et al. in coincidence with higher Ci values showing a peak at the end of waterlogging (2.2-fold higher) (Figure 1O). Drastic reductions in Pn were observed, showing values close to zero 1 week after waterlogging was imposed (Figure 1C). Such a poor performance during waterlogging was evidenced by a negative root RGR (Figure 4O). After water was removed, plants showed partial capacity to recover from the stress due to bud sprouting; gs and gm of new leaves attained 55% and similar values of controls, respectively (Figures 1G,K). Additionally, after 1 week of recovery, leaf greenness of the newly produced leaves reached higher values than controls (Figures 3C,G). Waterlogging Affected Species Differentially for Leaf Physiology and Growth During Early and Late Life-Cycle Stages Also, Fv/Fm and Ci values were restored (Figures 1O, 2C) and Pn attained 67% of controls near maturity (Figure 1C). However, as the time to recover from the late-waterlogging (late-stage) was shorter than for the early-stage, the recovery was lower. Shoot RGR represented 56% of controls while root RGR was low and similar between treatments in line with the end of the plants life-cycle (Figures 4K,O). waterlogged plants. Consequently, shoot RGR decreased during the stress (78% of controls) and plants presented negative root RGR (Figures 4L,P). Soon after waterlogging was removed, plants were not able to recover as revealed by the negative shoot and root RGR values (Figures 4L,P). Frontiers in Plant Science \| www.frontiersin.org Dry Mass and Seed Mass Responses Are Affected by Early- and Late- Waterlogging Seed mass of stressed plants represented 85% of controls (Table 3). Conversely, late-waterlogging caused a drastic reduction in dry masses of both roots and shoots (stressed plants attained 7 and 35% of controls, respectively), and these plants produced seed mass about 32% of controls (Table 3). Dry Mass and Seed Mass Responses Are Affected by Early- and Late- Waterlogging In barley, early-waterlogged plants attained 69% of controls in root dry mass, but shoots were unaﬀected. Seed mass of stressed plants represented 85% of controls (Table 3). Conversely, late-waterlogging caused a drastic reduction in dry masses of both roots and shoots (stressed plants attained 7 and 35% of controls, respectively), and these plants produced seed mass about 32% of controls (Table 3). In rapeseed, early-waterlogging compromised root dry mass (60% of controls) along with shoot dry mass (84% of controls). Seed yield showed values of 79% of controls (Table 3). Late- waterlogging had a greater impact on growth than early-, as root and shoot dry masses represented 48 and 54% of controls, respectively. Seed yield was reduced to 26% of controls (Table 3). Field pea was the most adv waterlogging. Early-waterlogging root and shoot mass (plants attai respectively) (Table 3). Late-w components to 29 and 31% of c respectively (Table 3). Seed p reduced by both waterlogging t late-waterlogged plants had only compared to controls (Table 3). DISCUSSION This study provides a compr progressive eﬀects of 2 weeks o late-stages on wheat, barley, rap an integrated analysis of root carbon ﬁxation and its regulators Field pea was the most adversely impacted species by waterlogging. Early-waterlogging provoked great losses of root and shoot mass (plants attained 10 and 15% of controls, respectively) (Table 3). Late-waterlogging reduced these components to 29 and 31% of controls for roots and shoots, respectively (Table 3). Seed production was considerably reduced by both waterlogging treatments, where early- and late-waterlogged plants had only 4.4 and 9.5% of seed mass compared to controls (Table 3). Field pea was the most adversely impacted species by waterlogging. Early-waterlogging provoked great losses of root and shoot mass (plants attained 10 and 15% of controls, respectively) (Table 3). Late-waterlogging reduced these components to 29 and 31% of controls for roots and shoots, respectively (Table 3). Seed production was considerably reduced by both waterlogging treatments, where early- and late-waterlogged plants had only 4.4 and 9.5% of seed mass compared to controls (Table 3). of controls (Table 3). In contrast, late-waterlogging signiﬁcantly reduced both root and shoot dry mass as they attained 75% of controls, and there was a reduction in seed mass (71% of controls) (Table 3). In barley, early-waterlogged plants attained 69% of controls in root dry mass, but shoots were unaﬀected. Dry Mass and Seed Mass Responses Are Affected by Early- and Late- Waterlogging In wheat, waterlogging at the early-stage did not impact on shoot or root dry mass, but seed per plant produced was 86% TABLE 2 \| F-values of two-way ANOVA (factors: ‘species’ and ‘treatment’) for aerenchyma percentages in adventitious root cross sections as treatment responses of wheat, barley, rapeseed, and ﬁeld pea. Early-waterlogging Late-waterlogging Species (Sp) Treatment (T) Sp × T Species (Sp) Treatment (T) Sp × T Total 22.67∗∗∗ 59.18∗∗∗ 20.24∗∗∗ 25.14∗∗∗ 68.09∗∗∗ 25.14∗∗∗ A ∗∗∗: P = 0.001. Analyses were conducted separately on data at the end of each waterlogging period (control vs. early-waterlogged plants; control vs. late- waterlogged plants). ADegrees of freedom for each source of variation were: 3 (‘species’), 1 (‘treatment’), 3 (Sp × T), and 24 (‘error’). December 2018 \| Volume 9 \| Article 1863 9 Ploschuk et al. Waterlogging of Winter Crops FIGURE 5 \| Adventitious root cross sections of control (A,D,G,J), early (B,E,H,K) and late waterlogged (C,F,I,L) plants of wheat (A–C), barley (D–F), rapeseed (G–I) and ﬁeld pea (J–L). Cross-sections were taken at 20 mm from the root apex of 25–40-mm-roots. Asterisks denote examples of aerenchyma lacunae. Scale bars represent 100 µm. Values of aerenchyma percentage for wheat were 2.0 ± 0.6%, 20.4 ± 4.4%, and 22.2 ± 2.9% for control, early- and late-waterlogged plants, respectively. Barley presented values of 1.5 ± 0.5%, 18.9 ± 3.6%, and 13.9 ± 3.3% for control, early- and late-waterlogged plants, respectively. Rapeseed and ﬁeld pea did not show any signs of aerenchyma formation under any treatment. FIGURE 5 \| Adventitious root cross sections of control (A,D,G,J), early (B,E,H,K) and late waterlogged (C,F,I,L) plants of wheat (A–C), barley (D–F), rapeseed (G–I) and ﬁeld pea (J–L). Cross-sections were taken at 20 mm from the root apex of 25–40-mm-roots. Asterisks denote examples of aerenchyma lacunae. Scale bars represent 100 µm. Values of aerenchyma percentage for wheat were 2.0 ± 0.6%, 20.4 ± 4.4%, and 22.2 ± 2.9% for control, early- and late-waterlogged plants, respectively. Barley presented values of 1.5 ± 0.5%, 18.9 ± 3.6%, and 13.9 ± 3.3% for control, early- and late-waterlogged plants, respectively. Rapeseed and ﬁeld pea did not show any signs of aerenchyma formation under any treatment. of controls (Table 3). In contrast, late-waterlogging signiﬁcantly reduced both root and shoot dry mass as they attained 75% of controls, and there was a reduction in seed mass (71% of controls) (Table 3). DISCUSSION In rapeseed, early-waterlogging compromised root dry mass (60% of controls) along with shoot dry mass (84% of controls). Seed yield showed values of 79% of controls (Table 3). Late- waterlogging had a greater impact on growth than early-, as root and shoot dry masses represented 48 and 54% of controls, respectively. Seed yield was reduced to 26% of controls (Table 3). This study provides a comprehensive evaluation of the progressive eﬀects of 2 weeks of waterlogging at early- or late-stages on wheat, barley, rapeseed and ﬁeld pea through an integrated analysis of root aerenchyma formation, leaf carbon ﬁxation and its regulators (i.e., stomatal or non-stomatal This study provides a comprehensive evaluation of the progressive eﬀects of 2 weeks of waterlogging at early- or late-stages on wheat, barley, rapeseed and ﬁeld pea through an integrated analysis of root aerenchyma formation, leaf carbon ﬁxation and its regulators (i.e., stomatal or non-stomatal December 2018 \| Volume 9 \| Article 1863 10 Waterlogging of Winter Crops Ploschuk et al. TABLE 3 \| Shoot, root and seed dry mass (g per plant) of mature plants of wheat, barley, rapeseed and ﬁeld pea under control, and after early-waterlogging (Early wl) and late-waterlogging (Late wl) treatments followed by a recovery period. form aerenchyma in roots, as we also observed. The lack of aerenchyma in the adventitious roots of both rapeseed and ﬁeld pea would likely explain the greater impact of waterlogging on their physiological and growth performance as compared with barley and wheat. DISCUSSION Control Early wl Late wl Wheat Shoot 22.4 ± 0.4 a 23.3 ± 0.9 (104) a 16.9 ± 1.1 (75) b Root 5.3 ± 0.3 a 4.8 ± 0.02 (90) a 3.5 ± 0.3 (66) b Seed 8.9 ± 0.3 a 7.6 ± 0.5 (86) b 6.3 ± 0.4 (71) c Barley Shoot 29.9 ± 1.4 a 28.9 ± 1.6 (97) a 10.5 ± 1.3 (35) b Root 7.5 ± 0.6 a 5.1 ± 1.0 (69) b 0.5 ± 0.1 (7) c Seed 10.6 ± 0.4 a 9.0 ± 0.5 (85) b 3.4 ± 0.4 (32) c Rapeseed Shoot 19.3 ± 0.5 a 16.3 ± 0.7 (84) b 10.4 ± 1.6 (54) c Root 5.0 ± 0.3 a 3.0 ± 0.4 (60) b 2.5 ± 0.3 (50) b Seed 5.7 ± 0.2 a 4.5 ± 0.1 (79) b 1.5 ± 0.3 (26) c Field pea Shoot 13.7 ± 2.1 a 2.0 ± 0.3 (15) b 4.2 ± 1.0 (31) b Root 0.9 ± 0.1 a 0.1 ± 0.02 (10) b 0.3 ± 0.1 (29) b Seed 7.5 ± 0.8 a 0.3 ± 0.1 (4) b 0.6 ± 0.2 (8) b Values attained by plants following waterlogging and recovery periods are given as the percentage of controls in brackets. Different letters across a row denote signiﬁcant differences among treatments within a species based on Fisher’s LSD test (P = 0.05). Values are means ± standard errors of 6 replicates. Waterlogging Impacts on Carbon Fixation Differentially Among Winter Crops Through Stomatal and Non-stomatal Limitations Wheat presented no diﬀerences compared to controls in its leaf physiological parameters during early- and late-waterlogging. So, photosynthesis, stomatal and mesophyll conductances, as well as chlorophyll ﬂuorescence were not aﬀected by 14 days of waterlogging. These results concur with those reported by de San Celedonio et al. (2017) for 45-day-old plants subjected to 15 days of waterlogging where no diﬀerences in photosynthesis were found at the end of the stress. However, other authors have observed reductions in photosynthesis and stomatal conductance of 85 and 80%, respectively, in 21-day-old plants after 14 days of waterlogging, successfully reestablishing values to controls after a 14-day-recovery (Malik et al., 2001). Moreover, despite the similar physiological performances for early- and late-waterlogged plants of wheat and controls in the present study, a recent review by Herzog et al. (2016) indicated variability in the responses of wheat to waterlogging in their analysis of experiments that involved 23 diﬀerent cultivars exposed to a range of waterlogging treatments from 7 to 34 days (data were from 15 separate studies), showing a wide range in photosynthesis reductions from 7 to 85% of controls. Values attained by plants following waterlogging and recovery periods are given as the percentage of controls in brackets. Different letters across a row denote signiﬁcant differences among treatments within a species based on Fisher’s LSD test (P = 0.05). Values are means ± standard errors of 6 replicates. Values attained by plants following waterlogging and recovery periods are given as the percentage of controls in brackets. Different letters across a row denote signiﬁcant differences among treatments within a species based on Fisher’s LSD test (P = 0.05). Values are means ± standard errors of 6 replicates. limitations), combined with responses in root and shoot growth monitored during the stress and the recovery periods, along with seed production at maturity. Wheat and Barley Developed Substantial Aerenchyma in Adventitious Roots While Rapeseed and Field Pea Did Not Barley showed reductions in photosynthesis (58% of controls) during early-waterlogging attributed to stomatal limitations, as revealed by a lower gs (43% of controls) along with falls in internal CO2 (Ci; 72% of controls). Partial closing of stomata during waterlogging for barley was also found by Pang et al. (2004), where gs followed a similar pattern to that of photosynthesis in 4-leaf stage plants waterlogged for 21 days. Nevertheless, no data on Ci of waterlogged plants for barley were available to match with gs, so this study demonstrates that stomata closing can indeed constrain the Ci available for photosynthesis during early-waterlogging in barley. In line with our results where no damage to PSII (i.e., low Fv/Fm) was detected during waterlogging, Zeng et al. (2013) reported only a slight decrease in Fv/Fm (ca. 10%) in 1-leaf stage seedlings of barley waterlogged for 14 days. Regarding to recovery, a study by Pang et al. (2004) showed a successful recovery for 5 out of 6 genotypes analyzed 1 week after water subsided; which agrees with plants fully reestablishing gs and Pn after a 3-day-recovery in our work. During late- waterlogging, photosynthesis of waterlogged barley was further reduced down to values representing only 25% of controls. This was ﬁrstly caused by partial stomatal closure (gs 32% of controls) that resulted in a lower Ci (71% of controls). After 1 week of waterlogging, non-stomatal constraints to photosynthesis were also evident, such as reduced gm along Wheat and barley produced adventitious roots with considerable aerenchyma. In wheat, aerenchyma occupied 20% of the root cross sections in early-waterlogged plants and 22% in late- waterlogged plants, being comparable with 19–30% aerenchyma in adventitious roots of wheat when waterlogged for 17 days commencing at the 3-leaf stage (Huang et al., 1994), and concurring with the range of values summarized by Herzog et al. (2016). In barley, root aerenchyma of waterlogged plants was slightly lower than in wheat (19 and 13%, for early- and late-waterlogging) but in line with those reported in previous works (e.g., Pang et al., 2004; de San Celedonio et al., 2017). The presence of substantial root aerenchyma is known to facilitate tissue aeration (Armstrong, 1979), thereby it could be related to a lessened impact of root- zone hypoxia on plants’ physiological performance Colmer and Greenway, 2011), as for wheat and barley in the present study. Frontiers in Plant Science \| www.frontiersin.org The Effect of Waterlogging on Plant Growth and Seed Production Is Determined by Species and Growth Stage with damage to the PSII (drops in Fv/Fm), which derived in a rise in Ci (3.2-fold of controls) by the end of waterlogging. Damage to PSII in waterlogged barley plants resulting in decreased rates of Pn were also registered by Pang et al. (2007). Regarding recovery, physiological variables were fully restored 2 weeks after water had drained from the soil, near maturity, which suggests that barley is able to recover the leaf physiological functioning even at advanced plant stages. with damage to the PSII (drops in Fv/Fm), which derived in a rise in Ci (3.2-fold of controls) by the end of waterlogging. Damage to PSII in waterlogged barley plants resulting in decreased rates of Pn were also registered by Pang et al. (2007). Regarding recovery, physiological variables were fully restored 2 weeks after water had drained from the soil, near maturity, which suggests that barley is able to recover the leaf physiological functioning even at advanced plant stages. Wheat showed similar performance to controls in shoot and root growth during early-waterlogging and its recovery. During late- waterlogging, only root RGR was aﬀected. However, despite that growth inhibition when waterlogged, root RGR increased during recovery until reaching 63% of the controls, and indicating ability of wheat for root growth resumption after the stress. Similar to our results, Malik et al. (2001) found a substantial decline in root growth due to 14 days of waterlogging on 21- day-old wheat plants (root RGR 26% of controls), but also a subsequent root growth recovery after water drainage. Seed production per plant varied between waterlogging treatments (as in de San Celedonio et al., 2014), being 86 and 71% of controls for early- and late-waterlogged plants, respectively. Lesser yield losses in wheat (near 92% of controls) were reported in Li et al. (2011), probably because of a shorter waterlogging (6 days) divided in three periods of 2 days each, at 7- and 9-leaf stage, and also in heading (i.e., spikes appearance). Nevertheless, the tolerance to waterlogging regarding seed production depends on the combination of environmental conditions and considered genotypes, apart from growth stage and duration of waterlogging (Herzog et al., 2016). As examples, 14-day-waterlogged plants of wheat at 22 days after sowing (cv. Wyalkatchem) showed reductions in seed weight per plant to 32% of controls (Robertson et al., 2009); while 15 days of waterlogging during ﬂowering (cv. The Effect of Waterlogging on Plant Growth and Seed Production Is Determined by Species and Growth Stage Karasu-90) carried reductions to 62% of controls in seed production (Olgun et al., 2008). g In rapeseed, waterlogging had a substantial impact on leaf physiological parameters during the early-stage. Pn reached values close to zero after 1 week of waterlogging, mainly related to non-stomatal factors, such as highly diminished gm and lower Fv/Fm (56% of controls), which led to an increase in Ci values up to 1.9-fold of controls. Taken together, these results indicate that the reduction in carbon ﬁxation was not related to the lack of internal CO2 as substrate but associated with a damaged photosynthetic apparatus. High sensitivity to waterlogging exhibited by rapeseed was also found in 5-leaf stage plants waterlogged for 3 weeks, showing lower Pn and increased Ci (Leul and Zhou, 1998). All physiological parameters were fully restored in the new developed leaves when assessed 10 days after water subsided. These new leaves were the result of the ability of plants to produce new branches, as already reported for plants of rapeseed (cv. Avatar) after being waterlogged for 14 days at stem elongation stage (Wollmer et al., 2018). Late-waterlogging had a greater impact over physiology, since plants not only showed Pn values close to zero but also exhibited generalized senescence as even young leaves had low greenness. Afterwards, although generation of new leaves through branching was observed with 1 week of recovery, these leaves were not able to reach control values for the measured physiological variables. In barley, shoot RGR was unaﬀected during and after early- waterlogging, and although root growth was negatively impacted by waterlogging, such plants showed 7-fold greater root RGR than controls during recovery. Enhancement in root growth after water subsided was probably supported initially by consumption of carbon reserves (as seen by de San Celedonio et al., 2017 for 20-day-waterlogged plants), and subsequently aided by current assimilation after leaf physiological parameters were restored during the 2nd week of recovery. Late-waterlogging severely aﬀected growth as root RGR was negative (denoting tissue mortality) during and after waterlogging, and shoot RGR values were close to zero during recovery. Related to the latter, greenness of adult leaves was signiﬁcantly lower in waterlogged plants, leading to a detrimental carbon ﬁxation performance at the plant level, and contributing to (partially) explain such a poorer growth. Wheat and Barley Developed Substantial Aerenchyma in Adventitious Roots While Rapeseed and Field Pea Did Not Rapeseed and ﬁeld pea did not form aerenchyma in adventitious roots under any growing condition. The inability of rapeseed to generate aerenchyma concurs with one earlier study on seedlings in which the short adventitious roots formed when grown in agar had very low gas-ﬁlled porosity (Voesenek et al., 1999). For ﬁeld pea, Healy and Armstrong (1972) suggested that this species is not able (or has a minimal capacity) to December 2018 \| Volume 9 \| Article 1863 11 Ploschuk et al. Waterlogging of Winter Crops Frontiers in Plant Science \| www.frontiersin.org December 2018 \| Volume 9 \| Article 1863 CONCLUSION There were substantial diﬀerences among the four winter crop species in tolerance to waterlogging. Wheat leaf physiology and shoot growth were not signiﬁcantly aﬀected by waterlogging in early- or late-stages of growth, but seed production was reduced to 86 and 71% of controls when plants experienced early- and late-waterlogging, respectively. In barley and rapeseed the growth stage when the stress occurred was critical for determining the eﬀects on leaf physiological performance, dry mass responses and seed production (seed mass was on average 82 and 29% of controls in early- and late- waterlogging, respectively). For barley during early-waterlogging, photosynthesis was compromised by stomatal limitations and root growth was impacted; however, upon drainage plants recovered. During late-waterlogging of barley, photosynthesis was also initially reduced by stomatal limitations but then by non-stomatal (i.e., lower Fv/Fm and gm) eﬀects, and negative root RGR (indicating death of root tissue) was also observed; physiological parameters were restored only near maturity, leading to an unsuccessful growth recovery. In rapeseed, photosynthesis was compromised by non-stomatal limitations and root RGR was negative in both waterlogging treatments, and there were diﬀerences between growth stages in the ability (and available time) to recover from the stress, as plants could only show an acceptable recovery after water subsided in early-waterlogging. Field pea was severely aﬀected either by early- or late-waterlogging, attaining on average 6% of controls for seed mass. During both waterlogging treatments, photosynthesis was decreased by non- stomatal limitations and root and shoot growths were both also compromised, and recovery was poor. Therefore, this study contributes to the understanding of the diﬀerential tolerances to early- and late-waterlogging of wheat, barley, rapeseed and ﬁeld pea, by integrating leaf physiological variables related to carbon ﬁxation, the ability (or not) to form aerenchyma in adventitious roots, along with shoot and root growth during the stress and importantly also recovery post-waterlogging, and ultimately, the impact on plant seed production. Field pea waterlogged at early- and late-stages showed lower shoot RGR than controls, not only during the stress, but also after water subsided. During recovery, previously late- waterlogged plants presented negative shoot RGR, denoting leaf detachment. In relation with root growth, both waterlogging treatments resulted in root mortality (i.e., negative RGR) and values could not be restored, continuing with tissue death until maturity. These results contrast with those of Cannell et al. The Effect of Waterlogging on Plant Growth and Seed Production Is Determined by Species and Growth Stage Chlorosis of basal leaves of 1/2- week-old barley waterlogged for 14 days could be associated with nitrogen remobilisation (Pang et al., 2007; Zeng et al., 2013). The impact on seed production was higher in late- than in early-waterlogging, attaining values of 32 and 85% of controls, probably because early-waterlogging allowed for an eﬀective recovery of physiological and growth parameters by the time of the critical period for yield determination. Contrasting timing of waterlogging during the life-cycle can be a determining factor for barley yield, as also reported that waterlogging during tillering (25–45 days after emergence) caused 25% reduction in ﬁnal seed p y g Field pea exhibited a leaf physiological performance severely aﬀected by waterlogging at both growth stages, typical of waterlogging-sensitive species. One week after waterlogging, a fall in gs was observed, and photosynthesis reached values close to zero, being the latter caused more likely by non- stomatal constraints characterized by a diminished gm followed by an increase of Ci. In addition, damage to PSII was evident, reﬂecting another constraint for carbon ﬁxation per se; as well as clear symptoms of chlorophyll degradation due to lower SPAD values in both adult and young leaves, as compared to controls. Previous reports showed that gs resulted in being a variable very sensitive to waterlogging for ﬁeld pea, given the fact that 9-leaf-stage plants after 4 days of waterlogging showed values of 26 and 52% of controls for adult and young leaves, respectively (Jackson, 1979). Nevertheless, this study indicates that stomatal closure seems not to be the primary constraint for photosynthesis as Ci values increased in comparison to controls. The increase in Ci appears to be related to the damage to PSII and potentially in the activity of Rubisco as suggested by the low Fv/Fm along with highly reduced gm. In line with this high sensitivity to waterlogging, for both early- and late-waterlogging, there was irreversible damage to leaf physiology as none of the variables were restored during recovery. December 2018 \| Volume 9 \| Article 1863 Frontiers in Plant Science \| www.frontiersin.org 12 Ploschuk et al. Waterlogging of Winter Crops mass while in pre-ﬂowering (65–85 days after emergence) led to a 75% reduction of seed mass (de San Celedonio et al., 2014). by the detrimental eﬀect on plant seed production, with values of 4 and 8% of controls for early- and late-waterlogging. CONCLUSION (1979) in which growth stage was important in determining the eﬀects of short-term (5-day) waterlogging on pea; as plants waterlogged at 24-day-old (vegetative stage) attained 41 and 47% of controls in shoot and seed dry mass, respectively, while when the stress was imposed on 47-day-old plants (ﬂowering stage), these plants attained 34 and 25% of controls in shoot and seed dry mass. In our study, 2 weeks of water excess had severe adverse eﬀects at both growth stages and so did not allow us to discriminate responses according to the growth stage. Similarly, another report showed dramatic reductions of shoot RGR (only 17% of controls) after exposing 35-day old plants of ﬁeld pea to 1-week waterlogging when assessed 10 days after water subsided (Solaiman et al., 2007). The sensitivity of ﬁeld pea to waterlogging is further highlighted The Effect of Waterlogging on Plant Growth and Seed Production Is Determined by Species and Growth Stage Even waterlogging for 5 days at the beginning of ﬂowering decreased seed production in ﬁeld pea to 38% of controls (Pampana et al., 2016b). Through the early-waterlogging period rapeseed showed positive shoot RGR but presented negative root RGR. Hypoxia-induced root mortality during waterlogging along with consumption of taproot reserves and low carbon ﬁxation at plant level due to leaf senescence (i.e., adult leaves turned purple and then senescent, see also Gutierrez Boem et al., 1996; Wollmer et al., 2018) would constrain root growth during the stress period. Importantly, root RGR exhibited 3.6-fold greater values compared to controls during recovery in coincidence with the restoration of the leaf physiological activity after the rapid sprouting of new leaves during the recovery. Concerning late-waterlogging, plants showed negative root RGR (i.e., root mortality) along with shoot RGR of 88% of controls during waterlogging, concurring with ﬁndings for 53-day-old rapeseed waterlogged for 14 days (Gutierrez Boem et al., 1996). During recovery, root growth was almost zero and shoot RGR was only 44% of controls. As an expected consequence of the adverse eﬀects of waterlogging on leaf physiology and growth, seed production was reduced but varied according to the growth stage at which waterlogging occurred; early-waterlogged plants attained 79% of the seed mass of controls, while the ones waterlogged at the late- stage showed values of 27% of controls. As in barley, leaf physiology and growth in early-waterlogged rapeseed might have been restored by the time yield deﬁnition occurred, so seed production was substantially less aﬀected compared with late-waterlogging. Observations by Wollmer et al. (2018) also found that the sensitivity of rapeseed to 14- day-waterlogging varied according to plants’ growth stage when waterlogged; as the stress applied at stem elongation led to a seed mass 75% of controls, while waterlogging at ﬂoral bud appearance caused a seed production of 85% of controls. Frontiers in Plant Science \| www.frontiersin.org REFERENCES Planta 103, 302–309. doi: 10.1007/ BF00386701 Herzog, M., Striker, G. G., Colmer, T. D., and Pedersen, O. (2016). Mechanisms of waterlogging tolerance in wheat–a review of root and shoot physiology. Plant Cell Environ. 39, 1068–1086. doi: 10.1111/pce.12676 Bohn, H. L. (1971). Redox potentials. Soil Sci. 112, 39–45. doi: 10.1097/00010694- 197107000-00007 Hirabayashi, Y., Mahendran, R., Koirala, S., Konoshima, L., Yamazaki, D., Watanabe, S., et al. (2013). Global ﬂood risk under climate change. Nat. Clim. Chang. 3:816. doi: 10.1038/nclimate1911 Cannell, R. Q., Gales, K., Snaydon, R. W., and Suhail, B. A. (1979). 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Plant Physiol. 130, 1992–1998. doi: 10.1104/pp. 008250 Healy, M. T., and Armstrong, W. (1972). The eﬀectiveness of internal oxygen transport in a mesophyte (Pisum sativum L.). FUNDING V. Gómez and Santiago L. Poggio for their advice on the cultivation of rapeseed and ﬁeld pea, respectively. RP was supported by a doctoral fellowship from CONICET (Argentina). This study was supported by the grants PIP-CONICET 11220150100041CO (National Research Council of Argentina) This study was supported by the grants PIP-CONICET 11220150100041CO (National Research Council of Argentina) and UBACyT 20020160100111BA (University of Buenos Aires). This study was supported by the grants PIP-CONICET 11220150100041CO (National Research Council of Argentina) and UBACyT 20020160100111BA (University of Buenos Aires). and UBACyT 20020160100111BA (University of Buenos Aires). ACKNOWLEDGMENTS The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2018.01863/ full#supplementary-material The authors would like to thank Lucía B. González for her technical assistance during the experiment, and Nora The authors would like to thank Lucía B. González for her technical assistance during the experiment, and Nora AUTHOR CONTRIBUTIONS All authors listed above made a substantial, direct, and intellectual contribution to this work. RP, DM, TC, and GS designed the experiments. EP discussed physiological measurements and trained RP to perform them. RP wrote the ﬁrst draft of the manuscript with GS. DM, EP, and TC made signiﬁcant comments and inputs to successive versions of the paper. All authors read and approved the text for publication. December 2018 \| Volume 9 \| Article 1863 Frontiers in Plant Science \| www.frontiersin.org 13 Ploschuk et al. 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A. C. J. (2017). Flooding and low oxygen responses in plants. Funct. Plant Biol. 44, iii–vi. doi: 10.1071/FPv44n9_FO Zeng, F., Shabala, L., Zhou, M., Zhang, G., and Shabala, S. (2013). Barley responses to combined waterlogging and salinity stress: separating eﬀects of oxygen deprivation and elemental toxicity. Front. Plant. Sci. 4:313. doi: 10.3389/fpls. 2013.00313 Ponnamperuma, F. N. (1972). “The chemistry of submerged soils,” in Advances in Agronomy, Vol. 24, ed. D. Sparks (Cambridge, MA: Academic Press), 29–96. Agronomy, Vol. 24, ed. D. Sparks (Cambridge, MA: Academic Press), 29–96. PRISM, GraphPad. 5 (2009). GraphPad Software. San Diego, CA: PRISM. Zhou, W., and Lin, X. (1995). Eﬀects of waterlogging at diﬀerent growth stages on physiological characteristics and seed yield of winter rape (Brassica napus L.). Field Crops Res. 44, 103–110. doi: 10.1016/0378-4290(95)00075-5 Robertson, D., Zhang, H., Palta, J. A., Colmer, T., and Turner, N. C. (2009). Waterlogging aﬀects the growth, development of tillers, and yield of wheat through a severe, but transient, N deﬁciency. Crop Pasture Sci. 60, 578–586. doi: 10.1071/CP08440 Setter, T. L., and Waters, I. (2003). Review of prospects for germplasm improvement for waterlogging tolerance in wheat, barley and oats. Plant Soil. 253, 1–34. Frontiers in Plant Science \| www.frontiersin.org REFERENCES doi: 10.1023/A:1024573305997 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. Shrestha, A. (2017). Variability in Mesophyll Conductance to CO2 in Grain Legumes. ph.D. thesis, University of Sidney, Sydney, NSW. Copyright © 2018 Ploschuk, Miralles, Colmer, Ploschuk and Striker. 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 © 2018 Ploschuk, Miralles, Colmer, Ploschuk and Striker. 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. Siddique, K. H. M., Walton, G. H., and Seymour, M. (1993). A comparison of seed yields of winter grain legumes in Western Australia. Aust. J. Exp. Agric. 33, 915–922. doi: 10.1071/EA9930915 Sinclair, T. R., and Horie, T. (1989). Leaf nitrogen, photosynthesis, and crop radiation use eﬃciency: a review. Crop Sci. 29, 90–98. doi: 10.2135/cropsci1989. 0011183X002900010023x December 2018 \| Volume 9 \| Article 1863 Frontiers in Plant Science \| www.frontiersin.org 15
https://openalex.org/W4388666256	https://journal.uokufa.edu.iq/index.php/ghjec/article/download/14030/11884	Arabic	null	تحليل الاداء المالي للقطاع المصرفي الخاص في ظل جائحة كورونا دراسة حالة لمصرفي (بغداد ، الاستثمار)	Maǧallaẗ al-ġabī	2,022	cc-by	12,888	44 7 - 25 7 P. P Ghari Journal of Faculty of Administration and Economics Vol. 18 (No.3) 2022 - Al 44 7 - 25 7 P. P Ghari Journal of Faculty of Administration and Economics Vol. 18 (No.3) 2022 - Al Al-Ghari Journal of Faculty of Administration and Economics حتليل االداء املايل للقطاع املصريف اخلاص يف ظل جائحة كورونا )دراسة حالة ملصريف (بغداد ، االستثمار أ. د حسين ديكان درويش الباحثة ابتسامه حسوني نور Al-Ghari Journal of Faculty of Administration and Economics حتليل االداء املايل للقطاع املصريف اخلاص يف ظل جائحة كورونا )دراسة حالة ملصريف (بغداد ، االستثمار أ. د حسين ديكان درويش الباحثة ابتسامه حسوني نور Analysis of the financial performance of the private banking sector during the Corona pandemic Case Study of a Banker (Baghdad, Investment) Analysis of the financial performance of the private banking sector during the Corona pandemic Case Study of a Banker (Baghdad, Investment) Analysis of the financial performance of the private banking sector during the Corona pandemic Case Study of a Banker (Baghdad, Investment) Researcher Ebtisama Hassooni Noor Prof. Dr. Hussein Diekan Darweesh Al- Dulaimi Researcher Ebtisama Hassooni Noor Abstract : The study aims to shed light on the special cognitive aspects of the analysis of the financial performance of a sample of private banks in during the Corona pandemic, and the study also seeks to give a clear picture of the effects and repercussions produced by the Corona pandemic in banking activity and how the financial performance of the banking sector is affected by those repercussions represented by the financial indicators of banks, and the importance of research lies from the novelty of the subject based on the novelty of the financial crisis The problem of the research is summarized in the main question :(What is the impact of the Corona pandemic on the financial performance of private banks), under the hypothesis (the Corona pandemic negatively affected the financial performance of the banks of the study sample compared to the period before the pandemic), and from perspective and to achieve the goal of the study, a sample of (2) Iraqi commercial banks was selected for a period of time It extended (6) years that were divided into two periods, the first was represented by the years (2016-2019) the period before the Corona pandemic and the second was represented by the years (2020-2021) period after the beginning of the pandemic. The study reached a set of conclusions, the most important of which are: The results of the financial analysis of the banks of the research sample showed a decrease in some indicators of financial performance for the period after the beginning of the Corona pandemic compared to the period before the pandemic. The most significant recommendations are to work to continue banking developments by directing and involving the management and staff of private banks through the association of private banks in advanced courses and under the supervision of the Central Bank of Iraq to familiarize them with the general guidelines that are being worked on to evaluate the bank, as well as to identify the basic and effective ways to overcome financial crises by contributing to banking conferences and seminars. Keywords: Financial Performance Analysis, Private Banking Sector, Corona Pandemic. :المستخلص تهدف الدراسة الى تسليط الضوء على الجوانب المعرفية الخاصة تحليل االداء المالي لعينة من المصارف الخاصة في ظل جائحة كورونا، كما تسعى الدراسة الى اعطاء صورة واضحة عن االثار والتداعيات التي افرزتها جائحة كورونا في النشاط المصرفي وكيف تاثر االداء المالي للقطاع المصرفي بتلك التداعيات المتمثل ة بالمؤشرات المالية ،للمصارف وتكمن اهمية البحث من حداثة الموضوع استنادا الى حداثة االزمة المالية التي تسببت بها جائحة كورونا (: على القطاعات االقتصادية والمصرفية، وتتلخص مشكلة البحث في التساؤل الرئيس االتي ما تأثير جائحة كورونا على االداء المالي للمصارف الخاصة ،) ( في ظل فرضية اثرت جائحة كورونا سلباً في االداء المالي للمصارف عينة الدراسة مقارنة بالمدة التي سبقت الجائحة ،) ومن ( هذا المنطلق ولتحقيق هدف الدراسة تم اختيار عينة مكونة من2 ) مصارف تجارية عراقية ( خاصة بفترة زمنية امتدت6 ) سنوات( تم تقسيمها الى فترتين االولى تمثلت بالسنوات2016 - 2019 ( ) فترة ما قبل جائحة كورونا والثانية تمثلت بالسنوات2020 - 2021 ) فترة بعد بداية .الجائحة : اذ توصلت الدراسة الى مجموعة من االستنتاجات اهمها اظهرت نتائج التحليل المالي للمصارف عينة البحث حصول انخفاض في بعض مؤشرات االداء المالي لفترة ما بعد بداية جائحة ك .ورونا مقارنة بالفترة السابقة للجائحة اما ابرز التوصيات العمل على مواكبة التطورات المصرفية من خالل توجيه واشراك ادارات ومالكات المصارف الخاصة عن طريق رابطة المصارف االهلية بدورات متقدمة وباشراف البنك المركزي العراقي لتعريفهم بالخطوط العامة التي 725 44 7 - 25 7 P. P Ghari Journal of Faculty of Administration and Economics Vol. 18 (No.3) 2022 - Al Al-Ghari Journal of Faculty of Administration and Economics يجري العمل بها لتقييم المصرف، كذلك التعرف على السبل االساسية والفاعلة لتخطي االزمات .المالية وذلك من خالل االسهام في المؤتمرات والندوات المصرفية : الكلمات المفتاحية تحليل ،االداء المالي ،القطاع المصرفي الخاص جائحة كورونا . المقدمة: ت ساهم المص ارف بشكل كبير ومؤثر في دعم نمو االقتصاد الوطني فهي تعد المؤسسات المالية التي تقوم بدور كبير في توفير السيولة النقدية الالزمة لتمويل المشر ات عو االستثمارية وتحتل المؤسسات المصرفية مكانة كبيرة في االقتصاديات المعاصرة فهي احدى المؤسسات المؤثرة بشكل مباشر في السياستين المالية والنقدية السيما في مجال االئتمان الداخلي والخارجي وكذلك تقوم بدور الوساطة بين المقرضين والمستثمرين فهي بذلك تقوم بإقتراض االم وال من 726 44 7 - 25 7 P. P Ghari Journal of Faculty of Administration and Economics Vol. 18 (No.3) 2022 - Al Al-Ghari Journal of Faculty of Administration and Economics المقرضين كحسابات جارية وودائع مقابل فائدة واقراضها للمستثمرين على شكل قروض او عن طريق المشاركة في رؤوس اموال المشر ات عو االستثمارية من اجل تنظيم الحركة المالية في السوق عبر سياسة اقتصادية كلية فاعلة لها ابعاد مؤثرة في العملية التنموية ونهوض االقتصاد .الوطني Al-Ghari Journal of Faculty of Administration and Economics 18 (No.3) 2022 - Al Al-Ghari Journal of Faculty of Administration and Economics تكمن مشكلة الدراسة في االجابة عن : التساؤالت اآلتية 1 - ما تأثير جائحة كورونا على االداء المالي للمصارف الخاصة ؟ 2 - ( ما مستوى اختالف درجة تأثير جائحة كوروناCOVID-19 ) ما بين المصارف عينة .الدراسة : فرضيات الدراسة :ولتحقيق اهداف البحث، صيغتْ الفرضية اآلتية 1 - اثرت جائحة كورونا سلباً في االداء المالي للمصارف عينة الدراسة مقارنة بالمدة ا لتي .سبقت الجائحة : اهداف الدراسة تهدف هذه الدراسة اساس ًا الى تحليل االداء المالي للقطاع المصرفي قبل وبعد انتش ار جائحة كورونا س عا يةً الى:تحقيق االهداف الفرعية االخرى واهمها 1 - تسليط الضوء عل.ى أهم مؤشرات االداء المالي للقطاع المصرفي العراقي 2 - بيان مدى تأثر المصارف عينة الدراسة فيما بينها في االثار المترتبة عن جائحة كورون ا ( COVID-19 . ) وكيفية السيطرة عليها من قبل االدارة المصرفية والتحكم بها 3 - مقارنة وترتيب كفاءة االداء للمصارف عينة الدراسة قبل.وبعد جائحة كورونا : حدود الدراسة :اما حدود الدراسة فهي ًاوال- : الحدود المكانية شملت الدراسة عينة من المصارف الخاصة (مصرف بغداد، مصرف ( االستثمار)، الجراء الجانب العملي من الدراسة، إذ تم اختيار2) مصارف لعينة ال دراسة وذلك لتوافر البيانات المالية والتقارير( السنوية للسنوات2016 - 2021 ) المنشورة ف ي .هيئة االوراق المالية العراقية خالل مدة البحث ًثانيا- ( الحدود الزمانية: وتمثلت بالمدة2016 - 2019) وهي المدة التي سبقت جائحة كورون ا ( COVID-19 ( ) والمدة2020 - 2021 ) اي مدة ما بعد بداية الجائحة، وحسب البيانات المالية المتا.حة من قبل الجهات الرسمية :أسلوب الدراسة استعملت الباحثة األسلوب الوصفي النظري لوصف االطار النظري لمفهوم تحليل االداء ااالمالي للجهاز المصرفي، فضالً عن األسلوب التحليلي المستند على استخراج النسب المالية لأ 44 7 - 25 7 P. P Ghari Journal of Faculty of Administration and Economics Vol. 18 (No.3) 2022 - Al 44 7 - 25 7 P. Ghari Journal of Faculty of Administration and Economics Vol. 18 (No.3) 2022 - Al Al-Ghari Journal of Faculty of Administration and Economics تكمن مشكلة الدراسة في االجابة عن : التساؤالت اآلتية 1 - ما تأثير جائحة كورونا على االداء المالي للمصارف الخاصة ؟ 2 - ( ما مستوى اختالف درجة تأثير جائحة كوروناCOVID-19 ) ما بين المصارف عينة .الدراسة : فرضيات الدراسة :ولتحقيق اهداف البحث، صيغتْ الفرضية اآلتية 1 - اثرت جائحة كورونا سلباً في االداء المالي للمصارف عينة الدراسة مقارنة بالمدة ا لتي .سبقت الجائحة Al-Ghari Journal of Faculty of Administration and Economics ( فمنذ أواخر عام2019 ) واجه العالم جائحة سلبية انتشرت عبر الدول اصطلح عل يها تسمي ة جائحة كورونا (COVID-19) المستجد(فايروس تاجي شديد العدوى واالنتقال بين البش ر يصيب الجهاز التنفسي مسببا الوفاة) ( 1 ) حيث اجبرت هذه الجائحة معظم دول العالم على اغالق حدودها لما لهذه الجائحة من انعكاسات سلبية على جميع القطاعات والمجاالت االقتصادية واالجتماعية والمالية وتمثل جائحة كورونا اكبر اختبار للنظام المالي حتى اآلن بعد اال زمة العالمية( 2008 ) حيث يشكل هذا الوباء صدمة اقتصادية كلية عالمية تؤثر في كبح النشاط اال قتصادي المحلي واالقليمي والعالمي من حيث استدامة حدة الركود االقتصادي. ويواجه ال نظام المالي التحدي(المزدوج) والمتمثل ب تراجع معدالت النمو االقتصادي وتدني مستويات تدفق ،االئتمان في ظل تزايد المخاطر، وقد ادت هذه الصدمة الى ركود النظام المالي كما ادى ني تد الن شاط االقتصادي الى اعادة تسعير وتنظيم هيكل األصول في االسواق المالية، اذ يفضل مق دمو التمويل زيادة االصول اآلمنة على المدى القصير من جهة ، ومن جهة اخرى ف أ ن المخاطر االئتمانية سترتفع ًكثيرا مما يؤدي الى ارتفاع حافز االحتفاظ برؤوس االموال وارتفاع م عدالت سيولة ا لنظام المصرفي . اهمية الدراسة: تتجلى اهمية الدراسة في حداثة الموضوع الذي سيشكل النواة للمزيد من الدراسات ال تي لها صلة بهذا الموضوع ، استنادا الى حداثة االزمة المالية التي تسببت بها جائحة كورونا في القطاعات االقتصادية والمصرفية كافة، فهو يعطينا فكرة واضحة عن اثر الجائحة في االداء المالي للقطاع المصرفي. فضالً عن اهمية الدراسة في ظل االزمة االقتصادية العالمية نتيجة جائحة كورونا ا لتي اثرت في االداء المالي لجميع القطاعات االقتصادية المكونة للناتج المحلي االجمالي ، كذل ك توفير الا دلة ال تجريبية من واقع بيانات المصارف الخاصة العاملة في العراق، بشأن مؤشرا ت االستقرار المالي قبل وبعد جائحة كورونا، وتقديم الفائدة للمستثمرين واالطراف ذات الع القة ح ول االثر الفعلي لجائحة كورونا في التقارير المالية للمصارف العراقية الخاصة. ة ة 727 44 7 - 25 7 P. P Ghari Journal of Faculty of Administration and Economics Vol. : اهداف الدراسة تهدف هذه الدراسة اساس ًا الى تحليل االداء المالي للقطاع المصرفي قبل وبعد انتش ار جائحة كورونا س عا يةً الى:تحقيق االهداف الفرعية االخرى واهمها تهدف هذه الدراسة اساس ا الى تحليل االداء المالي للقطاع المصرفي قبل وبعد انتش ار جائحة كورونا س عا يةً الى:تحقيق االهداف الفرعية االخرى واهمها 1 - تسليط الضوء عل.ى أهم مؤشرات االداء المالي للقطاع المصرفي العراقي 2 - بيان مدى تأثر المصارف عينة الدراسة فيما بينها في االثار المترتبة عن جائحة كورون ا ( COVID-19 . ) وكيفية السيطرة عليها من قبل االدارة المصرفية والتحكم بها 3 - مقارنة وترتيب كفاءة االداء للمصارف عينة الدراسة قبل.وبعد جائحة كورونا :حدود الدراسة :اما حدود الدراسة فهي ا ًاوال- : الحدود المكانية شملت الدراسة عينة من المصارف الخاصة (مصرف بغداد، مصرف ( االستثمار)، الجراء الجانب العملي من الدراسة، إذ تم اختيار2) مصارف لعينة ال دراسة وذلك لتوافر البيانات المالية والتقارير( السنوية للسنوات2016 - 2021 ) المنشورة ف ي .هيئة االوراق المالية العراقية خالل مدة البحث ًاوال- : الحدود المكانية شملت الدراسة عينة من المصارف الخاصة (مصرف بغداد، مصرف ( االستثمار)، الجراء الجانب العملي من الدراسة، إذ تم اختيار2) مصارف لعينة ال دراسة وذلك لتوافر البيانات المالية والتقارير( السنوية للسنوات2016 - 2021 ) المنشورة ف ي .هيئة االوراق المالية العراقية خالل مدة البحث اا ًثانيا- ( الحدود الزمانية: وتمثلت بالمدة2016 - 2019) وهي المدة التي سبقت جائحة كورون ا ( COVID-19 ( ) والمدة2020 - 2021 ) اي مدة ما بعد بداية الجائحة، وحسب البيانات المالية المتا.حة من قبل الجهات الرسمية اا ًثانيا- ( الحدود الزمانية: وتمثلت بالمدة2016 - 2019) وهي المدة التي سبقت جائحة كورون ا ( COVID-19 ( ) والمدة2020 - 2021 ) اي مدة ما بعد بداية الجائحة، وحسب البيانات المالية المتا.حة من قبل الجهات الرسمية ًثانيا- ( الحدود الزمانية: وتمثلت بالمدة2016 - 2019) وهي المدة التي سبقت جائحة كورون ا ( COVID-19 ( ) والمدة2020 - 2021 ) اي مدة ما بعد بداية الجائحة، وحسب البيانات المالية المتا.حة من قبل الجهات الرسمية :أسلوب الدراسة استعملت الباحثة األسلوب الوصفي النظري لوصف االطار النظري لمفهوم تحليل االداء المالي للجهاز المصرفي، فضالً عن األسلوب التحليلي المستند على استخراج النسب المالية المدعوم باألسلوب االحصائي لعمل بعض مقاييس التقييم السي )ما (الوسط الحسابي، الوسيط 728 44 7 - 25 7 P. P Ghari Journal of Faculty of Administration and Economics Vol. : اهداف الدراسة 18 (No.3) 2022 - Al Al-Ghari Journal of Faculty of Administration and Economics لدعم التحليل المالي لمؤشرات االداء المالي الالزمة لتحليل المصارف عينة الدراسة للمدة ما قبل .وبعد بداية جائحة كورونا : هيكلية البحث اا Al-Ghari Journal of Faculty of Administration and Economics لغرض دعم فرضية البحث فقد تم تقسيمه الى ثالث مباحث تضمن المبحث االول االطار النظري لمفاهيم متغيرات الدراسة التي تمثلت بتحليل األدا ء المالي في ظل جائحة كورونا، اما المبحث الثاني فقد خصص للجانب التحليلي لمتغيرات البحث من خالل محورين كان االول منها يختص بالتعريف عن المصارف عينة البحث، اما الثاني فقد اختص بتحليل ( االداء المالي للمصارف عينة البحث للمدة من2016 - 2019 ) قبل الجائحة، كما تم التركيز ف ي المبحث الثالث الذي قسم الى ثالث محاور أيضاً، اختص االول منها على تحليل االداء الما لي ( للمصارف للمدة2020 - 2021 ) بعد بداية جائحة كورونا، واشتمل المحور الثاني على اجراء مقارنة بتحليل المصارف بين الفترتين قبل وبعد بداية.الجائحة المبحث االول : االطار النظري لتحليل االداء المالي في ظل جائحة كورونا أوالا- مفهوم تحليل االداء المالي: يحتل مفهوم تحليل االداء مكان ة متميز ة في الوقت الحاضر ألهميته الكبيرة في الوق وف على كفاءة اداء المصرف و مدى تحقيقه ،ألهدافه ًفضال عن تحديد االيجابيات والسلبيات في االداء خالل مد ة محدد ة وتحليل اسبابها من اجل تدعيم الجوانب االيجابي ة وتجاوز العقبات ب ما ا يس ًهم في رفع مستوى االداء المصرفي مستقبال. ويع د مفهوم تحليل االداء المالي من المفاهيم المهم ة لمؤسسات االعمال بصف ة ة عام وللمصارف بصف ة خاص ،ة حيث يمكن من خالله التعرف على مدى نجاح او فشل تلك المؤسسات المالي ة والمصرفي ة من خالل قدرتها على الوفاء بالتزاماتها المالية في اوقاتها المحد د ،ة كذلك في توفير السيول ة وتحقيق الربح وتقوي ة مركزها االئتمان.ي و يمثل االداء مخرجات االنشطة التي تتشكل داخل الوحد ة( 2 ) ،وهن اك من يرى ان عملية تحليل االداء هي االدوات التي تستعمل للتعرف على نشاط الوحدة االقتصادية بهدف قياس النتائج المتحققة وتحليلها ومقارنتها ب ًاالهداف المحددة مسبقا من اجل الوقوف على االن حرافات وتشخيص .أسبابها مع اتخاذ االجراءات الكفيلة لتجاوز تلك االنحرافات وفي االغلب تكون ًالمقارنة بين ما تحقق فعال وما هو مستهدف في نهاية مدة زمنية محددة عادة ما تكون سنة ( 3 ). المبحث االول : االطار النظري لتحليل االداء المالي في ظل جائحة كورونا أوالا- مفهوم تحليل االداء المالي: يحتل مفهوم تحليل االداء مكان ة متميز ة في الوقت الحاضر ألهميته الكبيرة في الوق وف على كفاءة اداء المصرف و مدى تحقيقه ،ألهدافه ًفضال عن تحديد االيجابيات والسلبيات في االداء خالل مد ة محدد ة وتحليل اسبابها من اجل تدعيم الجوانب االيجابي ة وتجاوز العقبات ب ما ا يس ًهم في رفع مستوى االداء المصرفي مستقبال. Al-Ghari Journal of Faculty of Administration and Economics of Administration and Economics ثانياا- العوامل المؤثرة في االداء المالي : يواجه المصرف خالل القيام بنشاطاته عدة مشكال ت وصعوبات قد تحول بينه وبين اداء وظائفه بالوجه المطلو ب حيث يتأثر االداء المالي ل لمصرف بمجموعة من العوامل الداخ لية والخارجية و يمكن الحد من تأثيرها او ال تقليل من حدتها كونها تقع تحت سيطرة ادارة المصر ف ، ا ام العوامل الخارجية فال يمكن ال سيطر ة عليها او حتى تقليلها بوصفها تقع خارج قدرات اإل ،دارة وفيما يأتي بيان لتلك العوامل : 1 - العوامل الداخلية: وتتمثل هذه العوامل باالتي: أ- حجم المصر ف: يعد حجم المصرف من العوامل المؤثرة في االداء المالي وقد يكون تأثيره ًايجابيا ًاو سلبيا ، إذ يعزى تصنيف ه من حيث حجم ه ًصغيرا ًاو متوسطا او كب ير،ًا ًفضال عن وجود عدة مقاييس لحجم ا لمصرف منها: اجمالي ر أ س المال او اجمالي الموجودات او عدد فروع المصرف( 4 ) . ثانياا- العوامل المؤثرة في االداء المالي : ثانيا العوامل المؤثرة في االداء المالي : يواجه المصرف خالل القيام بنشاطاته عدة مشكال ت وصعوبات قد تحول بينه وبين اداء وظائفه بالوجه المطلو ب حيث يتأثر االداء المالي ل لمصرف بمجموعة من العوامل الداخ لية والخارجية و يمكن الحد من تأثيرها او ال تقليل من حدتها كونها تقع تحت سيطرة ادارة المصر ف ، ا ام العوامل الخارجية فال يمكن ال سيطر ة عليها او حتى تقليلها بوصفها تقع خارج قدرات اإل ،دارة وفيما يأتي بيان لتلك العوامل : 1ال ا ل ثل ذ ال ا ل ة ال ا ل اال يا أ- حجم المصر ف: يعد حجم المصرف من العوامل المؤثرة في االداء المالي وقد يكون تأثيره ًايجابيا ًاو سلبيا ، إذ يعزى تصنيف ه من حيث حجم ه ًصغيرا ًاو متوسطا او كب ير،ًا ًفضال عن وجود عدة مقاييس لحجم ا لمصرف منها: اجمالي ر أ س المال او اجمالي الموجودات او عدد فروع المصرف( 4 ) . ب- :الكفاءة المالية يمكن تحقيق الكفاءة المالية عن طريق السيطرة على التكاليف و ذل ك ك السيطرة على استعمال ا ا لمو در المتاحة لدى المصرف ب صورة فاعلة فضالً عن السيطرة على مصادر م لت ا ويل التي يعتمد عليها المصرف ، إذ تعد الكفاءة المالية احد اهم المقاييس المعتمدة في قياس االداء المالي للم صرف، و تشير الكفاءة المالية الى وجو د عالقة وثيقة بين المدخالت والمخرجات، و ًتؤدي دورا ًمهما في العديد من القرارات ا ليومي ة التي تمارسها ادارة المصرف( 5 ). Al-Ghari Journal of Faculty of Administration and Economics Al-Ghari Journal of Faculty of Administration and Economics ثانياا- العوامل المؤثرة في االداء المالي : يواجه المصرف خالل القيام بنشاطاته عدة مشكال ت وصعوبات قد تحول بينه وبين اداء وظائفه بالوجه المطلو ب حيث يتأثر االداء المالي ل لمصرف بمجموعة من العوامل الداخ لية والخارجية و يمكن الحد من تأثيرها او ال تقليل من حدتها كونها تقع تحت سيطرة ادارة المصر ف ، ا ام العوامل الخارجية فال يمكن ال سيطر ة عليها او حتى تقليلها بوصفها تقع خارج قدرات اإل ،دارة وفيما يأتي بيان لتلك العوامل : 1 - العوامل الداخلية: وتتمثل هذه العوامل باالتي: أ- حجم المصر ف: يعد حجم المصرف من العوامل المؤثرة في االداء المالي وقد يكون تأثيره ًايجابيا ًاو سلبيا ، إذ يعزى تصنيف ه من حيث حجم ه ًصغيرا ًاو متوسطا او كب ير،ًا ًفضال عن وجود عدة مقاييس لحجم ا لمصرف منها: اجمالي ر أ س المال او اجمالي الموجودات او عدد فروع المصرف( 4 ) . ب- :الكفاءة المالية يمكن تحقيق الكفاءة المالية عن طريق السيطرة على التكاليف و ذل ك ك السيطرة على استعمال ا ا لمو در المتاحة لدى المصرف ب صورة فاعلة فضالً عن السيطرة على مصادر م لت ا ويل التي يعتمد عليها المصرف ، إذ تعد الكفاءة المالية احد اهم المقاييس المعتمدة في قياس االداء المالي للم صرف، و تشير الكفاءة المالية الى وجو د عالقة وثيقة بين المدخالت والمخرجات، و ًتؤدي دورا ًمهما في العديد من القرارات ا ليومي ة التي تمارسها ادارة المصرف( 5 ). ج- التكنولوجي:ا المستخدمة ا دز ادت اهمية استعمال المعلومات والمعرفة من اجل الحصول على الميزة التنافسية من خالل االساليب واالدوات التي قدمتها تكنولوجيا المعلوم ات( 6 ) والتي ادت الى تحسين قدر ة المصرف وتعظيم كفاءته من خالل الربط الداخلي والخارجي عن طريق تدفق البيانات والمعلومات وانسيابي اهت ومن ثم تحسين عملية صنع القرار والذي بدوره يؤدي الى تحسين االداء المالي( 7 ). 2 - العوامل:الخارجية تتمثل العوامل الخارجية المؤثرة في االداء المالي باال تي( 8 ). أ- ال عوامل القانونية :والسياسية نو عني بها الظروف السياسي ة للبلد الذي يقع فيه ال مصرف ت وال ي تجبره على الخضوع ،لها مع قيود القوانين الحاكمة ل تحركاته او التي يكون ع لى علم تام بها عند تعا ،مله مع الزبون أو الخصم ًفضال عن التحكم السياسي والقانوني من قبل الدول االخرى ب إجراءات المصرف المتخذة عند تعامله مع ا ال ستثمارات ال خارجية ف ي تلك الدولة. المبحث االول : االطار النظري لتحليل االداء المالي في ظل جائحة كورونا أوالا- مفهوم تحليل االداء المالي: اأ ا يا ع م ي ويع د مفهوم تحليل االداء المالي من المفاهيم المهم ة لمؤسسات االعمال بصف ة ة عام وللمصارف بصف ة خاص ،ة حيث يمكن من خالله التعرف على مدى نجاح او فشل تلك المؤسسات المالي ة والمصرفي ة من خالل قدرتها على الوفاء بالتزاماتها المالية في اوقاتها المحد د ،ة كذلك في توفير السيول ة وتحقيق الربح وتقوي ة مركزها االئتمان.ي و يمثل االداء مخرجات االنشطة التي تتشكل داخل الوحد ة( 2 ) ،وهن اك من يرى ان عملية تحليل االداء هي االدوات التي تستعمل للتعرف على نشاط الوحدة االقتصادية بهدف قياس النتائج المتحققة وتحليلها ومقارنتها ب ًاالهداف المحددة مسبقا من اجل الوقوف على االن حرافات وتشخيص .أسبابها مع اتخاذ االجراءات الكفيلة لتجاوز تلك االنحرافات وفي االغلب تكون ًالمقارنة بين ما تحقق فعال وما هو مستهدف في نهاية مدة زمنية محددة عادة ما تكون سنة ( 3 ). ويع د مفهوم تحليل االداء المالي من المفاهيم المهم ة لمؤسسات االعمال بصف ة ة عام وللمصارف بصف ة خاص ،ة حيث يمكن من خالله التعرف على مدى نجاح او فشل تلك المؤسسات المالي ة والمصرفي ة من خالل قدرتها على الوفاء بالتزاماتها المالية في اوقاتها المحد د ،ة كذلك في توفير السيول ة وتحقيق الربح وتقوي ة مركزها االئتمان.ي يو ي و ير يق ربح و وي و ر ز ال ي و يمثل االداء مخرجات االنشطة التي تتشكل داخل الوحد ة( 2 ) ،وهن اك من يرى ان عملية تحليل االداء هي االدوات التي تستعمل للتعرف على نشاط الوحدة االقتصادية بهدف قياس النتائج المتحققة وتحليلها ومقارنتها ب ًاالهداف المحددة مسبقا من اجل الوقوف على االن حرافات وتشخيص .أسبابها مع اتخاذ االجراءات الكفيلة لتجاوز تلك االنحرافات وفي االغلب تكون ًالمقارنة بين ما تحقق فعال وما هو مستهدف في نهاية مدة زمنية محددة عادة ما تكون سنة ( 3 ). 729 44 7 - 25 7 P. P Ghari Journal of Faculty of Administration and Economics Vol. 18 (No.3) 2022 - Al Al-Ghari Journal of Faculty of Administration and Economics ج- التكنولوجي:ا المستخدمة ا دز ادت اهمية استعمال المعلومات والمعرفة من اجل الحصول على الميزة التنافسية من خالل االساليب واالدوات التي قدمتها تكنولوجيا المعلوم ات( 6 ) والتي ادت الى تحسين قدر ة المصرف وتعظيم كفاءته من خالل الربط الداخلي والخارجي عن طريق تدفق البيانات والمعلومات وانسيابي اهت ومن ثم تحسين عملية صنع القرار والذي بدوره يؤدي الى تحسين االداء المالي( 7 ). 2 - العوامل:الخارجية تتمثل العوامل الخارجية المؤثرة في االداء المالي باال تي( 8 ). أ- ال عوامل القانونية :والسياسية نو عني بها الظروف السياسي ة للبلد الذي يقع فيه ال مصرف ت وال ي تجبره على الخضوع ،لها مع قيود القوانين الحاكمة ل تحركاته او التي يكون ع لى علم تام بها عند تعا ،مله مع الزبون أو الخصم ًفضال عن التحكم السياسي والقانوني من قبل الدول االخرى ب إجراءات المصرف المتخذة عند تعامله مع ا ال ستثمارات ال خارجية ف ي تلك الدولة. 730 44 7 - 25 7 P. P Ghari Journal of Faculty of Administration and Economics Vol. 18 (No.3) 2022 - Al Al-Ghari Journal of Faculty of Administration and Economics Al-Ghari Journal of Faculty of Administration and Economics ب- العوامل:االقتصادية وهي العوامل المتعلقة باالقتصاد ،الكلي فهناك مجموعة واسعة من االدبيات التي تدعم ت أ ثير عوامل االقتصاد الكلي في اداء المصرف ، إذ يعد استقرار سياسة االقتصاد الكلي والناتج المحلي اإلجمالي من ناحية والت ضخم وعدم االستقرار االقتصادي من ناحية ،أخرى من متغيرات االقتصاد الكلي التي تؤثر في االداء المالي ،للمصرف تدعم معظم االدبيات الت ثير أ االيجابي للنمو االقتصادي في اداء المصرف المر تبط بحواجز موجهة عند الدخول الى السوق المصرفية وهي ضرورية لرفع معدالت ارباح المصارف، اما التضخم فله تأثيره وه ًايضا ويعد احد العوامل المهمة المؤثرة في االدا ء المصرفي و ي تميز في كونه ًمتفاعال مع المصرف فكالهما يؤثر في االخر ،وبشكل عام ترتب ط معدالت التضخم ا لعالية ارتف ب اع معدالت الفائدة االسمية على القروض( 9 ). ي هناك العديد من وجهات النظر حول مفهوم المصرف من اهمها : ا لكالسيكية والحديثة فقد ع فر ال مصرف حسب المفهوم الكالسيكي( بانه منظمة مالية تتوسط بين فئتين الفئة االو لى لديها فائض نقدي وترغب ب استثماره او الحفاظ عليه والفئة الثانية دي ل ها عجز نقدي وترغب في الحصول على التمويل لغرض سداد عجز مؤقت او لغرض االستثمار ،)ا ام المفهوم الحديث في عرف المصرف بصفته (مجموعة من االشخاص يطلق عليهم تسمي( ة(Brokers يقومون بوظيفة الوساطة المالية عن طريق قبوله م ودائع تحت الطلب او ودائع آل جال محددة وتمارس عمليات التمويل الداخلي والخارجي لتحقيق اهداف خطة التنمية وسياسة الدولة ودعم االقتص اد القومي) ( 10 ) . ويعرف الجهاز المصرفي بانه المؤسسات المصرفية واالنظمة والقوانين التي تتالف من ها وتعمل في ظلها هذه المؤسسات ( 11 ). وتقوم ًايضا المصارف بدفع الودائع الجارية للعمالء باستعمال الشيكات ، اذن هي مؤسسات تقوم بصناعة اال ئتمان وتسهيل تبادل االموال بين االفراد والمؤسسات المالية اال خرى بطر ق آم نة( 12 ). والمصارف تمثل حلقة من حلقات التطور االقتصادي والمالي ألي بلد وهو انعكاس ألنظمة البلد اال قتصادية والمالية النها عماد االقتصاد ومحور ه وان الدول ال تتقدم بد ون نظام مصرفي سليم وذلك لما يوفره من امكانات من االنشطة والعمليات التي ت قوم بها كونها عماد ب ناء االقتصاد في اي بلد مهما كانت طبيعة نظامه االقتصادي والسياسي وان الم ص ف ار تحتل اهمية جوهرية كون ها من اكبر المؤسسات المالية في ذلك االقتصاد( 13 ). 731 731 44 7 - 25 7 P. P Ghari Journal of Faculty of Administration and Economics Vol. Al-Ghari Journal of Faculty رابعاا- اهداف المصارف وجوانب:تأثيرها يمكن تقسيم االهداف التي تسعى المصارف الى تحقيقها :كاالتي( 14 ) 1 - اهداف مالية: منها قيام المصرف الى زيادة االرباح وتعظيم ثروة المالكين وزيادة م عدالت العائد على االستثمار والمحافظة على نسبة سيولة محددة. 2 - اهداف تسويق:ية منها تعظيم القيمة السوقية للمصرف وذلك لزيادة المنافسة السوقية عن طريق ج ودة الخدمات المالية المقدمة وباقل التكاليف لتحسين سمعة المصرف وزيادة قاعدة العمالء. يمكن تقسيم االهداف التي تسعى المصارف الى تحقيقها :كاالتي( 14 ) 1 - اهداف مالية: منها قيام المصرف الى زيادة االرباح وتعظيم ثروة المالكين وزيادة م عدالت العائد على االستثمار والمحافظة على نسبة سيولة محددة. 2 - اهداف تسويق:ية منها تعظيم القيمة السوقية للمصرف وذلك لزيادة المنافسة السوقية عن طريق ج ودة الخدمات المالية المقدمة وباقل التكاليف لتحسين سمعة المصرف وزيادة قاعدة العمالء. 3 - اهداف عامة: تشمل تقديم الخدمات لإلفراد والشركات والمؤسسات الحكومية لخدمة التنمية االقتصادية واالجتماعية والسياسية. اا كما ان المصارف تتميز بالتأثير في نواحي مختلفة من االقتصاد من خالل : ( 15 ) 1 - تأثيرها في كمية النقود لقدرتها على توفير النقود والتأثير في كميتها المعروضة ءوا س عمل كل مصرف على حد ة ك او مجموعة مصارف من خالل منح القروض مقابل قبول الودا ئع. ااا اا كما ان المصارف تتميز بالتأثير في نواحي مختلفة من االقتصاد من خالل : ( 15 ) 1 - تأثيرها في كمية النقود لقدرتها على توفير النقود والتأثير في كميتها المعروضة ءوا س عمل كل مصرف على حد ة ك او مجموعة مصارف من خالل منح القروض مقابل قبول الودا ئع. ااا 2 - تؤثر في االقتصاد الوطني بشكل مباشر عن طريق منح التسهيالت المصرفية واالئتمان للجمهور مقابل ايداع اكبر قدر ممكن من الودائع. خامساا- :االطار المفاهيمي لجائحة كورونا 2 - تؤثر في االقتصاد الوطني بشكل مباشر عن طريق منح التسهيالت المصرفية واالئتمان للجمهور مقابل ايداع اكبر قدر ممكن من الودائع. خامساا- :االطار المفاهيمي لجائحة كورونا 2 - تؤثر في االقتصاد الوطني بشكل مباشر عن طريق منح التسهيالت المصرفية واالئتمان للجمهور مقابل ايداع اكبر قدر ممكن من الودائع. Al-Ghari Journal of Faculty of Administration and Economics 18 (No.3) 2022 - Al Al-Ghari Journal of Faculty of Administration and Economics رابعاا- اهداف المصارف وجوانب:تأثيرها يمكن تقسيم االهداف التي تسعى المصارف الى تحقيقها :كاالتي( 14 ) 1 - اهداف مالية: منها قيام المصرف الى زيادة االرباح وتعظيم ثروة المالكين وزيادة م عدالت العائد على االستثمار والمحافظة على نسبة سيولة محددة. 2 - اهداف تسويق:ية منها تعظيم القيمة السوقية للمصرف وذلك لزيادة المنافسة السوقية عن طريق ج ودة الخدمات المالية المقدمة وباقل التكاليف لتحسين سمعة المصرف وزيادة قاعدة العمالء. Al-Ghari Journal of Faculty خامساا- :االطار المفاهيمي لجائحة كورونا ( اطلقت منظمة الصحة العالميةWHO ) على الساللة الجديدة من فايروس كورونا االسم ( العلميnCov-19 ) وبعد ذلك اطلقت عليه اللجنة الدولية لتصنيف الفيروسات مصطلح ( SARA-Cov-2 ) لتميزه عن بقية السالالت االخرى التي سبقته بالظهور والتفشي وقد أعلنت ( منظمة الصحة العالمية في12 من شهر آذار للسنة2020 ) عن وباء الفيروس التاجي (كورونا) على انه جائحة مع إن هذا االعالن ال عالقة له بخصائص المرض وال بخطورته بل باالنتشار الجغرافي والتزايد غير المتوقع في حاالت االصابة ، وكذلك الو فيات في معظم ا نحاء .العالم فضالً عن قصور الدول في السيطرة على تفشي الفيروس( 16 ) ا فالوباء الصحي يعرف بانه ذلك المرض الذي يقتصر انتشاره على دولة دون اخرى، اما الجائحة فهي الوباء الذي ينتشر في معظم دول العالم في الوقت نفسه، فضالً عن عدم تمتع االشخاص بالحصانة ضده ، وهذا االنتشار يفوق التوقعات( 17 ) ، ويمكن تعريف فايروس كورونا بأنه: ساللة واسعة من الفيروسات التي تتسبب للحيوان واالنسان بامراض تنفسية تتراوح حدتها ما بين نزالت البرد الشائعة الى االمراض االشد وخامة مثل (متالزمة الشرق االوسط التنفسية 732 44 7 - 25 7 P. P Ghari Journal of Faculty of Administration and Economics Vol. 18 (No.3) 2022 - Al Al-Ghari Journal of Faculty of Administration and Economics ( MERS) والمتالزمة التنفس" ية الحادة الوخيمةSARS ً") ويسبب فيروس كورونا المكتشف مؤخرا ( ما يسمى بمرض19 - COVID . Al-Ghari Journal of Faculty ) ( 18 ) ( أما تعريف مرضCOVID-19 ) فهو مرض ناتج عن الساللة الواسعة لفيروسات كورونا المستجد المسمى فيروس (كورونا– سارس2 ) وقد اكتشفت منظمة الصحة العالمية الفيروس ( المستجد ألول مرة في يوم31 كانون االول لعام2019) وذلك بعد االبالغ عن فئة من حاال ت االلتهاب الرئوي الفيروسي من مدينة ووهان لدولة الصين الشعبية، وتتراوح معظم تقديرات ف ترة (حضانة المرض ما بين يوم واحد و14) يوم اً وأن االسم االنكليزي للمرض هو مشتق كاالتي ( CO ( ) وهما أول حرفين من كلمة كوروناCorona () وVI ) هما اول حرفين من كلمة فايروس ( Virus ( )، أما حرفD ( ) فهو اول حرف من كلمة مرضDisease .) ( 19 ) ًسادسا- :التحديات التي تواجه القطاع المصرفي في اعقاب انتشار فيروس كورونا ه نالك العديد من التحديات التي ربما تواجه عمل البنوك المركزية خالل الفترة الق ادمة :ويمكن استعراض اهم هذه التحديات في االتي( 20 ) 1 - .اضعاف قدرة القطاع المصرفي على تمويل االقتصاد في المستقبل 2 - مشكلة طبع النقد وتحديات التضخم واالنكماش التي تتفرع الى مشكلتين فرعتين وهم:ا أ- .مشكلة التضخم الناجم عن ضخ االموال في االقتصاد العالمي ب- .مشكلة احتمالية الوقوع في فخ االنكماش 3 - .تفاقم ازمة الديون العالمية 4 - :تحديات أخرى محتملة تشمل المشكالت االتية أ- .حرب العمالت ب- .االثر السلبي للفوائد السلبية للسندات االوربية ت- ارتفاع معدالت الفائدة االمريكية .وازمة االسواق الناشئة :اما التحديات الرئيسية التي يواجها القطاع المصرفي فهي تتلخص باالتي( 21 ) 1 - .)انخفاض ربحية المصارف (انخفاض االيرادات 4 - :تحديات أخرى محتملة تشمل المشكالت االتية Al-Ghari Journal of Faculty االستثمار العراقي BIBI https://www.ibi-bankiraq.com/ 100 مليون دينار عراقي 22 فرعاا اعداد الباحثة اعتماداا على التقارير السنوية للمصارف عينة الدراسة مصرف بغداد هو أحد المصارف التجارية العاملة في القطاع الخاص والمدرجة في هيئة األوراق الما لية العراقية، أنشئ مصرف بغداد كشركة مساهمة خاصة وبرأس مال أسمي قدره ( 100 ) مليون ( دينار عراقي بموجب شهادة التأسيس المرقمة4512 ) بتأريخ( 18 / 12 / 1992 )، والصادرة من ( دائرة التسجيل العقاري بموجب قانون البنك المركزي العراقي رقم2ل ) ل سنة( 1992 )، وهو أ ول مصرف أ هلي افتتح في العراق، وباشر عملياته المصرفية ب تاريخ( 12 / 9 / 1992 )، وفي ( 25 / 9 / 1997 ) أجرى تعديل عقد التأسيس وذلك بممارسة الصيرفة الشاملة، وبلغ عدد الفروع ( 41 ) ًفرعا موزعاً في بغداد والمحافظات اما عدد العاملين فهو( 1079 ( ) شخصاً منهم265 ) في اإلدارة (العامة و814 ) موزعاً في الفروع( 23 ). :المحور األول: نبذه تعريفية عن المصارف عينة الدراسة نظراً الى أهمية المصارف الخاصة في هيكل عمل القطاع المصرفي العراقي تم ( اختيارها مجتمعاً للدراسة وذلك بتوظيف2 ) مصارف منها بصفتها عينة الدراسة بسبب توفر بيانات هذه المصارف التي تلبي فرضية واهداف الدراسة ومتطلباتها وفيما يلي عرض موجز لعينة الدراسة يتضمن التعريف بمصارف العينة ومقدار رؤوس اموالها وعدد فروعها كما يبين لنا ( الجدول1 :) اآلتي آ ( الجدول1) المصارف عينة الدراسة ت اسم المصرف رمز المصرف الموقع اإللكتروني رأس المال التأسيسي المصرح به فروع المصرف 1 . بغداد BBOB https://www.bankofbaghdad.com.iq/ 100 مليون دينار عراقي 37 فرعاا 2 . االستثمار العراقي BIBI https://www.ibi-bankiraq.com/ 100 مليون دينار عراقي 22 فرعاا اعداد الباحثة اعتماداا على التقارير السنوية للمصارف عينة الدراسة ( الجدول1) المصارف عينة الدراسة ت اسم المصرف رمز المصرف الموقع اإللكتروني رأس المال التأسيسي المصرح به فروع المصرف 1 . بغداد BBOB https://www.bankofbaghdad.com.iq/ 100 مليون دينار عراقي 37 فرعاا 2 . Al-Ghari Journal of Faculty Al-Ghari Journal of Faculty of Administration and Economics ب- .مشكلة احتمالية الوقوع في فخ االنكماش 4 - :تحديات أخرى محتملة تشمل المشكالت االتية ب- .ااااالثر السلبي للفوائد السلبية للسندات االوربية 733 44 7 - 25 7 P. P Ghari Journal of Faculty of Administration and Economics Vol. 18 (No.3) 2022 - Al 44 7 - 25 7 P. P Ghari Journal of Faculty of Administration and Economics Vol. 18 (No.3) 2022 - Al 44 7 - 25 7 P. P Ghari Journal of Faculty of Administration and Economics Vol. 18 (No.3) 2022 - Al Al-Ghari Journal of Faculty of Administration and Economics وكنتيجة ط:بيعية لهذه التحديات سيكون على المصارف التركيز في المجاالت اآلتية( 22 ) 1 - .تحليل هيكل السيولة ورأس المال 2 - .التخطيط للسيناريوهات المستقبلية المحتملة 3 - .)التواصل مع اصحاب العالقة (التفاعل مع العمالء 4 - .بيع القروض المتعثرة 5 - .ترشيد اعداد الفروع :المبحث الثاني تحليل( االداء المالي للمصارف عينة البحث للمدة من2016 - 2019 ) قبل الجائحة :المحور األول: نبذه تعريفية عن المصارف عينة الدراسة نظراً الى أهمية المصارف الخاصة في هيكل عمل القطاع المصرفي العراقي تم ( اختيارها مجتمعاً للدراسة وذلك بتوظيف2 ) مصارف منها بصفتها عينة الدراسة بسبب توفر بيانات هذه المصارف التي تلبي فرضية واهداف الدراسة ومتطلباتها وفيما يلي عرض موجز لعينة الدراسة يتضمن التعريف بمصارف العينة ومقدار رؤوس اموالها وعدد فروعها كما يبين لنا ( الجدول1 :) اآلتي ( الجدول1) المصارف عينة الدراسة اسم المصرف رمز المصرف الموقع اإللكتروني رأس المال التأسيسي المصرح به فروع المصرف روع ر ي :المبحث الثاني تحليل( االداء المالي للمصارف عينة البحث للمدة من2016 - 2019 ) قبل الجائحة :المحور األول: نبذه تعريفية عن المصارف عينة الدراسة نظراً الى أهمية المصارف الخاصة في هيكل عمل القطاع المصرفي العراقي تم ( اختيارها مجتمعاً للدراسة وذلك بتوظيف2 ) مصارف منها بصفتها عينة الدراسة بسبب توفر بيانات هذه المصارف التي تلبي فرضية واهداف الدراسة ومتطلباتها وفيما يلي عرض موجز لعينة الدراسة يتضمن التعريف بمصارف العينة ومقدار رؤوس اموالها وعدد فروعها كما يبين لنا ( الجدول1 :) اآلتي ( الجدول1) المصارف عينة الدراسة ت اسم المصرف رمز المصرف الموقع اإللكتروني رأس المال التأسيسي المصرح به فروع المصرف 1 . بغداد BBOB https://www.bankofbaghdad.com.iq/ 100 مليون دينار عراقي 37 فرعاا 2 . Al-Ghari Journal of Faculty المحور الثاني: تحليل اداء المصارف عينة الدراسة قبل جائحة كورونا ( تم تحليل تقييم االداء المالي على المصارف عينة الدراسة البالغ عددها2) مصارف ( ولمدة4 ( ) سنوات من2016 - 2019 ) المدة التي تسبق حدوث وانتشار جائحة كورونا ( COVID-19) بحسب مؤشرات المالية، إذ تم تطبي ق المؤشرات واستخراج النسب لقياس االداء .المصرفي وتحليله وذلك اعتماداً على التقارير المالية السنوية لتلك المصارف 1 - ( تحليل نتائج تقييم اداء المصارف عينة الدراسة للمدة2016 - 2019 :) تم تقييم اداء المصارف عينة الدراسة وفقاً لمؤشرات االداء المالي وتحليل تلك ال م ؤشرا ت :كاالتي Al-Ghari Journal of Faculty of Administration and Economics ( تأسس مصرف االستثمار العراقي كشركة مساهمة برأس مال قدره100 ) مليون دينار /عراقي بموجب شهادة التأسيس المرقمة (م.ش5236 ( ) المؤرخة في13 / 7 / 1993 ) الصادرة عن دائرة تسجيل الشركات في وزارة التجارة بموجب قانون الشركات( النافذ آنذاك المرقمة36 ) للعام ( 1983 /) المعدل وأجازة ممارسة الصيرفة بكتابها المرقم (ص10 / 3 / 942 / 5 ) والمؤرخ في ( 28 / 9 / 1993 ( ) وباشر المصرف نشاطه فعلياً منذ تاريخ21 / 7 / 1994 ) وحتى يومنا هذا وتطور ( رأسماله حتى وصل إلى250 ( ) مليار دينار منذ تاريخ19 / 2 / 2014 ) ( 24 ). المحور الثاني: تحليل اداء المصارف عينة الدراسة قبل جائحة كورونا ( تم تحليل تقييم االداء المالي على المصارف عينة الدراسة البالغ عددها2) مصارف ( ولمدة4 ( ) سنوات من2016 - 2019 ) المدة التي تسبق حدوث وانتشار جائحة كورونا ( COVID-19) بحسب مؤشرات المالية، إذ تم تطبي ق المؤشرات واستخراج النسب لقياس االداء .المصرفي وتحليله وذلك اعتماداً على التقارير المالية السنوية لتلك المصارف ي ي ى ي 1 - ( تحليل نتائج تقييم اداء المصارف عينة الدراسة للمدة2016 - 2019 :) ( جدول2 ) ( تطور النسب المالية للمصارف الخاصة عينة البحث للمدة2016 - 2019 ) المصرف ت النسب المالية 2016 % 2017 % 2018 % 2019 % Average بغداد 1 . نسبة االستثمارات الى راس المال واالحتياطيات السلي مة 36.51 33.89 34.14 37.43 35.49 2 . نسبة نمو االرباح 254.18 -69.76 -32.18 75.77 57.00 3 . نسبة العائد الى اجمالي الموجودات 1.69 0.56 0.37 0.64 0.82 4 . نسبة اجمالي االئتمان الى اجمالي الودائع 16.55 15.40 24.84 23.44 20.06 االستثمار العراقي 1 . نسبة االستثمارات الى راس المال واالحتياطيات السلي مة 5.86 8.39 9.14 1.70 6.27 2 . نسبة نمو االرباح -41.83 -60.75 -91.51 49.93 -36.04 3 . Al-Ghari Journal of Faculty االستثمار العراقي BIBI https://www.ibi-bankiraq.com/ 100 مليون دينار عراقي 22 فرعاا اعداد الباحثة اعتماداا على التقارير السنوية للمصارف عينة الدراسة هو أحد المصارف التجارية العاملة في القطاع الخاص والمدرجة في هيئة األوراق الما لية العراقية، أنشئ مصرف بغداد كشركة مساهمة خاصة وبرأس مال أسمي قدره ( 100 ) مليون ( دينار عراقي بموجب شهادة التأسيس المرقمة4512 ) بتأريخ( 18 / 12 / 1992 )، والصادرة من ( دائرة التسجيل العقاري بموجب قانون البنك المركزي العراقي رقم2ل ) ل سنة( 1992 )، وهو أ ول مصرف أ هلي افتتح في العراق، وباشر عملياته المصرفية ب تاريخ( 12 / 9 / 1992 )، وفي ( 25 / 9 / 1997 ) أجرى تعديل عقد التأسيس وذلك بممارسة الصيرفة الشاملة، وبلغ عدد الفروع ( 41 ) ًفرعا موزعاً في بغداد والمحافظات اما عدد العاملين فهو( 1079 ( ) شخصاً منهم265 ) في اإلدارة (العامة و814 ) موزعاً في الفروع( 23 ). 734 44 7 - 25 7 P. P Ghari Journal of Faculty of Administration and Economics Vol. 18 (No.3) 2022 - Al Al-Ghari Journal of Faculty of Administration and Economics مصرف االستثمار العراقي: ( تأسس مصرف االستثمار العراقي كشركة مساهمة برأس مال قدره100 ) مليون دينار /عراقي بموجب شهادة التأسيس المرقمة (م.ش5236 ( ) المؤرخة في13 / 7 / 1993 ) الصادرة عن دائرة تسجيل الشركات في وزارة التجارة بموجب قانون الشركات( النافذ آنذاك المرقمة36 ) للعام ( 1983 /) المعدل وأجازة ممارسة الصيرفة بكتابها المرقم (ص10 / 3 / 942 / 5 ) والمؤرخ في ( 28 / 9 / 1993 ( ) وباشر المصرف نشاطه فعلياً منذ تاريخ21 / 7 / 1994 ) وحتى يومنا هذا وتطور ( رأسماله حتى وصل إلى250 ( ) مليار دينار منذ تاريخ19 / 2 / 2014 ) ( 24 ). Al-Ghari Journal of Faculty المحور الثاني: تحليل اداء المصارف عينة الدراسة قبل جائحة كورونا ( تم تحليل تقييم االداء المالي على المصارف عينة الدراسة البالغ عددها2) مصارف ( ولمدة4 ( ) سنوات من2016 - 2019 ) المدة التي تسبق حدوث وانتشار جائحة كورونا ( COVID-19) بحسب مؤشرات المالية، إذ تم تطبي ق المؤشرات واستخراج النسب لقياس االداء .المصرفي وتحليله وذلك اعتماداً على التقارير المالية السنوية لتلك المصارف 1 - ( تحليل نتائج تقييم اداء المصارف عينة الدراسة للمدة2016 - 2019 :) تم تقييم اداء المصارف عينة الدراسة وفقاً لمؤشرات االداء المالي وتحليل تلك ال م ؤشرا ت :كاالتي ( جدول2 ) ( تطور النسب المالية للمصارف الخاصة عينة البحث للمدة2016 - 2019 ) المصرف ت النسب المالية 2016 % 2017 % 2018 % 2019 % verage بغداد 1 . نسبة االستثمارات الى راس المال واالحتياطيات السلي مة 36.51 33.89 34.14 37.43 5.49 2 . نسبة نمو االرباح 254.18 -69.76 -32.18 75.77 7.00 3 . نسبة العائد الى اجمالي الموجودات 1.69 0.56 0.37 0.64 0.82 4 . نسبة اجمالي االئتمان الى اجمالي الودائع 16.55 15.40 24.84 23.44 20.06 االستثمار العراقي 1 . نسبة االستثمارات الى راس المال واالحتياطيات السلي مة 5.86 8.39 9.14 1.70 6.27 2 . نسبة نمو االرباح -41.83 -60.75 -91.51 49.93 36.04 3 . نسبة العائد الى اجمالي الموجودات 1.76 0.70 0.06 0.003 0.63 4 . نسبة اجمالي االئتمان الى اجمالي الودائع 45.32 53.01 64.03 86.44 2.20 إعداد الباحثة باالعتماد على البيانات المالية السنوية للمصارف عينة البحث أ- نسبة االستثمارات الى رأس المال واالحتياطيات السليمة للمصارف عينة الدراسة للمد ة ( 2016 - 2019 ( ) كما هو موضح في الجدول2 :) وكاالتي 44 7 - 25 7 P. P Ghari Journal of Faculty of Administration and Economics Vol. 18 (No.3) 2022 - Al y Al-Ghari Journal of Faculty of Administration and Economics مصرف االستثمار العراقي: ( تأسس مصرف االستثمار العراقي كشركة مساهمة برأس مال قدره100 ) مليون دينار /عراقي بموجب شهادة التأسيس المرقمة (م.ش5236 ( ) المؤرخة في13 / 7 / 1993 ) الصادرة عن دائرة تسجيل الشركات في وزارة التجارة بموجب قانون الشركات( النافذ آنذاك المرقمة36 ) للعام ( 1983 /) المعدل وأجازة ممارسة الصيرفة بكتابها المرقم (ص10 / 3 / 942 / 5 ) والمؤرخ في ( 28 / 9 / 1993 ( ) وباشر المصرف نشاطه فعلياً منذ تاريخ21 / 7 / 1994 ) وحتى يومنا هذا وتطور ( رأسماله حتى وصل إلى250 ( ) مليار دينار منذ تاريخ19 / 2 / 2014 ) ( 24 ). Al-Ghari Journal of Faculty المحور الثاني: تحليل اداء المصارف عينة الدراسة قبل جائحة كورونا ( تم تحليل تقييم االداء المالي على المصارف عينة الدراسة البالغ عددها2) مصارف ( ولمدة4 ( ) سنوات من2016 - 2019 ) المدة التي تسبق حدوث وانتشار جائحة كورونا ( COVID-19) بحسب مؤشرات المالية، إذ تم تطبي ق المؤشرات واستخراج النسب لقياس االداء .المصرفي وتحليله وذلك اعتماداً على التقارير المالية السنوية لتلك المصارف 1 - ( تحليل نتائج تقييم اداء المصارف عينة الدراسة للمدة2016 - 2019 :) تم تقييم اداء المصارف عينة الدراسة وفقاً لمؤشرات االداء المالي وتحليل تلك ال م ؤشرا ت :كاالتي ( جدول2 ) ( تطور النسب المالية للمصارف الخاصة عينة البحث للمدة2016 - 2019 ) المصرف ت النسب المالية 2016 % 2017 % 2018 % 2019 % rage بغداد 1 . نسبة االستثمارات الى راس المال واالحتياطيات السلي مة 36.51 33.89 34.14 37.43 49 2 . نسبة نمو االرباح 254.18 -69.76 -32.18 75.77 00 3 . نسبة العائد الى اجمالي الموجودات 1.69 0.56 0.37 0.64 82 4 . نسبة اجمالي االئتمان الى اجمالي الودائع 16.55 15.40 24.84 23.44 06 االستثمار العراقي 1 . نسبة االستثمارات الى راس المال واالحتياطيات السلي مة 5.86 8.39 9.14 1.70 27 2 . نسبة نمو االرباح -41.83 -60.75 -91.51 49.93 .04 3 . نسبة العائد الى اجمالي الموجودات 1.76 0.70 0.06 0.003 63 4 . نسبة اجمالي االئتمان الى اجمالي الودائع 45.32 53.01 64.03 86.44 20 إعداد الباحثة باالعتماد على البيانات المالية السنوية للمصارف عينة البحث أا أا Al-Ghari Journal of Faculty of Administration and Economics مصرف االستثمار العراقي: ( تأسس مصرف االستثمار العراقي كشركة مساهمة برأس مال قدره100 ) مليون دينار /عراقي بموجب شهادة التأسيس المرقمة (م.ش5236 ( ) المؤرخة في13 / 7 / 1993 ) الصادرة عن دائرة تسجيل الشركات في وزارة التجارة بموجب قانون الشركات( النافذ آنذاك المرقمة36 ) للعام ( 1983 /) المعدل وأجازة ممارسة الصيرفة بكتابها المرقم (ص10 / 3 / 942 / 5 ) والمؤرخ في ( 28 / 9 / 1993 ( ) وباشر المصرف نشاطه فعلياً منذ تاريخ21 / 7 / 1994 ) وحتى يومنا هذا وتطور ( رأسماله حتى وصل إلى250 ( ) مليار دينار منذ تاريخ19 / 2 / 2014 ) ( 24 ). Al-Ghari Journal of Faculty of Administration and Economics 1 - حقق مصرف بغداد اعلى نسبة لالستثمارات الى رأس المال واالحتياطيات السليمة في ( 2019 ( )اذ بلغت37.43 ) ويرجع سبب ارتفاع النسبة الى ارتفاع مبلغ االستثمارات حيث ( بلغت102,42 ( ) مليون دينار، في حين بلغت ادنى نسبة33.89 ( ) في سنة2017 ) وذلك لقيام ادارة المصرف بتخفيض ا( الستثمارات الى8,923) مليون دينار، كما بلغ متوسط هذ ه ( النسبة خالل المدة اعاله35.49 .) 2 - ( سجل مصرف االستثمار العراقي اعلى نسبة له في عام2018 ( ) اذ بلغت9.14 ) بسبب ( قيام المصرف بزيادة االستثمارات الى439 ) مليار دينار، في حين بلغت ادنى نسبة له ف ي (سنة2019) وهي ( 1.70 ( ) بسبب ارتفاع رأس المال واالحتياطيات الى260 ،) مليار دينار ( كما وبلغ متوسط هذه النسبة6.27 .) ب ( معدل نمو األرباح: نالحظ في الجدول2 :) االتي 1 . اتصفت نسبة نمو األرباح في مصرف بغداد بالتفاوت من سنة الى أخرى حيث بلغت أعلى ( نسبة254.18 ( ) في سنة2016 ) وذلك ير جع إلى ارتفاع صافي الربح بعد الضريبة في ( هذه السنة مقارنة بالسنوات عينة الدراسة حيث بلغت20,200 ) مليون دينار، في حين ( بلغت أدنى نسبة69.76 - ( ) في سنة2017 ) لكونه حقق في هذه السنة خسارة مقدارها ( 6,100 ( ) مليون دينار، وكان متوسط هذه النسبة قد بلغ57.00 .) 2 . شهدت النسبة في مصرف االستثمار انخفاضات متتالية من سنة ألخرى حتى ارتفعت في ( سنة2019 ( ) إذ بلغت49.93 ) وذلك يرجع إلى ارتفاع األرباح بعد الضريبة إلى ( 17,216 ( ) مليون دينار، في حين كانت أدنى نسبة له41.83 - ( ) في سنة2016 ) وذلك ( بسبب تحقيقه لخسارة مقدارها10,179 ) مليو ن دينار، كما بلغ متوسط هذه النسبة ( 36.04 - .) ت- ( تحليل نسبة العائد إلى إجمالي الموجودات: نالحظ في الجدول2 :) االتي 1 . ( حقق مصرف بغداد أعلى نسبة للعائد إلى إجمالي الموجودات بلغت1.69 ) في سنة ( 2016 ( ) ويرجع سبب ذلك إلى ارتفاع صافي العوائد في هذه السنة اذ بلغ20,200 ) مليون ( دينار، كما بلغت أدنى نسبة0.37 ( ) في سنة2018 ) بسبب انخفاض صافي العوائد إلى ( 4,100 ( ) مليون دينار، أيضاً بلغ متوسط هذه النسبة0.82 .) 2 . Al-Ghari Journal of Faculty نسبة العائد الى اجمالي الموجودات 1.76 0.70 0.06 0.003 0.63 4 . نسبة اجمالي االئتمان الى اجمالي الودائع 45.32 53.01 64.03 86.44 62.20 إعداد الباحثة باالعتماد على البيانات المالية السنوية للمصارف عينة البحث أ- نسبة االستثمارات الى رأس المال واالحتياطيات السليمة للمصارف عينة الدراسة للمد ة ( 2016 - 2019 ( ) كما هو موضح في الجدول2 :) وكاالتي أ- نسبة االستثمارات الى رأس المال واالحتياطيات السليمة للمصارف عينة الدراسة للمد ة ( 2016 - 2019 ( ) كما هو موضح في الجدول2 :) وكاالتي أ- نسبة االستثمارات الى رأس المال واالحتياطيات السليمة للمصارف عينة الدراسة للمد ة ( 2016 - 2019 ( ) كما هو موضح في الجدول2 :) وكاالتي 735 44 7 - 25 7 P. P Ghari Journal of Faculty of Administration and Economics Vol. 18 (No.3) 2022 - Al 44 7 - 25 7 P. P Ghari Journal of Faculty of Administration and Economics Vol. 18 (No.3) 2022 - Al Al-Ghari Journal of Faculty of Administration and Economics 1 - حقق مصرف بغداد اعلى نسبة لالستثمارات الى رأس المال واالحتياطيات السليمة في ( 2019 ( )اذ بلغت37.43 ) ويرجع سبب ارتفاع النسبة الى ارتفاع مبلغ االستثمارات حيث ( بلغت102,42 ( ) مليون دينار، في حين بلغت ادنى نسبة33.89 ( ) في سنة2017 ) وذلك لقيام ادارة المصرف بتخفيض ا( الستثمارات الى8,923) مليون دينار، كما بلغ متوسط هذ ه ( النسبة خالل المدة اعاله35.49 .) 2 - ( سجل مصرف االستثمار العراقي اعلى نسبة له في عام2018 ( ) اذ بلغت9.14 ) بسبب ( قيام المصرف بزيادة االستثمارات الى439 ) مليار دينار، في حين بلغت ادنى نسبة له ف ي (سنة2019) وهي ( 1.70 ( ) بسبب ارتفاع رأس المال واالحتياطيات الى260 ،) مليار دينار ( كما وبلغ متوسط هذه النسبة6.27 .) ب- ( معدل نمو األرباح: نالحظ في الجدول2 :) االتي 1 . اتصفت نسبة نمو األرباح في مصرف بغداد بالتفاوت من سنة الى أخرى حيث بلغت أعلى ( نسبة254.18 ( ) في سنة2016 ) وذلك ير جع إلى ارتفاع صافي الربح بعد الضريبة في ( هذه السنة مقارنة بالسنوات عينة الدراسة حيث بلغت20,200 ) مليون دينار، في حين ( بلغت أدنى نسبة69.76 - ( ) في سنة2017 ) لكونه حقق في هذه السنة خسارة مقدارها ( 6,100 ( ) مليون دينار، وكان متوسط هذه النسبة قد بلغ57.00 .) 2 . Al-Ghari Journal of Faculty شهدت النسبة في مصرف االستثمار انخفاضات متتالية من سنة ألخرى حتى ارتفعت في ( سنة2019 ( ) إذ بلغت49.93 ) وذلك يرجع إلى ارتفاع األرباح بعد الضريبة إلى ( 17,216 ( ) مليون دينار، في حين كانت أدنى نسبة له41.83 - ( ) في سنة2016 ) وذلك ( بسبب تحقيقه لخسارة مقدارها10,179 ) مليو ن دينار، كما بلغ متوسط هذه النسبة ( 36.04 - .) ت- ( تحليل نسبة العائد إلى إجمالي الموجودات: نالحظ في الجدول2 :) االتي 1 . ( حقق مصرف بغداد أعلى نسبة للعائد إلى إجمالي الموجودات بلغت1.69 ) في سنة ( 2016 ( ) ويرجع سبب ذلك إلى ارتفاع صافي العوائد في هذه السنة اذ بلغ20,200 ) مليون (ة أ ك ا ل ا0 37 (ة ) ف2018ال ائ إل اف ا فا ) Al-Ghari Journal of Faculty ث- :تحليل نسبة إجمالي االئتمان إلى إجمالي الودائع( نالحظ في الجدول2 :) االتي 1 . ( سجل مصرف بغداد أعلى نسبة إلجمالي االئتمان إلى إجمالي الودائع بلغت24.84 ) في ( سنة2018 ) بسبب قيام المصرف بمنح قروض أكثر من بقية السنوات عينة الدراسة إذ ( بلغت161,954 ( ) مليون دينار، في حين سجل أدنى نسبة له بلغت15.40 ) في سنة ( 2017 ( ) وذلك بسبب انخفاض إجمالي االئتمان الممنوح من قبل المصرف إلى145,602 ) ( مليون دينار، أيضاً بلغ متوسط هذه النسبة20.06 .) 2 . شهدت النسبة في مصرف االستثمار العراقي ارتفاعاً متواصالً من سنة الى أخرى حيث ( بلغت أدنى نسبة له45.32 ( ) في سنة2016) وذلك بسبب زيادة إجما لي الودائع في هذه ( السنة إذ بلغت283,975 ( ) مليون دينار، في حين بلغت اعلى نسبة86.44 ) في سنة ( 2019 ( ) بسبب ارتفاع اجمالي االئتمان الممنوح من قبل المصرف والذي يبلغ146,505 ) ( مليون دينار وقد بلغ متوسط هذه النسبة62.20 .) :المبحث الثالث تحليل تقييم اداء المصارف عينة الدراسة بعد بداية جائحة كورونا ( تم تحليل تقييم االداء على المصارف عينة الدراسة البالغ عددها2 ) مصارف ولمدة ( سنتين2020 - 2021 ( ) مدة حدوث وانتشار جائحة كوروناCOVID-19 ) بحسب المؤشرات المالية واستخراج النسب لقياس االداء المالي وتحليله وذلك اعتماداً على التقارير الما لية ال سنوية . لتلك المصارف وباعتماد آلية ومنهجية التقييم ذاتها قبل جائحة كورونا ( الجدول3 ) ( تطور النسب المالية للمصارف الخاصة عينة البحث للمدة2020 - 2021 ) المصارف ت مؤشرات عنصر كفاية رأس المال 2020 % 2021 % Average ث- :تحليل نسبة إجمالي االئتمان إلى إجمالي الودائع( نالحظ في الجدول2 :) االتي 1 . ( سجل مصرف بغداد أعلى نسبة إلجمالي االئتمان إلى إجمالي الودائع بلغت24.84 ) في ( سنة2018 ) بسبب قيام المصرف بمنح قروض أكثر من بقية السنوات عينة الدراسة إذ ( بلغت161,954 ( ) مليون دينار، في حين سجل أدنى نسبة له بلغت15.40 ) في سنة ( 2017 ( ) وذلك بسبب انخفاض إجمالي االئتمان الممنوح من قبل المصرف إلى145,602 ) ( مليون دينار، أيضاً بلغ متوسط هذه النسبة20.06 .) 2 . Al-Ghari Journal of Faculty of Administration and Economics ( كما حقق مصرف االستثمار أعلى نسبة للعائد على الموجودات بلغت1.76 ( ) في2016 ) ويرجع سبب هذا االرتفاع إلى زيادة صافي الع( وائد في هذه السنة إذ بلغ10,179 ) مليون ( دينار، في حين بلغت أدنى نسبة له0.003 ( ) في عام2019 ) وذلك بسبب انخفاض 736 44 7 - 25 7 P. P Ghari Journal of Faculty of Administration and Economics Vol. 18 (No.3) 2022 - Al Al-Ghari Journal of Faculty of Administration and Economics ( إجمالي الموجودات في هذه السنة فكانت529,829 ) مليون دينار، كما بلغ متوسط هذه ( النسبة0.63 .) Al-Ghari Journal of Faculty اظهرات نتائج مصرف االستثمار انخفاضاً كبيراً في نسبة االستثمارات الى رأس المال واالحتياطيات السليمة ( للسنة2021 ( ) اذ بلغت1.92 ( ) مقارنة بالسنة االساس2020 ) ( البالغة27.71 ) وحصل هذا االنخفاض في النسبة بسبب انخفاض اجمالي االستثمارات الى ( 4,950,588 ( ) مليون دينار في حين كانت في سنة2020 ( ) قد بلغت71,394,000 ) ( مليون دينار، وقد بلغ متوسط هذه النسبة14.82 .) 2 . اظهرات نتائج مصرف االستثمار انخفاضاً كبيراً في نسبة االستثمارات الى رأس المال واالحتياطيات السليمة ( للسنة2021 ( ) اذ بلغت1.92 ( ) مقارنة بالسنة االساس2020 ) ( البالغة27.71 ) وحصل هذا االنخفاض في النسبة بسبب انخفاض اجمالي االستثمارات الى ( 4,950,588 ( ) مليون دينار في حين كانت في سنة2020 ( ) قد بلغت71,394,000 ) ( مليون دينار، وقد بلغ متوسط هذه النسبة14.82 .) (ب و ب غ يون ي ر و) ب. تح( ليل نتائج نسبة نمو االرباح للمصارف عينة الدراسة، حيث نالحظ في الجدول3 ) :اآلتي 1 . ( سجل مصرف بغداد انخفاضاً كبيراً في نسبة نمو االرباح للسنة2021 ( ) اذ بلغت48.42 ) ( مقارنة بالسنة االساس2020 ( ) البالغة176.71 ) وذلك بسبب انخفاض حجم االرباح التي حققها المصرف في ه( ذه السنة البالغة29,980 ) مليون دينار في حين بلغت ارباح السنة ( 2020 ( ) 20,200 ( ) مليون دينار، وقد بلغ متوسط هذه النسبة112.57 .) 2 . كذلك لم يحقق مصرف االستثمار أية ارباح خالل السنتين عينة الدراسة اذ بلغت نسب نمو ( االرباح72.86 - ، 80.26 - ( ) للسنتين2020 ، 2021 ) على التوالي وذلك بسبب تحقيق ( خسائر بمقدار4,673,583 ( ) مليون دينار للسنة2020 ( ) وبمقدار922,599 ) مليون ( دينار للسنة2021 ( ) نتيجة تاثير الوباء العالميCOVID-19 ) الذي ادى الى انخفاض العمليات اإليرادية المصرفية مقابل ارتفاع التكاليف والمصروفات، وقد بلغ متوسط هذه ( النسبة76.56 - .) ب. تح( ليل نتائج نسبة نمو االرباح للمصارف عينة الدراسة، حيث نالحظ في الجدول3 ) :اآلتي ا (ارب ح و ب بير ي ب ر ج(ب )) ( مقارنة بالسنة االساس2020 ( ) البالغة176.71 ) وذلك بسبب انخفاض حجم االرباح التي حققها المصرف في ه( ذه السنة البالغة29,980 ) مليون دينار في حين بلغت ارباح السنة ( 2020 ( ) 20,200 ( ) مليون دينار، وقد بلغ متوسط هذه النسبة112.57 .) 2 . Al-Ghari Journal of Faculty اظهرات نتائج مصرف االستثمار انخفاضاً كبيراً في نسبة االستثمارات الى رأس المال واالحتياطيات السليمة ( للسنة2021 ( ) اذ بلغت1.92 ( ) مقارنة بالسنة االساس2020 ) ( البالغة27.71 ) وحصل هذا االنخفاض في النسبة بسبب انخفاض اجمالي االستثمارات الى ( 4,950,588 ( ) مليون دينار في حين كانت في سنة2020 ( ) قد بلغت71,394,000 ) ( مليون دينار، وقد بلغ متوسط هذه النسبة14.82 .) ب. تح( ليل نتائج نسبة نمو االرباح للمصارف عينة الدراسة، حيث نالحظ في الجدول3 ) :اآلتي 1 . ( سجل مصرف بغداد انخفاضاً كبيراً في نسبة نمو االرباح للسنة2021 ( ) اذ بلغت48.42 ) ( مقارنة بالسنة االساس2020 ( ) البالغة176.71 ) وذلك بسبب انخفاض حجم االرباح التي حققها المصرف في ه( ذه السنة البالغة29,980 ) مليون دينار في حين بلغت ارباح السنة ( 2020 ( ) 20,200 ( ) مليون دينار، وقد بلغ متوسط هذه النسبة112.57 .) 2 . كذلك لم يحقق مصرف االستثمار أية ارباح خالل السنتين عينة الدراسة اذ بلغت نسب نمو ( االرباح72.86 - ، 80.26 - ( ) للسنتين2020 ، 2021 ) على التوالي وذلك بسبب تحقيق ( خسائر بمقدار4,673,583 ( ) مليون دينار للسنة2020 ( ) وبمقدار922,599 ) مليون ( دينار للسنة2021 ( ) نتيجة تاثير الوباء العالميCOVID-19 ) الذي ادى الى انخفاض العمليات اإليرادية المصرفية مقابل ارتفاع التكاليف والمصروفات، وقد بلغ متوسط هذه ( النسبة76.56 - .) ت. تحليل نتائج نسبة العائد الى اجمالي الموجودات للمصارف عينة الدراسة، حيث ن الحظ ف ي ( الجدول3 :) االتي 44 7 - 25 7 P. P Ghari Journal of Faculty of Administration and Economics Vol. 18 (No.3) 2022 - Al Al-Ghari Journal of Faculty of Administration and Economics أ. تحليل نتائج نسبة االستثمارات الى رأس المال واالحتياطيات السليمة، حيث نال حظ في ( الجدول3 :) اآلتي 1 . حقق مصرف بغداد ارتفاعاً كبيراً في نسبة االستثمارات الى رأس المال واالحتياطيات ا لسليم ة ( في سنة2021 ( ) إذ بلغت167.98 ( ) مقارنة بالسنة2020) التي بلغت ( 71.17 ) بسبب ( ارتفاع اجمالي االستثمارات الى445,816,981 ) مليون دينار في حين بلغت ( 168,593,729 ( ) مليون دينار في سنة2020 ( )، كما بلغ متوسط هذه النسبة119.58 .) 2 . Al-Ghari Journal of Faculty شهدت النسبة في مصرف االستثمار العراقي ارتفاعاً متواصالً من سنة الى أخرى حيث ( بلغت أدنى نسبة له45.32 ( ) في سنة2016) وذلك بسبب زيادة إجما لي الودائع في هذه ( السنة إذ بلغت283,975 ( ) مليون دينار، في حين بلغت اعلى نسبة86.44 ) في سنة ( 2019 ( ) بسبب ارتفاع اجمالي االئتمان الممنوح من قبل المصرف والذي يبلغ146,505 ) ( مليون دينار وقد بلغ متوسط هذه النسبة62.20 .) :المبحث الثالث تحليل تقييم اداء المصارف عينة الدراسة بعد بداية جائحة كورونا ( تم تحليل تقييم االداء على المصارف عينة الدراسة البالغ عددها2 ) مصارف ولمدة ( سنتين2020 - 2021 ( ) مدة حدوث وانتشار جائحة كوروناCOVID-19 ) بحسب المؤشرات المالية واستخراج النسب لقياس االداء المالي وتحليله وذلك اعتماداً على التقارير الما لية ال سنوية . لتلك المصارف وباعتماد آلية ومنهجية التقييم ذاتها قبل جائحة كورونا ( الجدول3 ) ( تطور النسب المالية للمصارف الخاصة عينة البحث للمدة2020 - 2021 ) المصارف ت مؤشرات عنصر كفاية رأس المال 2020 % 2021 % Average بغداد 1 . نسبة االستثمارات الى راس المال واالحتياطيات السلي مة 71.17 167.98 119.58 2 . نسبة نمو االرباح 176.71 48.42 112.57 3 . نسبة العائد الى اجمالي الموجودات 1.42 1.95 1.69 4 . نسبة اجمالي االئتمان الى اجمالي الودائع 13.21 14.57 13.89 االستثمار العراقي 1 . نسبة االستثمارات الى راس المال واالحتياطيات السلي مة 27.71 1.92 14.82 2 . نسبة نمو االرباح -72.86 -80.26 -76.56 3 . نسبة العائد الى اجمالي الموجودات 0.82 0.14 0.48 4 . نسبة اجمالي االئتمان الى اجمالي الودائع 51.61 128.52 90.07 إعداد الباحثة اعتماداا على البيانات المالية السنوية للمصارف عينة الدراسة 737 737 44 7 - 25 7 P. P Ghari Journal of Faculty of Administration and Economics Vol. 18 (No.3) 2022 - Al Al-Ghari Journal of Faculty of Administration and Economics أ. تحليل نتائج نسبة االستثمارات الى رأس المال واالحتياطيات السليمة، حيث نال حظ في ( الجدول3 :) اآلتي 1 . حقق مصرف بغداد ارتفاعاً كبيراً في نسبة االستثمارات الى رأس المال واالحتياطيات ا لسليم ة ( في سنة2021 ( ) إذ بلغت167.98 ( ) مقارنة بالسنة2020) التي بلغت ( 71.17 ) بسبب ( ارتفاع اجمالي االستثمارات الى445,816,981 ) مليون دينار في حين بلغت ( 168,593,729 ( ) مليون دينار في سنة2020 ( )، كما بلغ متوسط هذه النسبة119.58 .) 2 . Al-Ghari Journal of Faculty Al-Ghari Journal of Faculty of Administration and Economics Al-Ghari Journal of Faculty كذلك لم يحقق مصرف االستثمار أية ارباح خالل السنتين عينة الدراسة اذ بلغت نسب نمو ( االرباح72.86 - ، 80.26 - ( ) للسنتين2020 ، 2021 ) على التوالي وذلك بسبب تحقيق ( خسائر بمقدار4,673,583 ( ) مليون دينار للسنة2020 ( ) وبمقدار922,599 ) مليون ( دينار للسنة2021 ( ) نتيجة تاثير الوباء العالميCOVID-19 ) الذي ادى الى انخفاض العمليات اإليرادية المصرفية مقابل ارتفاع التكاليف والمصروفات، وقد بلغ متوسط هذه ( النسبة76.56 - .) ت. تحليل نتائج نسبة العائد الى اجمالي الموجودات للمصارف عينة الدراسة، حيث ن الحظ ف ي ( الجدول3 :) االتي 1 . ( حقق مصرف بغداد ارتفاعاً طفيفاً في نسبة العائد الى اجمالي الموجودات للسنة2021 ) اذ ت. تحليل نتائج نسبة العائد الى اجمالي الموجودات للمصارف عينة الدراسة، حيث ن الحظ ف ي ( الجدول3 :) االتي 1 . ( حقق مصرف بغداد ارتفاعاً طفيفاً في نسبة العائد الى اجمالي الموجودات للسنة2021 ) اذ ( بلغت1.95 ( ) مقارنة بالسنة األساس2020 ( ) البالغة1.42 ) وذلك بسبب ارتفاع اجمالي 44 7 - 25 7 P. P Ghari Journal of Faculty of Administration and Economics Vol. 18 (No.3) 2022 - Al Al-Ghari Journal of Faculty of Administration and Economics 18 (No.3) 2022 - Al Al-Ghari Journal of Faculty of Administration and Economics ال( جدول4 ( ) مقارنة نتائج تقييم االداء للمصارف عينة البحث للفترتين2016 - 2019 ) و ( 2020 - 2021 ) قبل وبعد بداية جائحة كورونا ت مؤشرات االداء المالي مصرف بغداد مصرف االستثمار العراقي متوسط النسب قبل الجائحة متوسط النسب بعد الجائحة التفاوت في معدالت النسب متوسط النسب قبل الجائحة متوسط النسب بعد الجائحة التفاوت في معدالت النسب 1 . نسبة االستثمارات الى راس المال واالحتياطيات السلي مة 35.49 119.58 84.09 6.27 14.82 8.55 2 . نسبة نمو االرباح 57.00 112.57 55.57 -36.04 -76.56 -40.52 3 . نسبة العائد الى اجمالي الموجودات 0.82 1.69 0.87 0.63 0.48 0.15 4 . نسبة اجمالي االئتمان الى اجمالي الودائع 20.06 13.89 6.17 62.20 90.07 27.87 إعداد الباحثة باالعتماد على البيانات المالية السنوية للمصارف عينة البحث :االستنتاجات والتوصيات :أوالا: االستنتاجات 1 - اظهرت نتائج التحليل المالي للمصارف عينة البحث حصول انخفاض في بعض مؤشرات االداء المالي لفترة ما بعد بداية جائحة كورونا مقارنة بالفترة السابقة ل لجائح.ة 2 - سجل مصرف بغداد تراجعاً في كل من (نسبة اجمالي االئتمان الى اجمالي الودائع) اال انه حقق تقدماً في كل من (نسبة االستثمارات الى رأس المال ونسبة نمو االرباح ون سبة العائد الى اجمالي الموجودات) خالل فترة جائحة كورونا بالمقارنة مع فترة ما قبل .الجائحة 3 - سجل مصرف االستثمار تراجعاً في كل من (نسبة نمو االرباح ونسبة العائد الى اجمال ي الموجودات) اال انه ح قق تقدماً في كل من (نسبة االستثمارات الى رأس المال ونسبة اجمالي االئتمان الى اجمالي الودائع) خالل فترة جائحة كورونا مقارنة بالفترة ال تي سب قت . حدوث وانتشار الجائحة :ثانياا: التوصيات 1 - العمل على مواكبة التطورات المصرفية من خالل توجيه واشراك ادارات ومالكات الم صارف الخاصة عن طريق رابطة المصارف االهلية بدورات متقدمة وباشراف البنك المركزي العراقي لتعريفهم بالخطوط العامة التي يجري العمل بها لتقييم المصرف، ك ذلك التعرف على السبل االساسية والفاعلة لتخطي االزمات المالية وذلك من خالل االسها م 44 7 - 25 7 P. P Ghari Journal of Faculty of Administration and Economics Vol. Al-Ghari Journal of Faculty of Administration and Economics ( سنة2020) كم( ا بلغ متوسط هذه النسبة1.69 .) 2 . كذلك سجل مصرف االستثمار انخفاضاً في نسبة العائد الى اجمالي الموجودات للسنة ( 2021 ( ) اذ بلغت0.14 ( ) مقارنة بالسنة السابقة2020 ( ) البالغة0.82 ) وذلك بسبب ( انخفاض اجمالي االرباح بعد الضريبة في هذه السنة الى922,599) مليون دينار ب عد أن ( كانت4,673,583 ( ) مليون دينار في سنة2020 ( )، وقد بلغ متوسط هذه النسبة0.48 .) ث. تحليل نتائج نسبة اجمالي االئتمان الى اجمالي الودائع للمصارف عينة الدراسة ، حيث ( نالحظ في الجدول3 :) االتي ا 1 . حقق مصرف بغداد ارتفاعاً طفيفاً في نسبة اجمالي االئتمان الى اجمالي ال ودائع للسنة ( 2021 ( ) اذ بلغت14.57 ( ) مقارنة بالسنة السابقة2020 ( ) البالغة13.21 ) وذلك بسبب ( ارتفاع اجمالي االئتمان في هذه السنة الى168,747 ) مليون دينار بعد ما كان ( 141,629 ( ) مليون دينار في سنة2020 ( )، كما بلغ متوسط هذه النسبة13.89 .) 1 . حقق مصرف بغداد ارتفاعاً طفيفاً في نسبة اجمالي االئتمان الى اجمالي ال ودائع للسنة ( 2021 ( ) اذ بلغت14.57 ( ) مقارنة بالسنة السابقة2020 ( ) البالغة13.21 ) وذلك بسبب ( ارتفاع اجمالي االئتمان في هذه السنة الى168,747 ) مليون دينار بعد ما كان ( 141,629 ( ) مليون دينار في سنة2020 ( )، كما بلغ متوسط هذه النسبة13.89 .) 2 . أيضاً حقق مصرف( االستثمار ارتفاعاً كبيراً في النسبة للسنة2021 ( ) اذ بلغت128.52 ) ( مقارنة بالسنة السابقة2020 ( ) البالغة51.61) وذلك بسبب ارتفاع اجمالي االئتمان ل دى ( المصرف لهذه السنة الى316,174 ( ) مليون دينار في حين كان138,827 ) مليون دينار ( في سنة2020)، اما متوسط هذه ال( نسبة فكان90.07 . ) 2 . أيضاً حقق مصرف( االستثمار ارتفاعاً كبيراً في النسبة للسنة2021 ( ) اذ بلغت128.52 ) ( مقارنة بالسنة السابقة2020 ( ) البالغة51.61) وذلك بسبب ارتفاع اجمالي االئتمان ل دى ( المصرف لهذه السنة الى316,174 ( ) مليون دينار في حين كان138,827 ) مليون دينار ( في سنة2020)، اما متوسط هذه ال( نسبة فكان90.07 . ) :مقارنة تقييم اداء المصارف قبل وبعد بداية جائحة كورونا ( سيتم اجراء مقارنة لتقييم أداء المصارف عينة البحث بين الفترتين216 - 2019 ) ما ل قب (جائحة كورونا و2020 - 2021) فترة ما بعد بداية الجائحة ليتسنى لنا معرفة مدى تأثير الجائحة على االداء الم.الي للمصارف كعينة للبحث 739 44 7 - 25 7 P. P Ghari Journal of Faculty of Administration and Economics Vol. Al-Ghari Journal of Faculty of Administration and Economics 18 (No.3) 2022 - Al y ( ) Al-Ghari Journal of Faculty of Administration and Economics ال( جدول4 ( ) مقارنة نتائج تقييم االداء للمصارف عينة البحث للفترتين2016 - 2019 ) و ( 2020 - 2021 ) قبل وبعد بداية جائحة كورونا ت مؤشرات االداء المالي مصرف بغداد مصرف االستثمار العراقي متوسط النسب قبل الجائحة متوسط النسب بعد الجائحة التفاوت في معدالت النسب متوسط النسب قبل الجائحة متوسط النسب بعد الجائحة التفاوت في معدالت النسب 1 . نسبة االستثمارات الى راس المال واالحتياطيات السلي مة 35.49 119.58 84.09 6.27 14.82 8.55 2 . نسبة نمو االرباح 57.00 112.57 55.57 -36.04 -76.56 -40.52 3 . نسبة العائد الى اجمالي الموجودات 0.82 1.69 0.87 0.63 0.48 0.15 4 . نسبة اجمالي االئتمان الى اجمالي الودائع 20.06 13.89 6.17 62.20 90.07 27.87 إعداد الباحثة باالعتماد على البيانات المالية السنوية للمصارف عينة البحث :االستنتاجات والتوصيات :أوالا: االستنتاجات Al-Ghari Journal of Faculty اا 1 - اظهرت نتائج التحليل المالي للمصارف عينة البحث حصول انخفاض في بعض مؤشرات االداء المالي لفترة ما بعد بداية جائحة كورونا مقارنة بالفترة السابقة ل لجائح.ة 2 - سجل مصرف بغداد تراجعاً في كل من (نسبة اجمالي االئتمان الى اجمالي الودائع) اال انه حقق تقدماً في كل من (نسبة االستثمارات الى رأس المال ونسبة نمو االرباح ون سبة العائد الى اجمالي الموجودات) خالل فترة جائحة كورونا بالمقارنة مع فترة ما قبل .الجائحة 1 - اظهرت نتائج التحليل المالي للمصارف عينة البحث حصول انخفاض في بعض مؤشرات االداء المالي لفترة ما بعد بداية جائحة كورونا مقارنة بالفترة السابقة ل لجائح.ة 2 - سجل مصرف بغداد تراجعاً في كل من (نسبة اجمالي االئتمان الى اجمالي الودائع) اال انه حقق تقدماً في كل من (نسبة االستثمارات الى رأس المال ونسبة نمو االرباح ون سبة العائد الى اجمالي الموجودات) خالل فترة جائحة كورونا بالمقارنة مع فترة ما قبل .الجائحة 3 - سجل مصرف االستثمار تراجعاً في كل من (نسبة نمو االرباح ونسبة العائد الى اجمال ي الموجودات) اال انه ح قق تقدماً في كل من (نسبة االستثمارات الى رأس المال ونسبة اجمالي االئتمان الى اجمالي الودائع) خالل فترة جائحة كورونا مقارنة بالفترة ال تي سب قت . حدوث وانتشار الجائحة 3 - سجل مصرف االستثمار تراجعاً في كل من (نسبة نمو االرباح ونسبة العائد الى اجمال ي الموجودات) اال انه ح قق تقدماً في كل من (نسبة االستثمارات الى رأس المال ونسبة اجمالي االئتمان الى اجمالي الودائع) خالل فترة جائحة كورونا مقارنة بالفترة ال تي سب قت . Al-Ghari Journal of Faculty of Administration and Economics حدوث وانتشار الجائحة 3 - سجل مصرف االستثمار تراجعاً في كل من (نسبة نمو االرباح ونسبة العائد الى اجمال ي الموجودات) اال انه ح قق تقدماً في كل من (نسبة االستثمارات الى رأس المال ونسبة اجمالي االئتمان الى اجمالي الودائع) خالل فترة جائحة كورونا مقارنة بالفترة ال تي سب قت . حدوث وانتشار الجائحة :ثانياا: التوصيات 1 - العمل على مواكبة التطورات المصرفية من خالل توجيه واشراك ادارات ومالكات الم صارف الخاصة عن طريق رابطة المصارف االهلية بدورات متقدمة وباشراف البنك المركزي العراقي لتعريفهم بالخطوط العامة التي يجري العمل بها لتقييم المصرف، ك ذلك التعرف على السبل االساسية والفاعلة لتخطي االزمات المالية وذلك من خالل االسها م في المؤتمرات والندوات المصرف.ية 1 - العمل على مواكبة التطورات المصرفية من خالل توجيه واشراك ادارات ومالكات الم صارف الخاصة عن طريق رابطة المصارف االهلية بدورات متقدمة وباشراف البنك المركزي العراقي لتعريفهم بالخطوط العامة التي يجري العمل بها لتقييم المصرف، ك ذلك التعرف على السبل االساسية والفاعلة لتخطي االزمات المالية وذلك من خالل االسها م في المؤتمرات والندوات المصرف.ية 740 44 7 - 25 7 P. P Ghari Journal of Faculty of Administration and Economics Vol. 18 (No.3) 2022 - Al 44 7 - 25 7 P. P Ghari Journal of Faculty of Administration and Economics Vol. 18 (No.3) 2022 - Al Al-Ghari Journal of Faculty of Administration and Economics 2 - العمل على تشجيع المصارف من قبل البنك المركزي في تعزيز او زيادة منح القروض الى االفراد والمؤسسات خالل فترات االزمات المالية واالوبئة التي من الممكن ان يتعرضوا لها من اجل تقديم الدعم الالزم ومساندة الشركات واالفراد لتفادي االثار المت رتبة عن تلك االزمات.واالوبئة هوامش البحث 2 - العمل على تشجيع المصارف من قبل البنك المركزي في تعزيز او زيادة منح القروض الى االفراد والمؤسسات خالل فترات االزمات المالية واالوبئة التي من الممكن ان يتعرضوا لها من اجل تقديم الدعم الالزم ومساندة الشركات واالفراد لتفادي االثار المت رتبة عن تلك االزمات.واالوبئة ( 1) سالم محمد عبود، ادارة االزمات ( والكوارث جائحة كورونا19 - COVID) أنموذجاً، دار الصادق للنشر، ط1 ، 2021، ص8 . 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Procedia – social and Behavioural sciences, 24, 1615 -1623, 2011,P1618. g , , , , (8) T.B lelissa, the Determinants of Ethiopian Commercial Banks Performance European Journal of Business and Management, 6 (14), PP:52-62, 2014, P55. ا g , , , , (8) T.B lelissa, the Determinants of Ethiopian Commercial Banks Performance European Journal of Business and Management, 6 (14), PP:52-62, 2014, P55. ا ( 9) معدل الفائدة االسمي = معدل الفائدة الحقيقي +معدل التضخم .للمزيد من التفاصيل ا نظر : عبد المنعم السيد علي ،نزار سعد الدين العيسى ، النقود والمصارف واالسواق المالية ،بدون دار النشر ،األردن، ط1 ، 2004 ، ص 298 . ( 10 ) محمد فتحي البديوي ،اداره ال ،بنوك المكتبة األكاديمية، ط1 ، 2012، ص16 .ا ي ب يوي ر ب و ب أي ي ص ( 11 ) زياد رمضان، محفوظ جوده، االتجاهات المعاصرة في اداره البنوك، دار وائل للنشر والطباعة ،ط4 ، 2006، ص3. (12) somashekar, N.T., "Banking" new age international pubished, USA, 2009, P1. 11 ) زياد رمضان، محفوظ جوده، االتجاهات المعاصرة في اداره البنوك، دار وائل للنشر والطباعة ،ط4 ، 2006، ص3. (12) somashekar N T "Banking" new age international pubished USA 2009 P1 ا (12) somashekar, N.T., "Banking" new age international pubished, USA, 2009, P1. g g p (13) Peter, Rose, commercial Bank Management, Measuring and Evaluting Bank performanc Mc Grow, Hill Irwin Edition, 2002, P22. ااا (13) Peter, Rose, commercial Bank Management, Measuring and Evaluting Bank performanc Mc Grow Hill Irwin Edition, 2002, P22. ن التفاصيل ا نظر :عبد المنعم السيد Al-Ghari Journal of Faculty of Administration and Economics ااا ( 14 ) رسالن عالء الدين ،التطوير التنظيمي ، دار رسالن للطباعة والنشر والتوزيع ،سوريا، ط1 ، 2012 ،ص 186 . ( 14 ) رسالن عالء الدين ،التطوير التنظيمي ، دار رسالن للطباعة والنشر والتوزيع ،سوريا، ط1 ، 2012 ، ص 186 . ( 15 ) حيدر عباس الجنابي ، التنبؤ بالفشل المالي للمصارف التجاري ة باستخدام نموذجsherrord (بحث تطبيقي على عين ة من المصارف في سوق العراق لألوراق المالي ة للمد ة 2011 - 2016 ) مجله االدار ة واالقتصاد/مجلد7 ، العدد28 ،ص6 . ( 16 ) ،أحمد بن البار و ،حمزة غربي و عيسى بدروني، دراسة قياسية ألثر جائحة كورونا على اال سواق المالية العربية– بورصة القاهرة أ نموذجاً، مجلة االجتهاد للدراسات القانونية واالقتصادية ، المجلد9 ، العدد4 ، 2020، ص425 . ( 17 ) حسن لطيف كاظم، االقتصاد العراقي في زمن كورونا فايروس: نتائج خفض االنفاق، شبكة االقتصاديين ،العراقيين، اوراق في السياسة المالية ،العراق2020، ص5 . 741 44 7 - 25 7 P. P Ghari Journal of Faculty of Administration and Economics Vol. 18 (No.3) 2022 - Al ( 18 ) ،محمد المهدي، أحمد عكاشة، كورونا الصمود في وجه الجائحة، دار الورد للطباعة والنشر والتوزيع ط1 ، 2020 ، ص11 . ( 19 ) علي حمود السعدي، واسماء حسن محمد، فعالية المرونة الجينية للفيروسات التاجية، مؤسسة دار الصادق الثقافية، ط1 ، 2021، ص7 . ( 18 ) ،محمد المهدي، أحمد عكاشة، كورونا الصمود في وجه الجائحة، دار الورد للطباعة والنشر والتوزيع ط1 ، 2020 ، ص11 . ( 19 ) علي حمود السعدي، واسماء حسن محمد، فعالية المرونة الجينية للفيروسات التاجية، مؤسسة دار الصادق الثقافية، ط1 ، 2021، ص7 . (20) Huw Jones, There are limits to boldly bank rules in corona virus pandemic, 15 APRIL, 2020 https//www.reuters/article/us-health-coronavirus. ا p ( 21 ) ( المشهد المصرفي في دولة قطر2020 :) متاح على الرابط االتيhome.kpmg/qa ( 22 ) .المصدر نفسه ( 23 ) مصرف بغداد االهلي، التقرير السنوي2021 . ( 24 ) مصرف االستثمار العراقي، التقرير السنوي، 2021 . المصادر والمراجع 1 . أحمد بن البار، وحمزة غربي، وعيسى بدروني، دراسة قياسية ألثر جائحة كورونا على االسواق المالية العربية– بورصة القاهرة أنموذجاً، مجلة االجتهاد للدراسات الق انونية واالقتصادية ، المجلد9 ، العدد4 ، 2020 . 1 . أحمد بن البار، وحمزة غربي، وعيسى بدروني، دراسة قياسية ألثر جائحة كورونا على االسواق المالية العربية– بورصة القاهرة أنموذجاً، مجلة االجتهاد للدراسات الق انونية واالقتصادية ، المجلد9 ، العدد4 ، 2020 . 2 . ،حسن لطيف كاظم، االقتصاد العراقي في زمن كورونا فايروس: نتائج خفض االنفاق ،شبكة االقتصاديين العراقيين، اوراق في السياسة المالية، العراق2020 . 2 . حسن لطيف كاظم، االقتصاد العراقي في زمن كورونا فايروس: نتائج خفض االنفاق ،شبكة االقتصاديين العراقيين، اوراق في السياسة المالية، العراق2020 . 3 . ،حيدر عباس الجنابي التنبؤ بالفشل المالي للمصارف التجاري ة باستخدام نموذج sherrord (بحث تطبيقي على عين ة من المصارف في سوق العراق لألوراق المالي ة للمد ة 2011 - 2016 ) مجله االدار ة واالقتصاد/مجلد7 ، العدد28 . 4 . رسالن عالء الدين، التطوير التنظيمي، دار رسالن للطباعة والنشر والتوزيع، س،وريا ط1 ، 2012 . 4 . رسالن عالء الدين، التطوير التنظيمي، دار رسالن للطباعة والنشر والتوزيع، س،وريا ط1 ، 2012 . 4 . رسالن عالء الدين، التطوير التنظيمي، دار رسالن للطباعة والنشر والتوزيع، س،وريا ط1 ، 2012 . 5 . زياد رمضان، محفوظ جوده، االتجاهات المعاصرة في اداره البنوك، دار وائل للنشر والطباعة ،ط4 ، 2006 . 5 . زياد رمضان، محفوظ جوده، االتجاهات المعاصرة في اداره البنوك، دار وائل للنشر 742 44 7 - 25 7 P. P Ghari Journal of Faculty of Administration and Economics Vol. 18 (No.3) 2022 - Al Al-Ghari Journal of Faculty of Administration and Economics 6 . ( سالم محمد عبود، ادارة االزمات والكوارث جائحة كوروناCOVID-19) أنموذجاً، دار الصادق للنشر، ط1 ، 2021 . 7 . عبد المنعم السيد علي ،نزار سعد الدين العيسى ، النقود والمصارف واالسواق المالية ، ،بدون دار النشر األردن، ط1 ، 2004 . 8 . ،علي حمود السعدي، واسماء حسن محمد، فعالية المرونة الجينية للفيروسات التاجية 8 . ،علي حمود السعدي، واسماء حسن محمد، فعالية المرونة الجينية للفيروسات التاجية مؤسسة دار الصادق الثقافية، ط1 ، 2021 . مؤسسة دار الصادق الثقافية، ط1 ، 2021 . 9 . محمد المهدي، أحمد عكاشة، كورونا الصمود في وجه الجائحة، دار الورد للطباعة 9 . محمد المهدي، أحمد عكاشة، كورونا الصمود في وجه الجائحة، دار الورد للطباعة والنشر والتوزيع، ط1 ، 2020 . 10 . المصادر والمراجع محمد فتحي البديوي ،اداره ال بنوك، المكتبة األكاديمية، ط1 ، 2012 . 11 . ( المشهد المصرفي في دولة قطر2020 :) متاح على الرابط االتيhome.kpmg/qa 12 . مصرف االستثمار العراقي، التقرير السنوي، 2021 . 13 . مصرف بغداد االهلي، التقرير السنوي2021 . 11 . ( المشهد المصرفي في دولة قطر2020 :) متاح على الرابط االتيhome.kpmg/qa 12 . مصرف االستثمار العراقي، التقرير السنوي، 2021 . 13 .مصرف بغداد االهلي، التقرير السنوي2021 . 13 . مصرف بغداد االهلي، التقرير السنوي2021 . 13 . مصرف بغداد االهلي، التقرير السنوي2021 . 14. Champa Ramkrishna parab , credit Risk and impact on the performance of select public and private Banks in india, Doctoral dissertation, Goa univer 2019, P138. 15. David f, concept of strategic management 8th ed, prentice Hall, inc, 2001, P308. 16. Gragg Ross Dawson , financial Effficency in school, Research Report, Department for Educat 2010, P20. 743 44 7 - 25 7 P. P Ghari Journal of Faculty of Administration and Economics Vol. 18 (No.3) 2022 - Al Al-Ghari Journal of Faculty of Administration and Economics 17. Huw Jones, There are limits to boldly bank rules in corona virus pandemic, 15 APRIL, 2020. https//www.reuters/article/us-health- coronavirus. 18. Mohammad fakhrul Alamonik , Business value of Information Technology:A complex Adaptive system theory view, Doctral Dissertation, Queensland university of Technology, 2019،P150. 19. Peter, Rose, commercial Bank Management, Measuring and Evaluting Bank performanc Mc Grow, Hill Irwin Edition, 2002, P22. 20. Rose. S. Peter. commercial Bank management, Irw MCG raw- Hill, London. 1999, P179. 21. somashekar, N.T., "Banking" new age international pubished, USA, 2009, P1. 22. T. colakoglu, The problematic of competitive intelligence: How to Evaluate & Develop competitive intelligence? Procedia – social and Behavioural sciences, 24, 1615 -1623, 2011, P1618. 23. T.B lelissa, the Determinants of Ethiopian Commercial Banks Performance European Journal of Business and Management, 6 (14), PP:52-62, 2014, P55. 22. T. colakoglu, The problematic of competitive intelligence: How to Evaluate & Develop competitive intelligence? Procedia – social and Behavioural sciences, 24, 1615 -1623, 2011, P1618. 744
W4313031385.txt	https://aprp.msal.ru/jour/article/download/3514/1994	en		Relationship between Participation and Governance in the System of Corporate Relations	Aktualʹnye problemy rossijskogo prava	2,022	cc-by	5,024	ПРЕДПРИНИМАТЕЛЬСКОЕ И КОРПОРАТИВНОЕ ПРАВО DOI: 10.17803/1994-1471.2022.142.9.125-135 В. Л. Слесарев, В. Д. Кравец* Отношения участия и управления в системе корпоративных отношений Аннотация. Актуальность темы исследования обуславливается изменениями положений ст. 2 Гражданского кодекса РФ, в соответствии с которыми к предмету гражданско-правового регулирования отнесены отношения, связанные с участием в корпоративных организациях или с управлением ими (корпоративные отношения). Работа посвящена исследованию правовой природы отношений участия в корпоративной организации и управления ею, определению места данных отношений в системе корпоративных отношений. Авторы приходят к выводу, что отношения участия и управления являются организационно-предпосылочными, носят общерегулятивный характер. Содержанием данных правоотношений является специальная корпоративная правосубъектность как субъективное право и противостоящая ему обязанность иных лиц признавать управомоченное лицо участником корпоративных отношений, учитывать особую связанность корпорации и участника. Общерегулятивные отношения участия и управления являются предпосылкой возникновения конкретных корпоративных отношений, определяют их содержание и динамику развития. Ключевые слова: предмет гражданского права; субъекты гражданского права; юридические лица; корпоративные отношения; организационные отношения; участие; управление; корпорация; правосубъектность; корпоративная правосубъектность. Для цитирования: Слесарев В. Л., Кравец В. Д. Отношения участия и управления в системе корпоративных отношений // Актуальные проблемы российского права. — 2022. — Т. 17. — № 9. — С. 125–135. — DOI: 10.17803/1994-1471.2022.142.9.125-135. © Слесарев В. Л., Кравец В. Д., 2022 * ** Слесарев Владимир Львович, доктор юридических наук, профессор, начальник отдела договорного права Исследовательского центра частного права имени С.С. Алексеева при Президенте Российской Федерации ул. Ильинка, д. 8, стр. 2, г. Москва, Россия, 103132 kalikin1948@gmail.com Кравец Виктория Дмитриевна, кандидат юридических наук, доцент кафедры гражданского права Московского государственного юридического университета имени О.Е. Кутафина (МГЮА) Садовая-Кудринская ул., д. 9, г. Москва, Россия, 125993 vdkravec@msal.ru Актуальные проблемы российского права. 2022. Т. 17. № 9 (142) сентябрь 125 Предпринимательское и корпоративное право Relationship between Participation and Governance in the System of Corporate Relations Vladimir L. Slesarev, Dr. Sci. (Law), Professor, Head of the Department of Contract Law, Private Law Research Centre (PLRC) ul. Ilinka, d. 8, str. 2, Moscow, Russia, 103132 kalikin1948@gmail.com Viktoria D. Kravets, Cand. Sci. (Law), Associate Professor, Department of Civil Law, Kutafin Moscow State Law University (MSAL) ul. Sadovaya-Kudrinskaya, d. 9, Moscow, Russia, 125993 vdkravec@msal.ru Abstract. The relevance of the research topic is determined by the changes in the provisions of Art. 2 of the Civil Code of the Russian Federation stating that the subject of civil law regulation includes relations involving participation in corporate organizations or their governance (corporate relations). The paper is devoted to the study of the legal nature of the relations of participation in a corporate organization and its management, determining the place of these relations in the system of corporate relations. The authors conclude that the relationship between participation and governance are organizational and prerequisite relations, they are of a general regulatory nature. The content of these legal relations is a special corporate legal personality as a subjective right and the opposing obligation of other persons to recognize the authorized person as a participant in corporate relations, to take into account the special connection between the corporation and the participant. General regulatory relations of participation and governance are a prerequisite for the emergence of specific corporate relations, and determine their content and development dynamics. Keywords: subject of civil law; subjects of civil law; legal entity; corporate relations; organizational relationships; participation; control; corporation; legal personality; corporate legal personality. Cite as: Slesarev VL, Kravets VD. Otnosheniya uchastiya i upravleniya v sisteme korporativnykh otnosheniy [Relationship between Participation and Governance in the System of Corporate Relations]. Aktualʹnye problemy rossijskogo prava. 2022;17(9):125-135. DOI: 10.17803/1994-1471.2022.142.9.125-135. (In Russ., abstract in Eng.). Общие положения Изменение ст. 2 ГК РФ путем фиксации положения о том, что гражданское законодательство регулирует, наряду с иными, отношения, связанные с участием в корпоративных организациях или с управлением ими (корпоративные отношения), основанные на равенстве, автономии воли и имущественной самостоятельности участников, значительно усилило интерес к исследованию не только природы и содержания корпоративных отношений, но и места, которое 1 2 126 они занимают в системе российского права в целом, и гражданского права в частности1. Необходимо подчеркнуть, что первоначально предлагалась несколько иная редакция данной статьи. В Концепции развития гражданского законодательства Российской Федерации (одобренной решением Совета при Президенте РФ по кодификации и совершенствованию гражданского законодательства от 07.10.2009)2 в разд. 2 (п. 1.2) была выражена следующая позиция: «С точки зрения предмета гражданского законодательства необходимо включить в п. 1 См. подробнее: Тягай Е. Д. «Участие» в корпоративных организациях и «управление» ими: проблемы легального закрепления и дифференциации корпоративных отношений // Lex russica. 2016. № 11 (120), ноябрь. С. 26. См.: Вестник ВАС РФ. 2009. № 11, ноябрь. Актуальные проблемы российского права. 2022. Т. 17. № 9 (142) сентябрь Слесарев В. Л. Отношения участия и управления в системе корпоративных отношений ст. 2 ГК еще одну группу отношений, регулируемых гражданским законодательством, а именно корпоративные отношения, под которыми следует понимать отношения, связанные с “правом участия” в корпорации, а также соответствующие обязательственные отношения между учредителями (участниками) и корпорацией. Нормы, регулирующие корпоративные отношения, могут также применяться на субсидиарной основе и к юридическим лицам некорпоративного типа, если иное не вытекает из существа таких отношений». Таким образом, Концепция связывала корпоративные отношения с правом участия в корпорации, а также с обязательственными отношениями между учредителями (участниками) и корпорацией. Действующая редакция ГК определяет их как отношения, связанные с участием в корпоративных организациях или с управлением ими. Определение корпоративных отношений, содержащееся в действующей статье ГК РФ, является более точным по ряду причин. Во-первых, нормативное предписание касается отношений участия как элемента предмета гражданскоправового регулирования, что первично, а не правоотношений участия (как результата регулирования), о чем говорилось в Концепции. Во-вторых, наряду с отношениями участия, в предмет гражданско-правового регулирования были включены также отношения по управлению корпоративными организациями. Не касаясь здесь соотношения терминов участия и управления, хотелось бы обратить внимание на разделительный союз «или», используемый в анализируемой формулировке: гражданское законодательство регулирует отношения, связанные с участием в корпоративных организациях или с управлением ими. Применение данной конструкции означает, что «участие» и «управление», являясь основными началами корпоративных отношений, не всегда одновременно в них присутствуют. Например, в 3 4 случаях, предусмотренных законодательством, корпорацией могут управлять лица, не являющиеся ее участниками. Речь идет о корпоративном управлении со стороны третьих лиц. Возможность такого управления предусмотрена, в частности, применительно к хозяйственному партнерству, когда порядок корпоративного управления может определяться соглашением об управлении партнерством с лицами, не являющимися его участниками3. С другой стороны, не всегда те или иные участники корпоративной организации наделены определенными полномочиями в рамках права управления. Как известно, в товариществе на вере (коммандитном товариществе), наряду с его участниками — полными товарищами, имеются и участникивкладчики (коммандитисты), которые не вправе участвовать в управлении и ведении дел товарищества на вере, выступать от его имени иначе как по доверенности (ст. 84 ГК РФ). Вместе с тем примеры указанного регулирования являются специальными правилами, общим же правилом выступает положение о принадлежности участнику корпорации как права участия, так и права управления. Поскольку легальных определений понятий «участие» и «управление» нет, в литературе даются их определения, зачастую значительно не совпадающие по своему содержанию. Более того, несмотря на представленную в ст. 2 ГК РФ структуру корпоративных отношений, в литературе было высказано мнение, что под корпоративными связями понимаются не отношения участия (членства), а исключительно отношения управления4. Категория «участие» в корпоративных отношениях Первостепенное значение при анализе корпоративных отношений имеет выделение среди них отношений участия, или членства (в дальнейшем — просто участия), субъектов корпорации См.: Федеральный закон от 03.12.2011 № 380‑ФЗ «О хозяйственных партнерствах», ч. 1 ст. 6 // СЗ РФ. 2011. № 48 (ч. 5). Ст. 7058. См.: Гутников О. В. Корпоративная ответственность в гражданском праве. М. : ИЗиСП, Контракт, 2019. С. 90. Актуальные проблемы российского права. 2022. Т. 17. № 9 (142) сентябрь 127 Предпринимательское и корпоративное право в качестве необходимой предпосылки как отношений управления, так и иных корпоративных связей. Анализ законодательства показывает, что отношение участия является длящимся элементом корпоративного отношения между корпорацией и ее участником, оно признается законодателем как особое состояние связанности ее субъектов: участник корпорации имеет право считаться участником корпорации, а последняя обязана действовать в соответствии с этим правом. Применительно к участию необходимо различать его фактическое основание (деятельность по созданию корпорации — учредительство) и юридические последствия такой деятельности — правоотношения связанности участников и корпорации. Необходимо отметить, что статус участника корпорации, по общему правилу, трансформируется из статуса ее учредителя при наступлении определенных юридических фактов: принятии решения о создании юридического лица, утверждении учредительных документов и государственной регистрации организации. Конкретный юридический состав зависит от типа, статуса, организационно-правовой формы и вида будущей деятельности корпорации5. Что касается самого правоотношения связанности (участников и корпорации), то в литературе иногда утверждается, что «сложный характер правоотношения участия (членства) проявляется в том, что его содержание образуют многочисленные имущественные и неимущественные права, а также обязанности, которыми обладают друг по отношению к другу субъекты данного правоотношения»6. Мы полагаем, что правоотношения связанности никакие другие права, как имущественные, 5 6 7 8 9 10 128 так и неимущественные, в себя не включают. Критикуемый подход не учитывает сложную структуру корпоративных отношений, которые складываются из предпосылочных организационных отношений участия, а также иных, уже организуемых корпоративных отношений, которые могут возникать и развиваться лишь при наличии первых, не сливаясь с ними. Более того, организационно-предпосылочный и, следовательно, неимущественный характер отношений участия не позволяет характеризовать их как имущественные связи, в противном случае может создаваться крайне противоречивое определение отношений участия7. Например, в литературе высказана позиция о том, что правоотношение участия можно определить как урегулированное нормами права единое, сложное, внутриорганизационное общественное отношение имущественного характера, возникающее между корпорациями и их участниками в момент приобретения последними прав участия, опосредующих процесс их участия в деятельности корпорации8. На наш взгляд, правоотношение участия носит общий характер, а его содержанием является корпоративная правосубъектность как особое субъективное право9, которому противостоит обязанность всех иных лиц признавать управомоченное лицо участником корпоративных отношений, а также воздержаться от действий, которые препятствуют возможности лица признаваться участником корпорации. Таким образом, правоотношения участия отражают особую связанность участника и корпорации, присущую корпоративным отношениям, и являются предпосылкой возникновения иных корпоративных отношений (в литературе их называют производными), которые в той или иной мере Качалова А. В., Сойфер Т. В. Проблемы участия юридических лиц в гражданском обороте : учебное пособие для магистратуры. М. : Норма, Инфра‑М, 2021. С. 31. Корпоративное право : учебный курс : в 2 т. / отв. ред. И. С. Шиткина. М. : Статут, 2017. Т. 1. С. 828–829. Подробнее вопрос о концепции организационных отношений, выдвинутой О. А. Красавчиковым, будет изложен ниже. Корпоративное право : учебный курс. С. 828. О правосубъектности как содержании общерегулятивных отношений см. подробнее: Алексеев С. С. Проблемы теории права : курс лекций // Собрание сочинений : в 10 т. М. : Статут, 2010. Т. 3. С. 272–279. Корпоративное право : учебный курс. С. 816–828. Актуальные проблемы российского права. 2022. Т. 17. № 9 (142) сентябрь Слесарев В. Л. Отношения участия и управления в системе корпоративных отношений вбирают в себя некоторые свойства, присущие отношениям, казалось бы, далеким от корпоративных связей. Так, в литературе наиболее распространены вещно-правовая и обязательственно-правовая природа правоотношений участия10. Вещно-правовая концепция базируется, главным образом, на позиции расщепления собственности между корпорацией и ее участниками. Так, Н. Н. Пахомова характеризует данные отношения как собственность с множественным составом субъектов11. По мнению С. А. Зинченко, корпоративные права есть абсолютные имущественные права собственности особого, невещного характера12. Данный подход не получил поддержки в силу того, что здесь не учитываются различия экономической и юридической категорий собственности: право собственности не может охватывать все экономические отношения собственности, при необходимости допустимо использование и иных институтов вещного права13. Что касается концепции обязательственноправовой природы правоотношений участия, то в ее основу положена позиция о договорной природе корпорации: соглашение учредителей о ее создании направлено на объединение усилий для достижения общей цели, а все последующие акции (формирование ее органов, деятельность корпорации) производны от указанного соглашения14. Возражения по представленному подходу в основном сводятся к наличию особенностей отношений участия (членства) по сравнению с другими обязательственными правоотношениями. Это касается и набора их юридико-фактических оснований, и сроков их существования, например, что для определения содержания конкретных действий корпорации, которые она должна совершить по отношению к своим участникам (членам), требуются, помимо отноше11 12 13 14 ний участия, и другие юридически значимые действия, в частности принятие общим собранием акционеров решения о выплате дивидендов и пр. Мы полагаем, что решение вопроса о вещноправовой или обязательственно-правовой природе (свойствах) касается лишь производных корпоративных связей, поскольку корпоративное отношение участия имеет организационноправовую, предпосылочную природу и лежит в основе всех иных корпоративных отношений, определяя правосубъектность лица как участника корпорации. Таким образом, существующая система корпоративных отношений складывается из ее основы — организационных отношений участия (а как мы увидим дальше, и отношений управления) и из производных отношений — конкретных имущественных или неимущественных связей (прав и обязанностей) субъектов корпоративных отношений. Именно они, несмотря на их самостоятельность, в значительной мере близки к абсолютным отношениям собственности или относительным обязательственным отношениям. Корпоративные отношения так или иначе взаимодействуют и в определенной мере вбирают в себя признаки и качества иных гражданско-правовых отношений, в том числе отношений собственности и обязательственных отношений. Корпоративные отношения являются, по сути, модификацией, развитием этих отношений, что в конечном счете позволило установить специальные правила о возможности применения к корпоративным отношениям обязательственно-правовых положений ГК РФ. Так, в ст. 307.1 «Применение общих положений об обязательствах» указано, что, поскольку иное не установлено ГК РФ, иными законами или не вытекает из существа соответствующих отношений, общие положения об обязательствах Пахомова Н. Н. Цивилистическая теория корпоративных отношений. Екатеринбург : Налоги и финансовое право, 2005. С. 133. Зинченко С. А. Корпоративные отношения в реформируемом гражданском законодательстве России // Гражданское право. 2014. № 4. С. 8–12. Корпоративное право : учебный курс. С. 819. Корпоративное право : учебный курс. С. 821–827. Актуальные проблемы российского права. 2022. Т. 17. № 9 (142) сентябрь 129 Предпринимательское и корпоративное право (подразд. 1) применяются к требованиям, возникающим из корпоративных отношений. Здесь же следует указать и на правоотношения, направленные на абсолютную защиту нарушенных прав правообладателей, со счета которых были неправомерно списаны бездокументарные ценные бумаги, например акции как инструменты корпоративного права, и применение к указанным случаям мер, весьма близких к виндикационным требованиям (ст. 149.3–149.5 ГК РФ). Более того, в литературе концептуально обосновывается расширение сферы применения юридической конструкции виндикации: на истребование не только недвижимого имущества и ценных бумаг, но и корпоративных прав15. Правоотношения участия означают не только принадлежность субъекта к определенной организации, связанность указанных лиц избранными ими целями и задачами совместной деятельности. Главное в этих отношениях — быть организующим ядром, предпосылкой иных корпоративных отношений, зависимых от отношений участия. В связи с этим отношения участия (членства), поскольку они направлены на организацию иных корпоративных связей и являются их необходимой предпосылкой, должны быть отнесены, по классификации О. А. Красавчикова, к организационно-предпосылочным отношениям. Напомним, что указанный автор выделял следующие виды организационных отношений: предпосылочные, делегирующие, контрольные и информационные16. К числу предпосылочных относятся и отношения участия. 15 16 17 18 19 20 21 130 Категория «управление» в корпоративных отношениях Категория управления обычно выводится из понятий, лежащих далеко за пределами права. Так, в одном из толковых словарей отмечается, что управлять — значит быть хозяином, распорядителем чего-то17. Философы говорят об управлении как элементе, функции организованных систем различной природы, в том числе и социальных, обеспечивающей сохранение их определенной структуры, поддержание режима деятельности, реализацию программы, цели деятельности18. Под корпоративным управлением понимается совокупность способов воздействия или процесс, с помощью которого организуется деятельность корпорации как особой организационно-правовой формы юридического лица19. В Кодексе корпоративного управления20, одобренном 21 марта 2014 г. Советом директоров Банка России21, указано, что данное понятие охватывает систему взаимоотношений между исполнительными органами акционерного общества, его советом директоров, акционерами и другими заинтересованными сторонами. Оно является инструментом для определения целей общества и средств достижения этих целей, а также обеспечения эффективного контроля за деятельностью общества со стороны акционеров и других заинтересованных сторон. Как справедливо указал В. К. Андреев, модернизация гл. 4 ГК РФ позволяет говорить о формировании отечественной модели корпоративного управления хозяйственного общества, при которой структура и компетенция органов конкретного общества самостоятельно устанав- Мурзин Д. В. Виндикационная модель защиты абсолютных имущественных прав в российском гражданском праве : автореф. дис. … д-ра юрид. наук. Екатеринбург, 2022. С. 33. См.: Красавчиков О. А. Гражданские организационно-правовые отношения // Советское государство и право. 1966. № 10. С. 50–57. Даль В. И. Толковый словарь живого великорусского языка : в 4 т. М., 1994. Т. 4. С. 504. Философский энциклопедический словарь. М., 1983. С. 704. Корпоративное право : учебник / отв. ред. И. С. Шиткина. М. : Статут, 2019. С. 519. См. подробнее: Макарова О. А., Попондопуло В. Ф. Корпоративное право : учебник и практикум для вузов. М. : Юрайт, 2022. С. 281. СПС «КонсультантПлюс». Актуальные проблемы российского права. 2022. Т. 17. № 9 (142) сентябрь Слесарев В. Л. Отношения участия и управления в системе корпоративных отношений ливаются по единогласному решению его участников в рамках п. 3 ст. 66.3 ГК РФ и отражаются в его уставе22. И еще два момента, на которые хотелось бы обратить внимание. Первый касается негативного отношения некоторых авторов к категории «управление» как основания специальной характеристики корпоративных отношений. Так, Д. В. Ломакин отмечает, что выделение в ГК РФ особой группы правоотношений, связанных с управлением корпорацией, имеет лишь некоторые предпосылки прикладного характера. Однако их появление приводит, по сути, к отказу от выработки единого критерия классификации данных отношений, что затрудняет их унифицированное регулирование. Так, правоотношения по управлению корпорацией возникают в процессе осуществления участниками (членами) права голоса. Вместе с тем данные правоотношения обусловлены принадлежностью к корпорации, в связи с чем они одновременно являются и правоотношениями участия23. Второй момент: в литературе справедливо указывается на тесную, даже неразрывную связь отношений участия и управления. Так, В. Ф. Яковлев отмечал, что субъектами корпоративных отношений являются корпорации, а также участники, которые обладают правом членства, т.е. правом управления деятельностью юридических лиц24. Мы исходим из того, что управление, наряду с участием, названо в ст. 2 ГК РФ одним из двух основных качеств (элементов) корпоративных отношений как относительно обособленной части предмета гражданско-правового регулирования. Если категория «участие» означает особую связанность участника и корпорации как организационную основу всех иных отношений между 22 23 24 25 ними, то и «управление» корпорацией ее участником означает особое взаимодействие указанных субъектов, направленное на реализацию целей объединения участников в корпорацию и достижения ими желательного результата. Особый характер такого управления заключается в том, что оно основано не на власти и подчинении субъектов корпоративных отношений, а на координации их деятельности, вытекающей из равенства, автономии воли и имущественной самостоятельности ее участников. Более того, нам представляется, что особенностью управления в корпоративных отношениях будет еще одно начало, формирующее взаимоотношение корпорации и ее участников. Речь идет о характеристике такого управления, как самоуправление. На этот момент в литературе иногда обращается внимание, нам же он представляется узловым. Так, Ю. С. Харитонова указывает, что управление пронизывает все частное право. Оно является функцией, которая сопутствует частной инициативе, частной собственности, творчеству и предпринимательству. Управление в частном праве отличается от управления в публичном праве в соответствии с критериями проведения границ между частным и публичным правом. Управление в гражданском праве осуществляется в рамках принципа юридического равенства и автономии воли сторон. Более того, присущая и частному праву иерархия обладает особыми свойствами: она основана на началах координации и самоуправления25. В этом смысле данные отношения близки отношениям управления, базирующимся на экономических отношениях, — отношениям, возникающим при разделении и кооперации труда в процессе совместной деятельности ассоциированных собственников. Они базируются на заинтересованности их участников в полу- См.: Андреев В. К., Лаптев В. А. Корпоративное право современной России : монография. М. : Проспект, 2021. С. 120. Ломакин Д. В. Коммерческие корпорации как субъекты корпоративных правоотношений : учебное пособие. М. : Статут, 2020. С. 14. См.: Яковлев В. Ф. Модернизация Гражданского кодекса Российской Федерации // Кодификация российского частного права 2019 / под ред. Д. А. Медведева. М. : Статут, 2019. Харитонова Ю. С. Управление в гражданском праве: проблемы теории и практики. М. : Норма, Инфра‑М, 2011. Актуальные проблемы российского права. 2022. Т. 17. № 9 (142) сентябрь 131 Предпринимательское и корпоративное право чении оптимального конечного результата совместной деятельности. Эта заинтересованность и является основой возможности управления26. Продолжению этой мысли применительно к корпоративному управлению служат следующие высказывания В. К. Андреева. Опираясь на закрепленные в ГК положения, он отметил, что корпоративное управление определяется функциями и компетенцией органов корпорации, а также правами и обязанностями самих участников корпорации в управлении делами. Ее особенностью выступает тесное переплетение задач и целей как корпорации, так и ее участников. Это проявляется в том, что участники корпорации формируют, точнее — являются высшим ее органом, который устанавливает параметры предпринимательской или иной деятельности и обеспечивает их выполнение. Модернизация гл. 4 ГК РФ закрепляет вышеупомянутую отечественную модель корпоративного управления, при которой структура и компетенция органов конкретного общества самостоятельно устанавливаются по единогласному решению ее участников в рамках п. 3 ст. 66.3 ГК и отражаются в его уставе. Важной чертой корпоративного управления является организационное единство полномочий высшего и иных органов корпорации и прав и обязанностей ее участников (членов)27. Указанные положения позволяют сделать вывод о том, что управление само по себе не является эксклюзивным признаком корпорации28. Но последней присуще такое свойство, как самоуправление данного социального образования29. Именно оно будет отличать управление корпоративной организацией от управления унитарной организацией. Наличие отношений участия в корпоративной организации предопределяет необходимость согласования 26 27 28 29 132 воли участников корпорации на управление ею, в силу чего эти отношения требуют специального правового регулирования. Общие свойства, присущие корпоративному управлению, позволили законодателю закрепить в ст. 65.3 ГК РФ единые правила управления корпоративными организациями. Правоотношения управления, наряду с правоотношениями участия, носят общерегулятивный характер, определяя возможность лица быть субъектом корпоративных отношений. Например, избрание лица членом совета директоров является основанием возникновения правоотношений управления, содержанием которых будет специальная правосубъектность лица как члена совета директоров. Указанная правосубъектность, а также права и обязанности действовать от имени юридического лица и в его интересах, с учетом принципов добросовестности и разумности, характеризуют статус лица — члена совета директоров как участника корпоративных отношений. Таким образом, общерегулятивные отношения участия и управления будут определять содержание правового статуса лица как участника корпоративных отношений и являться предпосылкой возникновения конкретных корпоративных отношений, определять их содержание и динамику развития. Это конкретные корпоративные правоотношения в силу зависимости от основных корпоративных начал — участия и управления — выступают как особая форма гражданско-правовых отношений и могут быть, в зависимости от своего объекта, подразделены на имущественные и неимущественные. Потребность в особом регулировании имущественных отношений, связанных с участием и управлением, приводит к образованию таких См. подробнее: Виханский О. С., Наумов А. И. Менеджмент : учебник. М. : Магистр, Инфра‑М, 2012. С. 36–37. Андреев В. К., Лаптев В. А. Указ. соч. С. 116–120. Беляева О. А. Управление юридическими лицами корпоративного типа // Современное корпоративное право: актуальные проблемы теории и практики : монография. М. : Статут, 2021. С. 147–160 ; Оболонкова Е. В. Управление в унитарных организациях // Там же. С. 161–185. См. также: Терновая О. А. Понятие корпоративного управления: теоретические и практические аспекты // Современное корпоративное право: актуальные проблемы теории и практики. С. 92–114. Актуальные проблемы российского права. 2022. Т. 17. № 9 (142) сентябрь Слесарев В. Л. Отношения участия и управления в системе корпоративных отношений корпоративных прав участников, как, например, права на распределение и получение прибыли; права на ликвидационную квоту; иные имущественные права, опосредующие выход из корпорации; на приобретение акционерным обществом собственных размещенных акций; право участников ООО требовать от общества приобретения принадлежащих им долей; преимущественные права приобретения долей (паев, акций), отчуждаемых участниками корпорации, и др.30 Весьма значительными в составе корпоративных правоотношений являются такие неимущественные права участников корпорации, как право на участие в управлении делами корпорации, право контроля за ее финансово-хозяйственной деятельностью, информационные и другие права31. Среди обязанностей участников корпорации выделяются, например, такие: участвовать в образовании имущества корпорации; не разглашать конфиденциальную информацию о ее деятельности; участвовать в принятии корпоративных решений; информировать корпорацию об изменении своих данных; не совершать действий, заведомо направленных на причи- нение вреда корпорации, действий, которые существенно затрудняют или делают невозможным достижение целей, ради которых создана корпорация, и др.32 В литературе выделяются целые пласты отношений (существенные корпоративные действия), регулирование которых носит комплексный характер (с элементами публичного и частного порядка, имущественного и организационного свойства), например: реорганизация и ликвидация корпорации; приобретение 30 и более процентов акций публичного общества; совершение корпорацией экстраординарных сделок; увеличение и уменьшение уставного капитала; листинг (делистинг) и др.33 Задачами исследований в данной области являются установление адекватности правового регулирования указанных корпоративных отношений их основным началам — участию и управлению, анализ обоснованности разнородного регулирования типичных корпоративных связей при различных организационно-правовых формах корпораций, превращение корпоративного права в эффективный гражданско-правовой механизм регулирования корпоративных отношений. БИБЛИОГРАФИЯ 1. Алексеев С. С. Проблемы теории права : курс лекций // Собрание сочинений : в 10 т. — Т. 3. — М. : Статут, 2010. — 787 с. 2. Андреев В. К., Лаптев В. А. Корпоративное право современной России : монография. — М. : Проспект, 2021. — 239 с. 3. Беляева О. А. Управление юридическими лицами корпоративного типа // Современное корпоративное право: актуальные проблемы теории и практики : монография. — М., 2021. — С. 147–160. 4. Виханский О. С., Наумов А. И. Менеджмент : учебник. — М. : Магистр, Инфра‑М, 2012. — 656 с. 30 31 32 33 Корпоративное право : учебник / отв. ред. И. С. Шиткина. С. 374–397. Особое место в системе корпоративных прав занимают т.н. преимущественные права. Например, для участников ООО при переходе доли в уставном капитале общества к третьему лицу предусмотрено преимущественное право. На первый взгляд, природа этого права зависима от того, которое реализуется в преимущественном порядке. 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https://openalex.org/W3005956088	https://europepmc.org/articles/pmc7072481?pdf=render	English	null	Donor Heme Oxygenase-1 Promoter Gene Polymorphism Predicts Survival after Unrelated Bone Marrow Transplantation for High-Risk Patients	Cancers	2,020	cc-by	8,966	Donor Heme Oxygenase-1 Promoter Gene Polymorphism Predicts Survival after Unrelated Bone Marrow Transplantation for High-Risk Patients Luis Espinoza 3 , Yasuo Morishima 4,5,6, Yoshihisa Kodera 2,4, Makoto Onizuka 7 , Koichi Kashiwase 8, Takahiro Fukuda 9, Noriko Doki 10, Koichi Miyamura 11, Takehiko Mori 12, Shinji Nakao 13 and Akiyoshi Takami 1,2,* 1 Division of Hematology, Department of Internal Medicine, Aichi Medical University School of Medicine, Nagakute 480-1195, Japan; kuroro@aichi-med-u.ac.jp (T.H.); shohei@aichi-med-u.ac.jp (S.M.); ksakai@aichi-med-u.ac.jp (K.U.); hanamura@aichi-med-u.ac.jp (I.H.) jp jp 2 Hematopoietic Cell Transplantation Center, Aichi Medical University Hospital, Nagakute 480-1195, Japan ykodera@river.ocn.ne.jp 3 Department of Clinical Laboratory Science, Kanazawa University School of Medical Sciences, Kanazawa 920-0942, Japan; eriko86@staﬀ.kanazawa-u.ac.jp (E.M.); luis1.esp2.cd@outlook.com (J.L.E.) p jp p 4 Department of Promotion for Blood and Marrow Transplantation, Aichi Medical University School of Medicine, Nagakute 480-1195, Japan; ymorisim@aichi-cc.jp 4 Department of Promotion for Blood and Marrow Transplantation, Aichi Medical University School of Medicine, Nagakute 480-1195, Japan; ymorisim@aichi-cc.jp 5 Central Japan Cord Blood Bank, Seto 489-8555, Japan 6 Department of Hematology and Oncology, Nakagami Hospital, Okinawa 904-2195, Japan 7 7 Department of Hematology and Oncology, Tokai University School of Medicine, Isehara 259-1193, Japa moni5@mac.com 8 Japanese Red Cross Kanto-Koshinetsu Block Blood Center, Tokyo 135-8521, Japan; k k h k k bb 8 Japanese Red Cross Kanto-Koshinetsu Block Blood Center, Tokyo 135-8521, Japan; k-kashiwase@ktks.bbc.jrc.or.jp k-kashiwase@ktks.bbc.jrc.or.jp 9 j jp 9 Hematopoietic Stem Cell Transplantation Unit, National Cancer Center Hospital, Tokyo 104-0045, Japan; tafukuda@ncc.go.jp 9 Hematopoietic Stem Cell Transplantation Unit, National Cancer Center Hospital, Tokyo 104-0045, Japan; tafukuda@ncc.go.jp g j 10 Hematology Division, Tokyo Metropolitan Cancer and Infectious Diseases Center Komagome Hospital, Tokyo 113-8677, Japan; n-doki@cick.jp 11 Department of Hematology, Japanese Red Cross Nagoya First Hospital, Nagoya 453-8511, Japan; k-miyamura@nagoya-1st.jrc.or.jp 12 Division of Hematology, Department of Medicine, Keio University School of Medicine, Tokyo 160-8582, Japan; tmori@a3.keio.jp p jp Department of Hematology, Faculty of Medicine, Institute of Medical, Pharmaceutical and Health Sciences k ﬀk 13 Department of Hematology, Faculty of Medicine, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa 920-8641, Japan; snakao8205@staﬀ.kanazawa-u.ac.jp 13 Department of Hematology, Faculty of Medicine, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa 920-8641, Japan; snakao8205@staﬀ.kanazawa-u.ac.jp * Correspondence: takami-knz@umin.ac.jp; Tel.: +81-561-62-3311; Fax: +81-561-63-3401 * Correspondence: takami-knz@umin.ac.jp; Tel.: +81-561-62-3311; Fax: +81-561-63-3401 ceived: 10 January 2020; Accepted: 10 February 2020; Published: 12 February 2020 Received: 10 January 2020; Accepted: 10 February 2020; Published: 12 February 2020 Abstract: Heme oxygenase-1 (HO-1), an intracellular enzyme that catalyzes the degradation of heme into biliverdin, free iron, and carbon monoxide, exerts anti-inﬂammatory and cytoprotective eﬀects against endothelial cell injury. cancers cancers cancers cancers Donor Heme Oxygenase-1 Promoter Gene Polymorphism Predicts Survival after Unrelated Bone Marrow Transplantation for High-Risk Patients Tomohiro Horio 1,2 , Eriko Morishita 3 , Shohei Mizuno 1,2 , Kaori Uchino 1,2, Ichiro Hanamura 1,2, J. Luis Espinoza 3 , Yasuo Morishima 4,5,6, Yoshihisa Kodera 2,4, Makoto Onizuka 7 , Koichi Kashiwase 8, Takahiro Fukuda 9, Noriko Doki 10, Koichi Miyamura 11, Takehiko Mori 12, Shinji Nakao 13 and Akiyoshi Takami 1,2,* Tomohiro Horio 1,2 , Eriko Morishita 3 , Shohei Mizuno 1,2 , Kaori Uchino 1,2, Ichiro Hanamura 1,2, J. Luis Espinoza 3 , Yasuo Morishima 4,5,6, Yoshihisa Kodera 2,4, Makoto Onizuka 7 , Koichi Kashiwase 8, Takahiro Fukuda 9, Noriko Doki 10, Koichi Miyamura 11, Takehiko Mori 12, Shinji Nakao 13 and Akiyoshi Takami 1,2,* , , , , Ichiro Hanamura 1,2, J. Keywords: HO-1; unrelated donor; bone marrow transplantation; single nucleotide polymorphism Keywords: HO-1; unrelated donor; bone marrow transplantation; single nucleotide polymorphism Keywords: HO-1; unrelated donor; bone marrow transplantation; single nucleotide polymorphism 1. Introduction Allogeneic stem cell transplantation (allo-HSCT) is an important therapeutic option to cure advanced non-malignant and malignant hematologic diseases. However, its eﬀectiveness has been limited by mortality and morbidity, due to transplant-related complications, such as conditioning regimen-related organ and tissue damage, severe infection, and graft-versus-host disease (GVHD). Several clinical and experimental studies [1–6] have shown that these complications, along with calcineurin inhibitors, activate and damage endothelial cells, potentially leading to organ dysfunction and subsequently multiorgan dysfunction syndrome. Thus, the modulation of endothelial cell injury could help to prevent or treat serious organ dysfunction after allo-HSCT. Heme oxygenase-1 (HO-1), also known as a 32 kDa heat-shock protein, is an intracellular enzyme that catalyzes the degradation of heme into biliverdin, ferrous iron, and carbon monoxide (CO), with biliverdin being subsequently catabolized into bilirubin [7–11]. HO-1 is highly induced in response to various stress signals, such as free heme and hemoglobin, inﬂammatory cytokines, ischemia, endotoxins, irradiation, and mucosal damage [9–16]. HO-1 thus exerts cytoprotective eﬀects on endothelial cells through ant-oxidative, anti-inﬂammatory, anti-apoptotic, and anti-thrombotic eﬀects, which are coordinated with heme metabolites of CO and bilirubin. The HO-1 gene, also called the HMOX1 gene, is mainly expressed in monocytes/macrophages, natural killer cells, endothelial cells, and the heart [17]. One important single nucleotide polymorphism (SNP) in the promoter region of the HO-1 gene, rs2071746 (-413A>T), is functional, and the major A allele is reported to be associated with higher expression of HO-1 than the minor T allele [18,19]. There is growing evidence to support that non-human leukocyte antigen (HLA) genetic polymorphism represents a signiﬁcant determinant of outcomes after allo-HSCT [20–26]. These ﬁndings prompted us to investigate the impact of the rs2071746 SNP in the HO-1 gene on the clinical outcomes of patients undergoing allogeneic bone marrow transplantation (BMT), using an HLA allele-matched, unrelated donor through the Japan Marrow Donor Program (JMDP). Donor Heme Oxygenase-1 Promoter Gene Polymorphism Predicts Survival after Unrelated Bone Marrow Transplantation for High-Risk Patients The HO-1 promoter gene has one important single-nucleotide polymorphism (SNP) rs2071746 (-413A>T) that is functional, and the A allele has been reported to be associated with higher HO-1 expression levels than the T allele. We investigated the inﬂuence of the HO-1 rs2071746 SNP on the transplant outcomes in 593 patients with hematological malignancies undergoing unrelated, human leukocyte antigen (HLA)-matched, T-cell-replete bone marrow transplantation (BMT) through the Japan Donor Marrow Program. In patients with high-risk diseases, the donor A/A or A/T genotype was associated with better 5 year overall survival (35% vs. 25%; p = 0.03) and 5 year disease-free survival (35% vs. 22%; p = 0.0072), compared to the donor T/T genotype. These eﬀects were not observed in patients with low-risk diseases. The current ﬁndings therefore indicate that HO-1 rs2071746 genotyping could be useful for selecting donors and tailoring transplant strategies for patients with high-risk hematologic malignancies. www.mdpi.com/journal/cancers www.mdpi.com/journal/cancers Cancers 2020, 12, 424; doi:10.3390/cancers12020424 2 of 13 Cancers 2020, 12, 424 2.1. The Frequencies of HO-1 Genotypes The HO-1 rs2071746 SNP was analyzed in 593 HLA-A, -B, -C, -DRB1, -DQB1, and -DPB1 allele-matched, unrelated BMT donor-transplant recipient pairs (Table 1). Based on the disease status and other risk factors that inﬂuence post-transplant outcomes, as previously reported [22,27–29], standard-risk disease was deﬁned as acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), or multiple myeloma (MM) in the ﬁrst complete remission; malignant lymphoma (ML) in any complete remission; or myelodysplastic syndrome (MDS) or chronic myeloid leukemia (CML) in the chronic phase. Both ALL and AML, which were in the second complete remission, were included in the high-risk group, according to recent reports [27,29] indicating that patients with AML and ALL in the second complete remission have worse post-transplant outcomes than those in the ﬁrst complete remission. All other conditions were classiﬁed as high-risk disease. The genotype frequencies of A/A, A/T, and T/T were 21%, 52%, and 27%, respectively, in recipients, and 23%, 52%, and 25% in donors. These results were in accordance with the Hardy-Weinberg equilibrium (p = 0.49), and were similar to the HapMap data in the Japanese population (27%, 50%, and 23%, respectively) [30]. 3 of 13 Cancers 2020, 12, 424 Table 1. Characteristics and heme oxygenase-1 (HO-1) genotypes of recipients and donors, according to the disease risk. to the disease risk. 2.1. The Frequencies of HO-1 Genotypes Variable All High-Risk Standard-Risk Value Value Value p Number of cases 593 232 361 Recipient age, years, median (range) 33 (1–67) 34 (1–67) 34 (1–65) Donor age, years, median (range) 34 (20–57) 34 (21–50) 34 (20–57) Year of HSCT, median (range) 2000 (1993–2007) 2000 (1993–2007) 2000 (1993–2007) Recipient HO-1 genotype, n (%) 0.03 A/A 125 (21) 40 (17) 85 (24) A/T 306 (52) 135 (58) 171 (47) T/T 162 (27) 57 (25) 105 (29) Donor HO-1 genotype, n (%) 0.96 A/A 134 (23) 51 (22) 83 (23) A/T 308 (52) 122 (53) 186 (52) T/T 151 (25) 59 (25) 92 (25) Recipient sex, n (%) 0.55 Male 352 (59) 134 (58) 218 (60) Female 241 (41) 98 (42) 143 (40) Donor sex, n (%) 0.43 Male 374 (63) 142 (61) 232 (64) Female 218 (37) 90 (39) 128 (36) Recipient/Donor sex match, n (%) 0.54 Sex-matched 387 (65) 158 (68) 229 (64) Female/Male 114 (19) 41 (18) 73 (20) Male/Female 92 (16) 33 (14) 59 (16) Disease, n (%) 0.01 AML 197 (33) 112 (48) 85 (24) ALL 145 (24) 72 (31) 73 (20) MDS 82 (14) 0 (0) 82 (23) ML 64 (11) 29 (13) 35 (9) CML 101 (17) 16 (7) 85 (24) MM 4 (1) 3 (1) 1 (0) ABO matching, n (%) 0.97 ABO-matched 359 (61) 137 (59) 222 (62) Major mismatch 115 (19) 46 (20) 69 (19) Minor mismatch 95 (16) 40 (17) 55 (15) Bidirectional 16 (3) 6 (3) 10 (3) Missing 8 (1) 3 (1) 5 (1) Conditioning regimen, n (%) 0.86 Myeloablative 517 (87) 204 (88) 313 (87) Reduced intensity 69 (12) 26 (11) 43 (12) Missing 7 (1) 2 (1) 5 (1) Pretransplantation CMV serostatus, n (%) 0.23 CMV-positive recipient 373 (63) 142 (61) 231 (64) CMV-negative recipient 102 (17) 36 (16) 66 (18) Missing 118 (20) 54 (23) 64 (18) TNC, ×108/kg, median (range) 7.7 (0.1–259) 7.7 (0.1–79.1) 7.7 (0.6–259) Abbreviations: AML, acute myeloid leukemia; ALL, acute lymphoblastic leukemia; MDS, myelodysplastic syndrome; ML, malignant lymphoma; CML, chronic myeloid leukemia; MM, multiple myeloma; HSCT, hematopoietic stem cell transplant; ABO, the “ABO” blood system; CMV, cytomegalovirus; TNC, total number of nucleated cells harvested. 2.1. The Frequencies of HO-1 Genotypes Abbreviations: AML, acute myeloid leukemia; ALL, acute lymphoblastic leukemia; MDS, myelodysplastic syndrome; ML, malignant lymphoma; CML, chronic myeloid leukemia; MM, multiple myeloma; HSCT, hematopoietic stem cell transplant; ABO, the “ABO” blood system; CMV, cytomegalovirus; TNC, total number of nucleated cells harvested. 2.2. Transplant Outcomes According to the HO-1 Genotype 2.2. Transplant Outcomes According to the HO-1 Genotype The analysis on the inﬂuence of the HO-1 on clinical outcomes after transplantation was stratiﬁed into standard-risk and high-risk disease groups to account for its prognostic signiﬁcance. In the high-risk disease group (n = 232), the donor A/A or A/T genotype was associated with signiﬁcantly better 5 year overall survival (OS; 35% vs. 25%, p = 0.033; Figure 1A) and signiﬁcantly better 5 year disease-free survival (DFS; 35% vs. 22%, p = 0.0072; Figure 1B), compared to the donor T/T genotype. There were no signiﬁcant diﬀerences between the donor A/A or A/T genotype and the donor T/T genotype with regard to the rates of relapse, transplant-related mortality (TRM), and graft-versus-host 4 of 13 ntly nor h Cancers 2020, 12, 424 signific better 5 disease (GVHD; see Tables S1 and S2). In the standard-risk disease group (n = 361), the donor genotype had no signiﬁcant eﬀect on the transplant outcomes (see Tables S1 and S2, Figure S1). The recipient genotype had no signiﬁcant eﬀects on the transplant outcomes regardless of the disease risk (see Tables S1 and S2). donor T/T genotype with regard to the rates of relapse, transplant-related mortality (TRM), and graft- versus-host disease (GVHD; see Tables S1 and S2). In the standard-risk disease group (n = 361), the donor genotype had no significant effect on the transplant outcomes (see Tables S1 and S2, Figure S1). The recipient genotype had no significant effects on the transplant outcomes regardless of the disease risk (see Tables S1 and S2). Figure 1. The Kaplan–Meier analysis of the overall survival (OS) rates (A) and the disease-free survival (DFS) rates (B) after transplantation, according to the donor HO-1 genotype in patients with high-risk disease. Solid lines represent the donor A/A or A/T genotype. Dashed lines represent the donor T/T genotype. An asterisk () denotes statistical significance (p < 0.05). Figure 1. The Kaplan–Meier analysis of the overall survival (OS) rates (A) and the disease-free survival (DFS) rates (B) after transplantation, according to the donor HO-1 genotype in patients with high-risk disease. 2.1. The Frequencies of HO-1 Genotypes Solid lines represent the donor A/A or A/T genotype. Dashed lines represent the donor T/T genotype. An asterisk () denotes statistical signiﬁcance (p < 0.05). 3 M lti i t A l i Figure 1. The Kaplan–Meier analysis of the overall survival (OS) rates (A) and the disease-free survival (DFS) rates (B) after transplantation, according to the donor HO-1 genotype in patients with high-risk disease. Solid lines represent the donor A/A or A/T genotype. Dashed lines represent the donor T/T genotype. An asterisk () denotes statistical significance (p < 0.05). Figure 1. The Kaplan–Meier analysis of the overall survival (OS) rates (A) and the disease-free survival (DFS) rates (B) after transplantation, according to the donor HO-1 genotype in patients with high-risk disease. Solid lines represent the donor A/A or A/T genotype. Dashed lines represent the donor T/T genotype. An asterisk () denotes statistical signiﬁcance (p < 0.05). Figure 1. The Kaplan–Meier analysis of the overall survival (OS) rates (A) and the disease-free survival (DFS) rates (B) after transplantation, according to the donor HO-1 genotype in patients with high-risk disease. Solid lines represent the donor A/A or A/T genotype. Dashed lines represent the donor T/T genotype An asterisk () denotes statistical significance (p < 0 05) e 1. The Kaplan–Meier analysis of the overall survival (OS) rates (A) and the disease-free survival rates (B) after transplantation, according to the donor HO-1 genotype in patients with high-risk se. Solid lines represent the donor A/A or A/T genotype. Dashed lines represent the donor T/T Figure 1. The Kaplan–Meier analysis of the overall survival (OS) rates (A) and the disease-free survival (DFS) rates (B) after transplantation, according to the donor HO-1 genotype in patients with high-risk disease. Solid lines represent the donor A/A or A/T genotype. Dashed lines represent the donor T/T genotype. An asterisk () denotes statistical significance (p < 0.05). Figure 1. The Kaplan–Meier analysis of the overall survival (OS) rates (A) and the disease-free survival (DFS) rates (B) after transplantation, according to the donor HO-1 genotype in patients with high-risk disease. Solid lines represent the donor A/A or A/T genotype. Dashed lines represent the donor T/T genotype. An asterisk (*) denotes statistical signiﬁcance (p < 0.05). 2.3. Multivariate Analysis Multivariate analyses were performed by adjusting for the cofounding variables, such as recipient, donor, and transplant characteristics, the details of which are described in Section 4.3. In the high-risk disease group, the donor A/A or A/T genotype showed signiﬁcantly better 5 year DFS (hazard ratio (HR) = 0.64; 95% conﬁdence interval (CI) = 0.44–0.93; p = 0.019; Table 2) and tended to show better 5 year OS (HR = 0.72; 95% CI = 0.50–1.04; p = 0.082) compared to the donor T/T genotype. In the standard-risk disease group, the donor genotype had no signiﬁcant eﬀect on the DFS or OS. The donor HO-1 genotype showed no signiﬁcant eﬀects on the TRM or GVHD rates in the multivariate analysis (Tables 2 and 3). The recipient HO-1 genotype did not signiﬁcantly inﬂuence the transplant outcomes. 5 of 13 Cancers 2020, 12, 424 Table 2. The results of the multivariate analysis of the association between the HO-1 genotype and the clinical outcomes after transplantation (ﬁrst part). Variable OS DFS TRM Relapse HR 95% CI p HR 95% CI p HR 95% CI p HR 95% CI p High-risk disease Donor HO-1 genotype, A/T or A/A vs. T/T 0.72 0.50–1.04 0.08 0.64 0.44–0.93 0.02 0.66 0.36–1.19 0.16 0.83 0.51–1.35 0.44 Recipient HO-1 genotype, A/T or A/A vs. T/T 0.91 0.62–1.32 0.61 0.99 0.68–1.45 0.98 1.03 0.57–1.85 0.92 0.99 0.60–1.64 0.98 Recipient age 0.99 0.97-0.99 0.004 0.99 0.98–1.00 0.02 0.98 0.96–0.99 0.006 Standard-risk disease Donor HO-1 genotype, A/T or A/A vs. T/T 1.05 0.71–1.54 0.80 0.91 0.56–1.47 0.70 1.04 0.59–1.82 0.89 0.71 0.40–1.22 0.20 Recipient HO-1 genotype, A/T or A/A vs. T/T 1.39 0.93–2.08 0.10 1.30 0.79–2.17 0.28 1.15 0.66–2.00 0.62 1.41 0.76–2.63 0.28 Recipient age 0.97 0.96–0.98 <0.001 1.00 0.97–1.01 0.24 0.96 0.94–0.98 0.11 0.91 0.56–1.47 0.70 Donor age 0.97 0.94–1.01 0.11 1.33 0.80–2.22 0.27 CMV-positive recipient 1.22 0.77–1.92 0.39 0.58 0.36–0.94 0.03 0.47 0.26–0.82 0.008 ABO major mismatch 1.04 0.54–200 0.90 ABO minor mismatch 0.59 0.39–0.88 0.01 0.50 0.28–0.89 0.02 ABO bidirectional 13513.5 5555.6–33333.3<0.001 Male donor/female recipient 1.00 0.58–1.75 0.99 Female donor/male recipient 2.27 1.05–5.00 0.04 2.22 1.03–4.76 0.04 Abbreviations: TRM, transplant-related mortality; HR, hazard ratio; CI, conﬁdence interval; HSCT, hematopoietic stem cell transplant. Table 2. The results of the multivariate analysis of the association between the HO-1 genotype and the clinical outcomes after transplantation (ﬁrst part). f the association between the HO-1 genotype and the clinical outcomes after transplantation (ﬁrst part). 3. Discussio 3. Discussion The current study showed that the donor A/A or A/T genotype of the HO-1 rs2071746 promoter SNP was associated with better DFS than the donor T/T genotype in patients with high-risk hematologic malignancies who received unrelated, HLA-matched BMT through the JMDP. Of note, individuals with the HO-1 rs2071746 A allele have been suggested to have higher promoter activity, which induces higher expression levels of HO-1 compared to those without the A allele [18,19], thus suggesting that donor-derived HO-1 may have beneficial effects on survival after allo-BMT. This is the first report to demonstrate that the HO-1 SNP may be involved in the survival outcomes after allo HSCT The current study showed that the donor A/A or A/T genotype of the HO-1 rs2071746 promoter SNP was associated with better DFS than the donor T/T genotype in patients with high-risk hematologic malignancies who received unrelated, HLA-matched BMT through the JMDP. Of note, individuals with the HO-1 rs2071746 A allele have been suggested to have higher promoter activity, which induces higher expression levels of HO-1 compared to those without the A allele [18,19], thus suggesting that donor-derived HO-1 may have beneﬁcial eﬀects on survival after allo-BMT. This is the ﬁrst report to demonstrate that the HO-1 SNP may be involved in the survival outcomes after allo-HSCT. allo-HSCT. The mechanisms through which the donor HO-1 A/A or A/T genotype (plausibly associated with the higher inducibility of HO-1) exerts its beneficial effects remains to be determined. HO-1 is only marginally produced in the resting state, but is rapidly and highly induced in response to various oxidative stresses (e.g., organ and tissue damage and infection), and acts as an anti-inflammatory and cytoprotective protein [7,9–11,15,31,32]. Evidence of HO-1 induction improving the survival outcomes after allo-HSCT has been demonstrated in previous studies using mouse models [31,33]. It was shown that the induction of HO-1 reduced serum levels of proinflammatory cytokines, prevented damage to the intestinal mucosa and subsequent bacterial invasion, and improved overall survival after allo-HSCT [33]. Another study [31], in which transplantation was performed from HO- 1 wild-type mice to HO-1 knockout mice, revealed that donor macrophages play pivotal roles in the recovery of the HO-1 function after allo-HSCT. In humans, HO-1 deficiency is associated with a distinctive feature: oxidative stress induces severe endothelial cell damage [10]. 2.3. Multivariate Analysis Table 3. The results of the multivariate analysis of the association between the HO-1 genotype and the clinical outcomes after transplantation (second part). The results of the multivariate analysis of the association between the HO-1 genotype and the clinical outco the association between the HO-1 genotype and the clinical outcomes after transplantation (second part). ltivariate analysis of the association between the HO-1 genotype and the clinical outcomes after transplanta Table 3. The results of the multivariate analysis of the association between the HO-1 genotype and the clinical outcomes after transplantation (second part). Variable Grades 2–4 acute GVHD Grades 3–4 acute GVHD Chronic GVHD HR 95% CI p HR 95% CI p HR 95% CI p High-risk disease Donor HO-1 genotype, A/T or A/A vs. T/T 1.10 0.61–1.96 0.76 1.03 0.41–2.78 0.88 0.91 0.51–1.59 0.72 Recipient HO-1 genotype, A/T or A/A vs. T/T 1.09 0.62–1.92 0.76 1.00 0.40–2.50 1.00 0.63 0.37–1.08 0.09 Recipient age Donor age Conditioning regimen, MAC vs. RIC 3.03 0.94–10.0 0.06 TNC 0.99 0.97–1.01 0.23 Year of HSCT 0.59 0.35–0.98 0.04 0.97 0.94–1.00 0.10 Standard-risk disease Donor HO-1 genotype, A/T or A/A vs. T/T 0.79 0.61–1.02 0.07 1.19 0.56–2.50 0.66 0.98 0.68–1.41 0.91 Recipient HO-1 genotype, A/T or A/A vs. T/T 1.09 0.80–1.47 0.60 1.96 0.85–4.35 0.11 1.00 0.70–1.41 1.00 Recipient age 0.99 0.97–1.00 0.02 CMV-positive recipient 1.49 1.16–1.92 0.002 Year of HSCT 1.61 1.14-2.27 0.008 Abbreviations: GVHD, graft-versus-host disease; MAC, myeloablative conditioning; RIC, reduced-intensity conditioning; TNC, total nucleated cell count harvested. 6 of 13 Cancers 2020, 12, 424 2.4. Main Causes of Death 2.4. Main Causes of Death 2.4. Main Causes of Death 2.4. Main Causes of Death When the main causes of death were analyzed according to the HO-1 genotype, although the donor A/A or A/T genotype showed relatively low cumulative incidence of fatal infection in the high-risk disease group (12% vs. 19%, p = 0.19; Figure 2), there were no statistically signiﬁcant diﬀerences. When the main causes of death were analyzed according to the HO-1 genotype, although the donor A/A or A/T genotype showed relatively low cumulative incidence of fatal infection in the high- risk disease group (12% vs. 19%, p = 0.19; Figure 2), there were no statistically significant differences. Figure 2. Main causes of death after transplantation, according to the donor HO-1 genotype in patients with high-risk disease. ARDS: acute respiratory distress syndrome; IP: interstitial pneumonia; disease prog: disease progression. Figure 2. Main causes of death after transplantation, according to the donor HO-1 genotype in patients with high-risk disease. ARDS: acute respiratory distress syndrome; IP: interstitial pneumonia; disease prog: disease progression. Figure 2. Main causes of death after transplantation, according to the donor HO-1 genotype in patients with high-risk disease. ARDS: acute respiratory distress syndrome; IP: interstitial pneumonia; disease prog: disease progression. Figure 2. Main causes of death after transplantation, according to the donor HO-1 genotype in patients with high-risk disease. ARDS: acute respiratory distress syndrome; IP: interstitial pneumonia; disease prog: disease progression. 3. Discussio 3. Discussion Vascular endothelial cell injury after allo-HSCT can potentially cause thrombotic microangiopathy, sinusoidal obstruction syndrome, capillary leak syndrome, and acute lung injury, leading to end-organ dysfunction [1–6]. These findings suggest that while organ and tissue damage from the conditioning regimen, infections, GVHD, and calcineurin inhibitors cause oxidative stress, which damages endothelial cells, the same can activate donor macrophages to produce HO-1, protecting against endothelial cell injury y The mechanisms through which the donor HO-1 A/A or A/T genotype (plausibly associated with the higher inducibility of HO-1) exerts its beneﬁcial eﬀects remains to be determined. HO-1 is only marginally produced in the resting state, but is rapidly and highly induced in response to various oxidative stresses (e.g., organ and tissue damage and infection), and acts as an anti-inﬂammatory and cytoprotective protein [7,9–11,15,31,32]. Evidence of HO-1 induction improving the survival outcomes after allo-HSCT has been demonstrated in previous studies using mouse models [31,33]. It was shown that the induction of HO-1 reduced serum levels of proinﬂammatory cytokines, prevented damage to the intestinal mucosa and subsequent bacterial invasion, and improved overall survival after allo-HSCT [33]. Another study [31], in which transplantation was performed from HO-1 wild-type mice to HO-1 knockout mice, revealed that donor macrophages play pivotal roles in the recovery of the HO-1 function after allo-HSCT. In humans, HO-1 deﬁciency is associated with a distinctive feature: oxidative stress induces severe endothelial cell damage [10]. Vascular endothelial cell injury after allo-HSCT can potentially cause thrombotic microangiopathy, sinusoidal obstruction syndrome, capillary leak syndrome, and acute lung injury, leading to end-organ dysfunction [1–6]. These ﬁndings suggest that while organ and tissue damage from the conditioning regimen, infections, GVHD, and calcineurin inhibitors cause oxidative stress, which damages endothelial cells, the same can activate donor macrophages to produce HO-1, protecting against endothelial cell injury and subsequently preventing organ dysfunction. BMT from donors with the HO-1 A/A or A/T genotype may be Cancers 2020, 12, 424 7 of 13 associated with improved survival outcomes in recipients by enhancing the beneﬁcial eﬀects of HO-1. However, this hypothesis is highly speculative, because the current study could not provide information on the inducibility of HO-1 or the degree of organ and endothelial cell damage, according to the HO-1 genotype. 3. Discussio 3. Discussion Further evidence that the HO-1 rs2071746 A/A or A/T genotype has the advantage of maintaining the organ function by protecting endothelial cells against oxidative stress may be seen in previous reports [18,19], which have shown that the HO-1 rs2071746 A/A genotype was associated with a reduced incidence of ischemic heart disease compared to the A/T or T/T genotypes in the Asian adult population, and that the donor HO-1 rs2071746 A/A or A/T genotype was associated with better graft survival and a better liver function after liver transplantation compared to the donor T/T genotype in the European adult population. On the other hand, one report noted that the HO-1 rs2071746 A/A and A/T genotypes were associated with an increased incidence of acute kidney injury in the American infant population [34]. A plausible reason for this discrepancy may be the negative eﬀect of hyperbilirubinemia, which possibly results from the higher inducibility of the HO-1 genotype (A/A or A/T) in neonates. Further studies using additional cohorts are needed to better understand this discrepancy. Recent reports [35–37] have suggested that the overexpression of HO-1 may promote the progression and relapse of various blood cancers, especially CML, as well as carcinogenesis, progression, and resistance to therapy in solid cancers. Unlike these observations, in the present study, the donor HO-1 A/A or A/T genotype (plausibly associated with the higher inducibility of HO-1) was associated with better survival outcomes without increasing the risk of disease relapse after allo-HSCT in patients with high-risk patients. However, allo-HSCT can exert a robust graft-versus-leukemia (GVL) eﬀect [38], which may eliminate the adverse eﬀect associated with the higher HO-1 inducibility to potentially to promote progression and relapse. This hypothesis may be supported by the current ﬁndings that post-transplant progression and relapse rates in patients with CML were comparable between the donor HO-1 A/A or A/T genotype vs. the donor HO-1 T/T genotype, with 33% vs. 33% (p = 0.82) in the high-risk group, and 21% vs. 42% (p = 0.14) in the standard-risk group. Studying the blood samples of donors and recipients would be useful for testing this hypothesis and deﬁning the functional roles of the HO-1 SNP in post-transplant survival and relapse. Unfortunately, no blood samples of donors or recipients were available in the present study, and the functional roles of HO-1 on post-transplant outcomes cannot be clariﬁed. 3. Discussio 3. Discussion It has been proposed that a higher HO-1 expression level prevents the progression of GVHD and acts prophylactically on sinusoid obstruction syndrome (SOS), while promoting relapse [39–41]. Although the results of this study suggest that the higher expression of HO-1 is associated with favorable survival outcomes after allo-HSCT, the higher expression of HO-1 has not been shown to aﬀect the risk of disease relapse or the development of GVHD. Furthermore, this cohort did not include information on the incidence of SOS, so the association between HO-1 and SOS is unknown. A plausible explanation for these diﬀerences may be due to the fact that the higher expression of HO-1—which was derived from the donor, not from the patient—appeared to inﬂuence the better survival outcomes after allo-HSCT. Namely, HO-1 derived from the vascular endothelium may be involved in the suppression of GVL eﬀects and GVHD development, whereas HO-1 derived from donor blood cells may be mainly responsible for its anti-inﬂammatory and cytoprotective eﬀects, leading to the prevention of organ damage. However, contrary to this hypothesis, a recent report from Denmark [39] showed that higher HO-1 expression derived from the donor may be associated with a higher risk of relapse after allo-HSCT, based on an investigation of the (GT)n repeat in the promotor region of donors and recipients receiving allo-HSCT. Diﬀerences between the Japanese and Danish ethnic groups might account for the role of HO-1 in the induction of the GVL eﬀect. These hypotheses are highly speculative and need to be clariﬁed by future research. In this study, the beneﬁcial eﬀects of the donor HO-1 A/A or A/T genotype were only observed in patients with high-risk diseases, and not in those with standard-risk diseases. Although it is Cancers 2020, 12, 424 8 of 13 unclear what causes this diﬀerence, patients with high-risk diseases are often heavily treated in an attempt to achieve remission, and may potentially be prone to endothelial cell damage, which may increase the importance of HO-1 in post-transplant survival. This hypothesis may be supported by previous reports showing that an advanced disease status was associated with an increased risk of endothelial cell damage, such as sinusoidal obstruction syndrome and thrombotic microangiopathy, after allo-HSCT [42,43]. One major limitation of this study is the lack of results that conclude the functional roles of the HO-1 SNP in survival outcomes after BMT. 4.1. Patients The HO-1 rs2071746 genotypes were determined on 593 recipients with hematological cancers who underwent transplant via the JMDP with T-cell-replete marrow from HLA-A, -B, -C, -DRB1, -DQB1, and -DPB1 allele-matched donors between January 1993 and December 2007, as well as their unrelated bone marrow donors. The HLA genotyping of recipients and donors was performed by the Luminex microbead method, as described previously (Luminex 100 System; Luminex, Austin, TX, United States) [45,46]. Although the Luminex microbead method does not bring unambiguous HLA four-digit typing for all genotypes, the JMDP has validated that this method can identify all HLA alleles with >0.1% frequency among the Japanese population [47]. Patients with a prior history of any transplantation have been excluded. The ﬁnal clinical survey of these patients was ﬁnalized by November 1, 2008. The diagnoses were ALL in 145 patients (24%), AML in 197 (33%), MDS in 82 (14%), ML in 64 (11%), CML in 101 (17%), and MM in 4 (1%). Cyclosporine- or tacrolimus-based regimens were administered to all patients for GVHD prophylaxis, and anti-T cell therapy (e.g., anti-thymocyte globulin and ex vivo T cell depletion) was administered to none of the patients. All patients and donors gave their written informed consent at the time of transplantation to participate in molecular studies of this nature, in accordance with the Declaration of Helsinki. This project was approved by the Institutional Review Boards of Aichi Medical University School of Medicine (2017-M002) and Kanazawa University, as well as the JMDP. 3. Discussio 3. Discussion The association of the surrogate markers for endothelial cell damage after allo-HSCT, such as serum levels of angiopoietin 2 and thrombomodulin [44], with the HO-1 SNP may oﬀer useful information on this issue. Unfortunately, no serum samples were available in the present study. A second major limitation of this study is that information on transplant-associated thrombotic microangiopathy (TAM) resulting from endothelial cell damage was also unavailable. A study to examine whether the HO-1 genotype and its expression levels contribute to the development of post-transplant complications (e.g., TAM, organ damage, and GVHD) and the graft-versus-tumor eﬀect is warranted. 4.2. HO-1 Genotyping The genotypes of HO-1 rs2071746 were determined using the TaqMan allelic discrimination method, as previously reported [20]. The genotyping assay was done in 96 well PCR plates, using speciﬁc TaqMan probes for the HO-1 gene single nucleotide variation rs2071746 (C__15869717_10), and in a StepOnePlus Real-Time PCR system (Applied Biosystems, Foster City, CA, United States). 4.3. Data Management and Statistical Analysis The JMDP collected data using a standardized report form [48,49]. Follow-up reports were submitted at 100 days, 1 year, and then annually after transplantation. The pre-transplant cytomegalovirus (CMV) serostatus was routinely checked in the patients only, and not in the donors. Engraftment was deﬁned by an absolute neutrophil count of >0.5 × 109/L for at least three consecutive days. The outcome classiﬁcation, including GVHD, did not change over time in the current study. After collecting the data, acute and chronic GVHD were diagnosed and graded based on conventionally 9 of 13 Cancers 2020, 12, 424 deﬁned criteria [50,51]—namely, acute GVHD was deﬁned as GVHD that developed within the ﬁrst 100 days post-transplant, while the manifestations of GVHD occurring after day 100 were classiﬁed as chronic GVHD. The data using the updated criteria for the assessment of GVHD [52,53] were unavailable in the present cohort. The OS rate was deﬁned as the number of days from transplantation to death from any cause. The DFS rate was deﬁned as the number of days from transplantation to death from any cause or disease relapse or disease progression. Disease relapse was deﬁned as the number of days from transplantation to disease relapse or disease progression. Transplant-related mortality (TRM) was deﬁned as death without relapse. ALL patients who were alive at the last follow-up date were censored. The data on the causative microbes of infections, supportive care (including prophylaxis for infection and therapy for GVHD given on an institutional basis), and postmortem change in the cause of death were unavailable for this cohort. The analysis was performed with Easy R (EZR) [54], which is a graphical user interface based on the R software program (The R Foundation for Statistical Computing, Vienna, Austria). The probability of OS was calculated using the Kaplan-Meier model and compared using the log-rank test. The probabilities of TRM, relapse, acute/chronic GVHD, and each cause of death were compared using the Gray test [55], and analyzed using a cumulative incidence model [56], considering relapse, death without relapse, death without acute GVHD, death without chronic GVHD, and death without each cause as respective competing risks. OS, TRM, relapse, grades 2–4 acute GVHD, grades 3–4 acute GVHD, and chronic GVHD were calculated by using a multivariate Cox method and stepwise selection at a signiﬁcance level of 5% to evaluate the HRs associated with the HO-1 genotype. 4.3. Data Management and Statistical Analysis The variables included the recipient’s age at the time of transplantation, sex, pre-transplant CMV serostatus, disease characteristics (disease type, disease lineage, and disease risk at transplant), transplant characteristics (conventional or reduced-intensity conditioning) [57], donor characteristics (age, sex, sex compatibility, and ABO compatibility), cyclosporine versus tacrolimus, total nucleated cell counts harvested per recipient weight (TNC), and year of transplantation. We used the median value as the cutoﬀpoint regarding continuous variables. The independence of the variables used in the multivariate models was conﬁrmed. The chi-squared test and Mann–Whitney test were used to compare between two groups. The Hardy–Weinberg equilibrium for the HO-1 gene variation was tested using the Haploview program [58]. For both the univariate and multivariate analyses, two-tailed p values of <0.05 were considered to indicate statistical signiﬁcance. Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6694/12/2/424/s1. Figure S1: The Kaplan–Meier analysis of the overall survival rates (A) and the disease-free survival rates (B) after transplantation, according to the donor HO-1 genotype in patients with standard-risk disease. Solid lines represent the donor A/A or A/T genotype. Dashed lines represent the donor T/T genotype. An asterisk denotes statistical signiﬁcance (p < 0.05). In the standard-risk disease group, the donor genotype had no signiﬁcant eﬀect on the transplant outcomes, Table S1: The results of the univariate analysis of the association between the HO-1 genotype and the clinical outcomes after transplantation (ﬁrst part), Table S2: The results of the univariate analysis of the association between the HO-1 genotype and the clinical outcomes after transplantation (second part). References 1. Biedermann, B.C.; Sahner, S.; Gregor, M.; Tsakiris, D.A.; Jeanneret, C.; Pober, J.S.; Gratwohl, A. Endothelial injury mediated by cytotoxic T lymphocytes and loss of microvessels in chronic graft versus host disease. Lancet 2002, 359, 2078–2083. [CrossRef] 2. Carreras, E.; Diaz-Ricart, M. The role of the endothelium in the short-term complications of hematopoietic SCT. Bone Marrow Transplant 2011, 46, 1495–1502. [CrossRef] [PubMed] 2. Carreras, E.; Diaz-Ricart, M. The role of the endothelium in the short-term complications of hematopoietic SCT. Bone Marrow Transplant 2011, 46, 1495–1502. [CrossRef] [PubMed] 3. Cooke, K.R.; Jannin, A.; Ho, V. The contribution of endothelial activation and injury to end-organ toxicity following allogeneic hematopoietic stem cell transplantation. Biol. Blood Marrow Transplant. J. Am. Soc. Blood Marrow Transplant. 2008, 14, 23–32. [CrossRef] [PubMed] 3. Cooke, K.R.; Jannin, A.; Ho, V. The contribution of endothelial activation and injury to end-organ toxicity following allogeneic hematopoietic stem cell transplantation. Biol. Blood Marrow Transplant. J. Am. Soc. Blood Marrow Transplant. 2008, 14, 23–32. [CrossRef] [PubMed] 4. Pagliuca, S.; Michonneau, D.; Sicre de Fontbrune, F.; Sutra Del Galy, A.; Xhaard, A.; Robin, M.; Peﬀault de Latour, R.; Socie, G. Allogeneic reactivity-mediated endothelial cell complications after HSCT: A plea for consensual deﬁnitions. Blood Adv. 2019, 3, 2424–2435. [CrossRef] [PubMed] 4. Pagliuca, S.; Michonneau, D.; Sicre de Fontbrune, F.; Sutra Del Galy, A.; Xhaard, A.; Robin, M.; Peﬀault de Latour, R.; Socie, G. Allogeneic reactivity-mediated endothelial cell complications after HSCT: A plea for consensual deﬁnitions. Blood Adv. 2019, 3, 2424–2435. [CrossRef] [PubMed] 5. Rodrigues-Diez, R.; Gonzalez-Guerrero, C.; Ocana-Salceda, C.; Rodrigues-Diez, R.R.; Egido, J.; Ortiz, A.; Ruiz-Ortega, M.; Ramos, A.M. Calcineurin inhibitors cyclosporine A and tacrolimus induce vascular inﬂammation and endothelial activation through TLR4 signaling. Sci. Rep. 2016, 6, e27915. [CrossRef] 5. Rodrigues-Diez, R.; Gonzalez-Guerrero, C.; Ocana-Salceda, C.; Rodrigues-Diez, R.R.; Egido, J.; Ortiz, A.; Ruiz-Ortega, M.; Ramos, A.M. Calcineurin inhibitors cyclosporine A and tacrolimus induce vascular inﬂammation and endothelial activation through TLR4 signaling. Sci. Rep. 2016, 6, e27915. [CrossRef] 6. Schmid, P.M.; Bouazzaoui, A.; Doser, K.; Schmid, K.; Hoﬀmann, P.; Schroeder, J.A.; Riegger, G.A.; Holler, E.; Endemann, D.H. Endothelial dysfunction and altered mechanical and structural properties of resistance arteries in a murine model of graft-versus-host disease. Biol. Blood Marrow Transplant. J. Am. Soc. Blood Marrow Transplant. 2014, 20, 1493–1500. [CrossRef] 6. Schmid, P.M.; Bouazzaoui, A.; Doser, K.; Schmid, K.; Hoﬀmann, P.; Schroeder, J.A.; Riegger, G.A.; Holler, E.; Endemann, D.H. 5. Conclusions Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. 5. Conclusions The ﬁndings of the present study suggested that the donor HO-1 rs2071746 A/A or A/T genotype in the promoter region, which is expected to be highly inducible by HO-1, may improve survival outcomes in patients with high-risk hematologic malignancies who are undergoing unrelated BMT. HO-1 rs2071746 genotyping in donors may therefore be a useful tool for evaluating pre-transplantation risk factors in patients with high-risk diseases that can form a basis for the appropriate tailoring transplantation strategies. Considering the plausible functional roles of this HO-1 promoter SNP, it may be a candidate for future prophylactic and therapeutic strategies for complications after allo-HSCT for high-risk patients. Further studies are needed to conﬁrm whether the ﬁndings of the present study can be extended to other stem cell sources or HLA-incompatible transplantation, as well as to the other ethnic groups. Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6694/12/2/424/s1. Figure S1: The Kaplan–Meier analysis of the overall survival rates (A) and the disease-free survival rates (B) after transplantation, according to the donor HO-1 genotype in patients with standard-risk disease. Solid lines represent the donor A/A or A/T genotype. Dashed lines represent the donor T/T genotype. An asterisk denotes statistical signiﬁcance (p < 0.05). In the standard-risk disease group, the donor genotype had no signiﬁcant eﬀect on the transplant outcomes, Table S1: The results of the univariate analysis of the association between the HO-1 genotype and the clinical outcomes after transplantation (ﬁrst part), Table S2: The results of the univariate analysis of the association between the HO-1 genotype and the clinical outcomes after transplantation (second part). Cancers 2020, 12, 424 10 of 13 10 of 13 Author Contributions: A.T. and E.M. designed the study. T.H. performed the experimental analyses under instruction by S.M., T.H. performed the statistical analysis, which S.M., K.U., and A.T. supported. T.H. and A.T. wrote the paper. I.H., J.L.E., Y.M., Y.K., M.O., K.K., T.F., N.D., K.M., T.M., S.N. contributed to data collection and sample management. All authors have read and agreed to the published version of the manuscript. 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https://openalex.org/W4285319149	https://repositorio.cepelin.org/index.php/repositorioppglintesesdissertaco/article/download/216/204	Portuguese	null	ANÁLISE COGNITIVA DOS GESTOS E DA DIREÇÃO DO OLHAR EM NARRATIVAS MULTIMODAIS DO PORTUGUÊS BRASILEIRO	Sygnum	2,021	cc-by	53,314	UNIVERSIDADE ESTADUAL DO SUDOESTE DA BAHIA (UESB) PROGRAMA DE PÓS-GRADUAÇÃO EM LINGUÍSTICA (PPGLIN) i ANÁLISE COGNITIVA DOS GESTOS E DA DIREÇÃO DO OLHAR EM NARRATIVAS MULTIMODAIS DO PORTUGUÊS BRASILEIRO Dissertação apresentada ao Programa de Pós- Graduação em Linguística (PPGLin), da Universidade Estadual do Sudoeste da Bahia (UESB), como requisito parcial e obrigatório para obtenção do título de Mestre em Linguística. Área de Concentração: Linguística Linha de Pesquisa: Texto, Significado e Discurso Orientadora: Maíra Avelar Miranda Orientadora: Maíra Avelar Miranda ii ii CDD: 410 Título em inglês: A cogntive analysis of gesture and gaze direction in multimodal narratives of Brazilia Portuguese Título em inglês: A cogntive analysis of gesture and gaze direction in multimodal narratives of Brazilian Portuguese Palavras-chave em inglês: Gesture. Gaze. Multimodal Narratives. Área de concentração: Linguística Titulação: Mestre em Linguística Banca examinadora: Profa. Dra. Maíra Avelar Miranda (Presidente/Orientadora - UESB); Prof. Dra. Adriana Fernandes Barbosa (UESB) e Profa. Dra. Lilian Vieira Ferrari (UFRJ) Data da qualificação: 26 de agosto de 2020 Data da defesa: 18 de fevereiro de 2021 Programa de Pós-Graduação: Programa de Pós-Graduação em Linguística Orcid ID: https://orcid.org/0000-0002-2906-3107 Lattes ID: http://lattes.cnpq.br/7357484967378886 Portuguese Palavras-chave em inglês: Gesture. Gaze. Multimodal Narratives. Área de concentração: Linguística Titulação: Mestre em Linguística Banca examinadora: Profa. Dra. Maíra Avelar Miranda (Presidente/Orientadora - UESB); Prof. Dra. Adriana Fernandes Barbosa (UESB) e Profa. Dra. Lilian Vieira Ferrari (UFRJ) Data da qualificação: 26 de agosto de 2020 Data da defesa: 18 de fevereiro de 2021 Programa de Pós-Graduação: Programa de Pós-Graduação em Linguística Orcid ID: https://orcid.org/0000-0002-2906-3107 Lattes ID: http://lattes.cnpq.br/7357484967378886 Palavras-chave em inglês: Gesture. Gaze. Multimodal Narratives. Palavras-chave em inglês: Gesture. Gaze. Multimodal Narratives. Área de concentração: Linguística Titulação: Mestre em Linguística Banca examinadora: Profa. Dra. Maíra Avelar Miranda (Presidente/Orientadora - UESB); Prof. Dra. Adriana Fernandes Barbosa (UESB) e Profa. Dra. Lilian Vieira Ferrari (UFRJ) Data da qualificação: 26 de agosto de 2020 Data da defesa: 18 de fevereiro de 2021 Programa de Pós-Graduação: Programa de Pós-Graduação em Linguística Área de concentração: Linguística Data da qualificação: 26 de agosto de 2020 Data da defesa: 18 de fevereiro de 2021 Orcid ID: https://orcid.org/0000-0002-2906-3107 Orcid ID: https://orcid.org/0000-0002-2906-3107 Lattes ID: http://lattes.cnpq.br/7357484967378886 Lattes ID: http://lattes.cnpq.br/7357484967378886 iii iii À Divina e Maravilhosa Trindade da minha vida: Nida, Ana e Ritinha. iv iv À Divina e Maravilhosa Trindade da minha vida: Nida, Ana e Ritinha. À Divina e Maravilhosa Trindade da minha vida: Nida, Ana e Ritinha. v 1 Forma padrão em conformidade com Portaria CAPES n° 206/2018 e esclarecimento do Ofício Circular nº 19/2018-CPG/CGSI/DPB/CAPES. AGRADECIMENTOS À Universidade Estadual do Sudoeste da Bahia (UESB) e ao Programa de Pós- Graduação em Linguística (PPGLin), pela oportunidade de realização da minha formação em nível de mestrado. À Capes: “O presente trabalho foi realizado com apoio da Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) - Código de Financiamento 001”. 1 À FAPESB, pela bolsa de estudos concedida. À minha orientadora, a Prof.ª Dr.ª Maíra Avelar Miranda, pela parceria de quase 7 anos. Por ter me ensinado tantas coisas que extrapolam o ambiente acadêmico e por me fazer compreender a mim mesmo como um sujeito pesquisador. Agradeço, ainda, por ter me dado as mãos nessa jornada pela qual viajamos durante esses anos de trabalho. À Prof.ª Dr.ª Adriana Fernandes Barbosa, quem tive o prazer de conhecer no período de execução deste trabalho, por ter aceitado avaliar a dissertação e por ter feito, tão sensivelmente, contribuições na minha qualificação e nas aulas da disciplina Tópicos em Estudos de Linguagem II. À Prof.ª Dr.ª Lilian Vieira Ferrari, por ser uma figura tão ímpar na minha trajetória (e também na trajetória de muitos pesquisadores) pela Linguística Cognitiva. Agradeço, também, por ter aceitado avaliar meu trabalho e pelas valiosas contribuições, não só na banca de qualificação como também nas aulas da disciplina Teoria dos Espaços Mentais. Às funcionárias do PPGLin, Vanêide e Luciana, por se mostrarem tão solícitas e engajadas com o fazer científico. Agradeço, ainda, por tratarem os alunos com muita gentileza e cuidado. À minha mãe, por me ajudar a definir a palavra AMOR. Por ter compreendido quando eu me mostrava ausente do mundo, mesmo dentro de casa. Por me considerar sua prioridade desde o final dos anos 1990. Por amar tanto...! E, principalmente, por sempre me ensinar a ser grato. À minha tia, por fazer tantos esforços para o bem-estar dos seus e dos próximos. Por me compreender acima de qualquer laço de família e por ser meu alívio racional. vi À minha avó, por compartilhar comigo o amor pela nossa cidade favorita no mundo. Por ter me apresentado a Praia de Ipanema, o Dedo de Deus e a barca Rio-Niterói. Minha avozinha, quando miro en tus pupilas sé que Diós no dejó de existir. A Matheus, por tecer ao meu lado tantos fios que ficaram presos em mim. Por todas as sessões de sushi-acarajé-pizza, quando tudo que eu precisava era me desconectar. AGRADECIMENTOS No que diz respeito à marcação de Espaços Mentais pelos gestos, utilizamos os parâmetros que relacionam os gestos com o segmento da narração em que determinada interação se aloca, seja ele parte de uma interação no aqui-agora (Ground) ou parte de uma interação no contexto narrativo (Espaço Narrativo). Já no que diz respeito à marcação de Espaços Mentais pela direção do olhar, utilizamos as categorizações que verificam se o olhar de um narrador representa o olhar de uma personagem (no Espaço Narrativo) ou o olhar do próprio narrador na interação (no Ground). Apenas nos casos em que os gestos marcaram o Espaço Narrativo, analisamos logo após, se eles se configuram como gestos do ponto de vista do observador (O-VPT) ou como gestos do ponto de vista da personagem (C-VPT). Ainda nesses casos, analisamos os gestos quanto à sua função dominante no interior da narrativa: representação, expressão ou apelo. Do mesmo modo, somente nos casos em que a direção do olhar marcou o Espaço Narrativo, analisamos, também, a sua função no interior da narrativa: i) encenação da personagem e ii) narração propriamente dita. Os resultados demonstraram que a marcação predominante de Espaços Mentais pelos gestos foi a marcação do Espaço Narrativo e que a marcação predominante de Espaços Mentais pela direção do olhar foi a marcação do Ground. AGRADECIMENTOS A Beatriz, por me lembrar que aqui na Bahia não usamos o artigo antes de nomes próprios. Por compartilhar o lado Frances-e-Sophie da vida comigo há 15 anos – inclusive uma graduação e um mestrado. Agradeço, ainda, por todos os “só vou se você for”, por todos “o que tem para amanhã?” e, especialmente, por todos “migo, tô aqui”. Aos meus amigos, principalmente os que me ouviram chorar enquanto escrevia ou dizia “meu deus, eu não consigo formatar essa tabela”. A Óscar, Bafta e Glória, meus amores não-humanos, pela companhia na madrugada e “lambeijos” da vida toda. Aos deuses e orixás. Ao universo. À Arte. À Ciência. vii Eu queria estar lá na hora em que a Shakira disse que nasceu dia 2 de fevereiro e o marido disse “eu também”. Eu queria estar em todas as coincidências do mundo porque é onde todo mundo está um pouco mais místico. Na hora das coincidências, todas as pessoas estão mais conectadas com uma excitação de isso aqui não ser só isso aqui. Eu choro com as coincidências. Eu quero estar em todas as coincidências. Letícia Novaes – Salve Poseidon viii RESUMO Nesta dissertação, analisamos o comportamento dos gestos e da direção do olhar em narrativas multimodais do português brasileiro. Partimos da hipótese de que dois articuladores multimodais – especificamente, os gestos e a direção do olhar – configuram-se de modo independente em Espaços Mentais diferentes. A fim de demonstrar como se configuram esses articuladores em contextos narrativos no português brasileiro, selecionamos uma narrativa pessoal proferida pela atriz Marisa Orth no programa semanal “Que História é Essa, Porchat?”, do canal GNT. Em termos metodológicos, dividimos a narrativa em 11 ocorrências multimodais presentes em 3 blocos narrativos: 3 ocorrências presentes no bloco “Exposição”, a apresentação dos fatos iniciais; 5 ocorrências do bloco “Clímax” que se refere ao conflito, o momento culminante da história e 3 ocorrências do bloco “Desfecho”, a conclusão do conflito. Em primeiro lugar, analisamos as escolhas linguísticas de cada ocorrência, e em seguida, partimos para a análise multimodal das ocorrências, com respaldo na identificação e descrição dos núcleos (strokes) gestuais e, posteriormente, identificamos a forma do gesto, levando em consideração os parâmetros do Sistema Linguístico de Anotação Gestual (LASG). AGRADECIMENTOS No que diz respeito à marcação de Espaços Mentais pelos gestos, utilizamos os parâmetros que relacionam os gestos com o segmento da narração em que determinada interação se aloca, seja ele parte de uma interação no aqui-agora (Ground) ou parte de uma interação no contexto narrativo (Espaço Narrativo). Já no que diz respeito à marcação de Espaços Mentais pela direção do olhar, utilizamos as categorizações que verificam se o olhar de um narrador representa o olhar de uma personagem (no Espaço Narrativo) ou o olhar do próprio narrador na interação (no Ground). Apenas nos casos em que os gestos marcaram o Espaço Narrativo, analisamos logo após, se eles se configuram como gestos do ponto de vista do observador (O-VPT) ou como gestos do ponto de vista da personagem (C-VPT). Ainda nesses casos, analisamos os gestos quanto à sua função dominante no interior da narrativa: representação, expressão ou apelo. Do mesmo modo, somente nos casos em que a direção do olhar marcou o Espaço Narrativo, analisamos, também, a sua função no interior da narrativa: i) encenação da personagem e ii) narração propriamente dita. Os resultados demonstraram que a marcação predominante de Espaços Mentais pelos gestos foi a marcação do Espaço Narrativo e que a marcação predominante de Espaços Mentais pela direção do olhar foi a marcação do Ground. Nesta dissertação, analisamos o comportamento dos gestos e da direção do olhar em narrativas multimodais do português brasileiro. Partimos da hipótese de que dois articuladores multimodais – especificamente, os gestos e a direção do olhar – configuram-se de modo independente em Espaços Mentais diferentes. A fim de demonstrar como se configuram esses articuladores em contextos narrativos no português brasileiro, selecionamos uma narrativa pessoal proferida pela atriz Marisa Orth no programa semanal “Que História é Essa, Porchat?”, do canal GNT. Em termos metodológicos, dividimos a narrativa em 11 ocorrências multimodais presentes em 3 blocos narrativos: 3 ocorrências presentes no bloco “Exposição”, a apresentação dos fatos iniciais; 5 ocorrências do bloco “Clímax” que se refere ao conflito, o momento culminante da história e 3 ocorrências do bloco “Desfecho”, a conclusão do conflito. Em primeiro lugar, analisamos as escolhas linguísticas de cada ocorrência, e em seguida, partimos para a análise multimodal das ocorrências, com respaldo na identificação e descrição dos núcleos (strokes) gestuais e, posteriormente, identificamos a forma do gesto, levando em consideração os parâmetros do Sistema Linguístico de Anotação Gestual (LASG). PALAVRAS-CHAVE Linguística Cognitiva. Gestos. Direção do Olhar. Narrativas Multimodais. ix ABSTRACT In this master’s thesis, I intend to analyse the performance of gesture and gaze direction in multimodal narratives of Brazilian Portuguese. I assume the hypothesis that two multimodal articulators, especifically gesture and gaze direction, are able to be located independently in different Mental Spaces. To do so, I selected a personal narrative told by the Brazilian actress Marisa Orth, in the weekly TV show “Que História é Essa, Porchat?”, from the TV channel “GNT”. Methodologically, I divided the narrative into 11 occurrences 3 narrative excerpts: 3 occurrences present in the “Exhibition” excerpt, the presentation of the initial facts; 5 occurrences of the “Climax” excerpt, the conflict itself, the culminating moment of the story and 3 occurrences of the “Outcome” excerpt, the conclusion of the conflict. First, I analysed the linguistic choices of each occurrence, and then, we initiated the multimodal analysis of the occurrences, with support in the identification and description of the gestural strokes and, later, we identified the form of the gesture, taking into account the parameters of the Linguistic Annotation System for Gestures (LASG). With regard to the marking of Mental Spaces by gestures, I used the parameters that relate the gestures to the segment of the narrative in which a given interaction is allocated, whether it is part of an interaction in the Base Space (Ground) or part of an interaction in the narrative context (Story Space). Regarding the marking of Mental Spaces by gaze direction, I used the categorisations that verify whether the narrator gaze represents a character gaze (in the Story Space) or the narrator own gaze in the interaction (in the Ground). Only in the cases where the gestures marked the Story, I analysed, right after, if they are configured as gestures from the observer viewpoint (O-VPT) or as gestures from character viewpoint (C-VPT). Still in these cases, I analysed the gestures regarding their dominant function within the narrative: representation, expression or appeal. Likewise, only in cases where the direction of the gaze marked the Story Space, I also analysed its function within the narrative: i) character enaction and ii) narratorial gaze as itself. The results have shown that the predominant marking of Mental Spaces by gestures was the Space Story and that the predominant marking of Mental Spaces by the direction of the eye was the marking of the Ground. KEYWORDS Cognitive Linguistics. Gestures. Gaze Direction. Multimodal Narratives. x LISTA DE FIGURAS LISTA DE FIGURAS Figura 1: Narrativa de Letícia Spiller ...................................................................................... 21 Figura 2: Representação dos Espaços Mentais – Narrativa Letícia Spiller ............................. 22 Figura 3: As sequências de evento e os níveis narrativos ....................................................... 39 Figura 4: Excursão Gestual ..................................................................................................... 45 Figura 5: Gestos Utilizados com Expressões Verbais Correspondentes ................................. 47 Figura 6: Gestos que Acompanham uma Expressão Verbal Não-correspondente .................. 48 Figura 7: Gestos como Especificadores Semânticos ............................................................... 49 Figura 8: Gestos como uma Amostra ...................................................................................... 50 Figura 9: Gestos que Representam Propriedades de um Objeto e Relações Espaciais ........... 51 Figura 10: Gestos como Objetos de Referência Dêitica .......................................................... 52 Figura 11: Continuum de Kendon (1988a) .............................................................................. 52 Figura 12: Gestos Icônicos ...................................................................................................... 55 Figura 13: Gestos Metafóricos ................................................................................................ 56 Figura 14: Gestos Rítmicos ..................................................................................................... 57 Figura 15: Gestos Coesivos ..................................................................................................... 59 Figura 16: Gestos Dêiticos ...................................................................................................... 60 Figura 17: Modo de Representação Gestual 1 – Encenar ........................................................ 62 Figura 18: Modo de Representação Gestual 2 – Corporificar ................................................. 62 Figura 19: Modo de Representação Gestual 3 – Moldar ......................................................... 63 Figura 20: Modo de Representação Gestual 4 – Desenhar ...................................................... 63 Figura 21: Desenvolvimento histórico dos gestos para os sinais ............................................ 65 Figura 22: Função Representacional 1: Ações, Objetos e Eventos concretos ......................... 66 Figura 23: Função representacional 2: Gestos Metafóricos .................................................... 67 Figura 24: Função Expressiva 1: Alegria ou Triunfo .............................................................. 68 Figura 25: Função Expressiva 2: Tristeza ou Sofrimento ....................................................... 68 Figura 26: Função Expressiva 3: Raiva ou Ira ........................................................................ 69 Figura 27: Função Apelativa 1: Público Amplo ...................................................................... 69 Figura 28: Função Apelativa 2: Interação Face a Face ........................................................... 70 Figura 29: Gesto “desenhar a linha” (draw the line) ............................................................... 72 Figura 30: Comparação entre o Olhar Humano e o Olhar de um Chimpanzé ........................ 75 Figura 31: Ilustração das narrativas “Certidão”, “Neve” e “Gatos” ....................................... 77 Figura 32: Segmentação Corporal nas narrativas Certidão(a), Neve (b) e Gatos (c) .............. 79 xi Figura 33: Narrativa “Certidão” .............................................................................................. 80 Figura 34: Narrativa “Gatos” ................................................................................................... 81 Figura 35: Narrativa “Neve” ................................................................................................... 82 Figura 36: Narrativa de Marisa Orth ....................................................................................... 85 Figura 37: Captura de tela do ELAN ....................................................................................... 86 Figura 38: Trilhas de Análise do Articulador Multimodal “Gestos” ...................................... 87 Figura 39: Trilhas de Análise do Articulador Multimodal “Direção do Olhar” ...................... 87 Figura 40: Movimento ao longo dos Eixos Horizontal e Vertical ........................................... 95 Figura 41: Movimentos ao longo do Eixo Sagital ................................................................... LISTA DE FIGURAS 96 Figura 42: Disposição Espacial da Narrativa de Marisa Orth ............................................... 103 Figura 43: Representação dos espaços mentais – Narrativa Marisa Orth ............................. 104 Figura 44: Representação multimodal da ocorrência 1 do bloco “Exposição” ..................... 108 Figura 45: Representação multimodal da ocorrência 2 do bloco “Exposição” ..................... 110 Figura 46: Representação multimodal da ocorrência 3 do bloco “Desfecho” ....................... 112 Figura 47: Representação multimodal da ocorrência 1 do bloco “Clímax” .......................... 118 Figura 48: Representação multimodal da ocorrência 2 do bloco “Clímax” .......................... 120 Figura 49: Representação multimodal da ocorrência 3 do bloco “Clímax” .......................... 121 Figura 50: Representação multimodal da ocorrência 4 do bloco “Clímax” .......................... 122 Figura 51: Representação multimodal da ocorrência 5 do bloco “Clímax” .......................... 124 Figura 52: Representação multimodal da ocorrência 1 do bloco “Desfecho” ....................... 129 Figura 53: Representação multimodal da ocorrência 2 do bloco “Desfecho” ....................... 131 Figura 54: Representação multimodal da ocorrência 3 do bloco “Desfecho” ....................... 132 xii LISTA DE GRÁFICOS Gráfico 1: Marcação gestual de Espaços Mentais ................................................................. 141 Gráfico 2: Marcação visual de Espaços Mentais ................................................................... 142 Gráfico 3: Simultaneidade na marcação de Espaços Mentais ............................................... 143 Gráfico 4: Pontos de Vista nos Gestos do Espaço Narrativo ................................................. 144 Gráfico 5: Funções dos Gestos no Interior da Narrativa ....................................................... 145 Gráfico 6: Função da Direção do Olhar no Interior da Narrativa .......................................... 146 xiii LISTA DE QUADROS Quadro 1: Trecho da Transcrição da narrativa de Letícia Spiller ........................................... 21 Quadro 2: Conceitos básicos da Gramática Cognitiva (Langacker, 1987) ............................. 27 Quadro 3: Termos Utilizados na Descrição de Ponto de Vista e Perspectiva ......................... 34 Quadro 4: Tipos de Núcleo Gestual ........................................................................................ 88 Quadro 5: Formato das Mãos .................................................................................................. 90 Quadro 6: Orientação das Palmas ........................................................................................... 91 Quadro 7: Orientação das Palmas em Relação ao Espaço Gestual ......................................... 93 Quadro 8: Tipos do Movimento .............................................................................................. 94 Quadro 9: Modos de Representação Gestual .......................................................................... 97 Quadro 10: Marcação dos Espaços Mentais pelos Gestos ...................................................... 98 Quadro 11: Marcação dos Pontos de Vista Gestuais na Narrativa .......................................... 99 Quadro 12: Função dos Gestos no Interior da Narrativa ......................................................... 99 Quadro 13: Marcação de Espaços Mentais pela direção do olhar ......................................... 101 Quadro 14: Função da Direção do Olhar no Interior da Narrativa ........................................ 102 Quadro 15: Escolhas linguísticas como evidências da estrutura da narrativa no bloco “Exposição” ............................................................................................................................ 106 Quadro 16: Marcação de Espaços Mentais nas ocorrências do bloco “Exposição” ............. 113 Quadro 17: Ponto de Vista dos Gestos Narrativos no bloco “Exposição” ............................ 114 Quadro 18: Função Gestual no Interior da Narrativa no bloco “Exposição” ........................ 114 Quadro 19: Função da Direção do Olhar no Interior da Narrativa no bloco “Exposição” ... 115 Quadro 20: Escolhas linguísticas como evidências da estrutura da narrativa no bloco “Clímax” ................................................................................................................................. 116 Quadro 21: Marcação de Espaços Mentais nas ocorrências do bloco “Clímax” .................. 125 Quadro 22: Ponto de Vista dos Gestos Narrativos no bloco “Clímax” ................................. 126 Quadro 23: Função Gestual no Interior da Narrativa no Bloco “Clímax” ............................ 126 Quadro 24: Função da Direção do Olhar no Interior da Narrativa no bloco “Clímax” ........ 127 Quadro 25: Escolhas linguísticas como evidências da estrutura da narrativa no bloco “Desfecho” .............................................................................................................................. 128 Quadro 26: Marcação de Espaços Mentais nas ocorrências do bloco “Desfecho” ............... 134 Quadro 27: Ponto de Vista dos Gestos Narrativos no bloco “Desfecho” ............................. 135 Quadro 28: Função Gestual no Interior da Narrativa no Bloco “Desfecho” ......................... 135 Quadro 29: Função da Direção do Olhar no Interior da Narrativa no bloco “Desfecho” ..... 136 xiv xiv SUMÁRIO 1 INTRODUÇÃO ................................................................................................................... 16 2 ESTRUTURA MULTIMODAL DA NARRATIVA ........................................................ 19 2.1 Espaços Mentais e Narrativa ........................................................................................... 19 2.2 Ponto de vista e perspectiva ............................................................................................. 23 2.3 Ponto de Vista e Narrativa: inter-relação entre fala e gestos ....................................... LISTA DE QUADROS 29 2.4 Organização multimodal da narrativa ........................................................................... 36 2.5 Reencenações de eventos passados .................................................................................. 40 3 O PAPEL DOS GESTOS MANUAIS E A DA DIREÇÃO DO OLHAR NA INTERAÇÃO .......................................................................................................................... 43 3.1 Breve histórico sobre os Estudos de Gesto na atualidade ............................................. 43 3.1.1 Kendon: ações visíveis como enunciados ...................................................................... 44 3.1.2 McNeill: o que os gestos revelam sobre o pensamento ................................................. 53 3.1.3 Müller: os gestos e seu potencial para a linguagem ..................................................... 60 3.1.4 Cienki: análise de metáfora nos gestos .......................................................................... 70 3.2 Gestos e outros articuladores independentes: o papel do olhar na interação ............. 73 3.3 As funções do olhar em narrativas: o estabelecimento de diferentes pontos de vista 77 4 PROCEDIMENTOS METODOLÓGICOS ...................................................................... 83 4.1 Procedimentos da Coleta de Dados ................................................................................. 84 4.2 Procedimentos Metodológicos de Análise ....................................................................... 85 4.2.1 Trilha 1: Trecho Narrativo ............................................................................................. 87 4.2.2 Trilha 2: Stroke (Núcleo) ............................................................................................... 88 4.2.3 Trilha 3: Transcrição ..................................................................................................... 89 4.2.4 Trilha 4: Formato das Mãos .......................................................................................... 89 4.2.5 Trilha 5: Orientação das Palmas ................................................................................... 91 4.2.6 Trilha 6: Posição do Movimento .................................................................................... 93 4.2.7 Trilha 7: Tipos do Movimento ........................................................................................ 94 4.2.8 Trilha 8: Direção do Movimento .................................................................................... 95 4.2.9 Trilha 9: Qualidade do Movimento ................................................................................ 96 4.2.10 Trilha 10: Modo de Representação Gestual ................................................................ 96 4.2.11 Trilha 11: Marcação dos Espaços Mentais (Gestos) ................................................... 98 4.2.12 Trilha 12: Ponto de Vista Gestual ................................................................................ 98 4.2.13 Trilha 13: Função dos Gestos no Interior da Narrativa ............................................. 99 xv 4.2.14 Trilha 14: Direção do Olhar ....................................................................................... 100 4.2.15 Trilha 15: Direção do Tronco .................................................................................... 100 4.2.16 Trilha 16: Marcação dos Espaços Mentais (Direção do Olhar) ............................... 101 4.2.17 Trilha 17: Função da Direção do Olhar no Interior da Narrativa ........................... 101 5 ANÁLISE DOS DADOS ................................................................................................... 103 5.1 Análise do bloco “Exposição” ........................................................................................ 106 5.1.1 Análise Multimodal da Ocorrência 1 do Bloco “Exposição” ..................................... 107 5.1.2 Análise multimodal da ocorrência 2 do bloco “Exposição” ....................................... 109 5.1.3 Análise multimodal da ocorrência 3 do bloco “Exposição” ....................................... 112 5.1.4 Discussão dos dados do bloco “Exposição” ................................................................ 113 5.2 Análise do bloco “Clímax” ............................................................................................. 116 5.2.1 Análise multimodal da ocorrência 1 do bloco “Clímax” ............................................ 118 5.2.2 Análise multimodal da ocorrência 2 do bloco “Clímax” ............................................ 120 5.2.3 Análise multimodal da ocorrência 3 do bloco “Clímax” ............................................ 121 5.2.4 Análise multimodal da ocorrência 4 do bloco “Clímax” ............................................ 122 5.2.5 Análise multimodal da ocorrência 5 do bloco “Clímax” ............................................ 1 INTRODUÇÃO Analisar narrativas a partir de uma perspectiva cognitivista requer que se leve em consideração o modo como elas constroem sentido. De acordo com Goodwin (1981), para que a construção de sentido seja efetiva, é necessário que os participantes não apenas produzam sentenças, mas que as coordenem de modo que haja a organização de sua fala com a fala dos outros indivíduos presentes na interação. Essa atividade, ainda de acordo com o autor, constitui uma forma de organização social humana pervasiva que estrutura as competências linguística e cultural das partes envolvidas, considerando, desse modo, que a emergência das narrações envolve um processo cognitivo que compreende várias faculdades mentais. O objetivo do nosso trabalho é investigar, a partir de dados multimodais, o comportamento dos gestos e da direção do olhar em narrativas do português brasileiro. Trata- se de uma pesquisa inédita, conforme a busca de dados realizadas no Portal CAPES. Partimos, então, para a nossa pergunta de pesquisa: Quais são as funções dos gestos manuais e da direção do olhar na marcação de Espaços Mentais em narrativas do Português Brasileiro? A partir da nossa pergunta de pesquisa, a hipótese de pesquisa é a seguinte: no português brasileiro, assim como no inglês americano (SWEETSER; STEC, 2016), o olhar e os gestos manuais podem marcar Espaços Mentais independentes em um mesmo momento interacional. Levando em consideração a nossa pergunta de pesquisa, chegamos aos seguintes objetivos específicos: i) analisar a marcação multimodal de Espaços Mentais; ii) verificar a relação entre os gestos e a direção do olhar na representação de Espaços Mentais; iii) realizar uma descrição da estrutura multimodal da narrativa, levando em consideração os gestos e a direção do olhar. As discussões presentes no Capítulo 2 demonstram que, desde a sua concepção, a Teoria dos Espaços Mentais (FAUCONNIER, 1994, 1997) tem sido considerada como um dos pilares da Linguística Cognitiva e possui como foco os modos com os quais o sentido é construído na mente a partir de diversos domínios da atividade humana. Essas atividades compreendem desde a compreensão de texto e do discurso até a construção de sentido em rituais religiosos e na música, conforme afirmam Hougaard e Oakley (2008). LISTA DE QUADROS 123 5.2.6 Discussão dos dados do bloco “Clímax” ...................................................................... 125 5.3 Análise do bloco “Desfecho” .......................................................................................... 127 5.3.1 Análise multimodal da ocorrência 1 do bloco “Desfecho” ......................................... 129 5.3.2 Análise multimodal da ocorrência 2 do bloco “Desfecho” ......................................... 131 5.3.3 Análise multimodal da ocorrência 3 do bloco “Desfecho” ......................................... 132 5.3.4 Discussão dos dados do bloco “Desfecho” .................................................................. 134 5.4 Discussão geral dos dados .............................................................................................. 136 6 CONSIDERAÇÕES FINAIS ............................................................................................ 147 REFERÊNCIAS .................................................................................................................... 151 ANEXOS ................................................................................................................................ 155 ANEXO A: TRANSCRIÇÃO DA NARRATIVA DE LETÍCIA SPILLER ................... 155 ANEXO B: TRANSCRIÇÃO DA NARRATIVA DE MARISA ORTH ......................... 157 ANEXO C – PARÂMETROS DE ANÁLISE .................................................................... 164 ANEXO D – TRILHAS DE ANÁLISE DO BLOCO “EXPOSIÇÃO” ........................... 165 ANEXO E – TRILHAS DE ANÁLISE DO BLOCO “CLÍMAX” ................................... 167 ANEXO F – TRILHAS DE ANÁLISE DO BLOCO “DESFECHO” ............................. 172 16 16 1 INTRODUÇÃO Esses autores afirmam, ainda, que o desenvolvimento de estudos, a partir da cognição, sobre o discurso e a interação humana e suas variantes, como por exemplo, a poesia, a conversação e as narrativas multimodais, são consequência da elaboração do quadro teórico proposto por Fauconnier (1994). Além disso, um Espaço Mental pode ser ativado por meio da recordação de um evento passado, retomado ao longo da conversação que ocorre na situação presente. Em um segundo momento, ainda no 17 capítulo 2, demonstramos que o ponto de vista é um elemento da cognição que impacta diretamente na visão da cena da ação e na estruturação multimodal da narrativa, determinando a estrutura linguística. No capítulo 3, traçamos um panorama a respeito dos Estudos de Gesto e da Direção do Olhar. No que diz respeito aos gestos, abordamos os trabalhos de quatro autores: Kendon (1980, 1986, 2004), McNeill (1979, 1995, 2005), Müller (2004, 2013, 2018) e Cienki (2008, 2017). Kendon (1980, 1986, 2004) expõe sobre o potencial linguístico dos gestos espontâneos. Além disso, o autor introduz a ideia de unidade gestual e como ela se relaciona com as unidades verbais. Complementarmente, McNeill (1979, 1995, 2005) relaciona a realização dos gestos com processos cognitivos na mente do falante. O autor propõe, ainda, a categorização das funções gestuais, incluindo o gesto metafórico. Müller (2004, 2013, 2018), por sua vez, aborda o gesto a partir de sua função representacional, com base no modelo de Bühler (1982), e de sua motivação cognitiva, a partir dos trabalhos de McNeill (1995, 2005). A autora propõe, além disso, os modos de representação gestual, que descrevem como os gestos representam o conteúdo da fala na interação. Por fim, Cienki (2008, 2017) explora as metáforas verbo-gestuais, elaborando, desse modo, métodos específicos para identificá-las no discurso. No que diz respeito ao olhar, descrevemos suas especificidades e a sua integração com os gestos manuais, particularmente quando se trata de atividades múltiplas e simultâneas. No capítulo 4, descrevemos, de forma panorâmica, a narrativa analisada e abordamos os procedimentos de coleta e análise dos dados. 1 INTRODUÇÃO Em seguida, tratamos de questões específicas que dizem respeito à análise de dados multimodais nas narrativas em questão, levando em consideração os dois articuladores multimodais: os gestos manuais e a direção do olhar No capítulo 5, descrevemos a narrativa com a qual trabalhamos, que, por sua vez, foi proferida pela atriz Marisa Orth, que relata, para o apresentador Fábio Porchat, para a plateia e para os outros convidados do programa – a atriz Samantha Schmütz e a cantora Ivete Sangalo –, o momento no qual, por indicação de suas amigas, sai para jantar com um homem e descobre que ele escondia o fato de ser casado. Ainda, tratamos de alguns elementos importantes que fazem parte da configuração espacial da narrativa com a qual trabalhamos. No que diz respeito à análise dos dados, partimos das escolhas linguísticas de cada ocorrência com base nos pressupostos de Dancygier (2011). Em seguida, seguimos para a análise multimodal das ocorrências, com respaldo na identificação e descrição dos núcleos (strokes) gestuais, conforme definição de Kendon (2004) e, posteriormente, identificamos a forma do gesto, levando em No capítulo 5, descrevemos a narrativa com a qual trabalhamos, que, por sua vez, foi proferida pela atriz Marisa Orth, que relata, para o apresentador Fábio Porchat, para a plateia e para os outros convidados do programa – a atriz Samantha Schmütz e a cantora Ivete Sangalo –, o momento no qual, por indicação de suas amigas, sai para jantar com um homem e descobre que ele escondia o fato de ser casado. Ainda, tratamos de alguns elementos importantes que fazem parte da configuração espacial da narrativa com a qual trabalhamos. No que diz respeito à análise dos dados, partimos das escolhas linguísticas de cada ocorrência com base nos pressupostos de Dancygier (2011). Em seguida, seguimos para a análise multimodal das ocorrências, com respaldo na identificação e descrição dos núcleos (strokes) gestuais, conforme definição de Kendon (2004) e, posteriormente, identificamos a forma do gesto, levando em –, o momento no qual, por indicação de suas amigas, sai para jantar com um homem e descobre que ele escondia o fato de ser casado. Ainda, tratamos de alguns elementos importantes que fazem parte da configuração espacial da narrativa com a qual trabalhamos. No que diz respeito à análise dos dados, partimos das escolhas linguísticas de cada ocorrência com base nos pressupostos de Dancygier (2011). 2 ESTRUTURA MULTIMODAL DA NARRATIVA Neste capítulo, pretendemos estabelecer uma correlação entre os estudos em Espaços Mentais, e a estrutura multimodal da narrativa. Especificamente, pretendemos demonstrar sua influência na criação de Espaços Narrativos a partir do Espaço-base (Ground). Para tanto, baseamo-nos no texto de Dancygier (2011) no qual ela aplica a Teoria dos Espaços Mentais (FAUCONNIER, 1994) nas análises das narrativas a partir de uma perspectiva cognitivista. Em seguida, buscamos articular os estudos em Ponto de Vista também com os estudos referentes à narrativa sob uma ótica multimodal, baseando-nos nas proposições de Verhagen (2010), Sweetser (2012) e Parrill (2009, 2012). Com base nas considerações realizadas por Parrill (2009), apresentamos uma descrição dos termos utilizados na descrição de ponto de vista e perspectiva, utilizando parâmetros dos estudos sobre a narrativa associados aos Estudos de Gesto e à Língua de Sinais Americana. Posteriormente, tratamos da organização multimodal da narrativa, com base nos estudos empreendidos por McNeill (1990, 1995). E, por fim, tratamos das reencenações (SIDNELL, 2006). 1 INTRODUÇÃO Em seguida, seguimos para a análise multimodal das ocorrências, com respaldo na identificação e descrição dos núcleos (strokes) gestuais, conforme definição de Kendon (2004) e, posteriormente, identificamos a forma do gesto, levando em 18 consideração os parâmetros descritos no Capítulo 4, que dizem respeito ao bloco 1 do Sistema Linguístico de Anotação Gestual (LASG) (BRESSEM; LADEWIG; MÜLLER, 2013). No que se refere à marcação de Espaços Mentais pelos gestos, utilizamos os parâmetros de Cassell e McNeill (1990) que relacionam os gestos com o segmento da narração em que determinada interação se aloca, seja ele parte de uma interação no aqui-agora (Ground) ou parte de uma interação no mundo da narrativa (Espaço Narrativo). No que diz respeito à marcação de Espaços Mentais pela direção do olhar, utilizamos como parâmetros de análise, os pressupostos de Sweetser e Stec (2016), que afirmam que o olhar de um narrador pode representar tanto o olhar de uma personagem (no Espaço Narrativo) como o olhar do próprio narrador na interação (no Ground). Somente nos casos em que os gestos marcaram o Espaço Narrativo, analisamos com base em McNeill (1995), ainda no Capítulo 5, se eles se configuram como gestos do ponto de vista do observador (O-VPT) ou como gestos do ponto de vista da personagem (C-VPT). Ainda nesses casos, analisamos os gestos quanto à sua função dominante no interior da narrativa: representação, expressão ou apelo, conforme Müller (2013), a partir dos trabalhos de Bühler (1982) sobre a teoria psicológica da linguagem e a teoria da expressão. Somente nos casos em que a direção do olhar marcou o Espaço Narrativo, analisamos, também, a sua função no interior da narrativa, de acordo com os pressupostos de Sweetser e Stec (2016): i) encenação da personagem e ii) narração propriamente dita. 19 2 I was really tired last night, but I’m better now so do tell me what you think about my book. (Tradução do autor. Todas as traduções cujos trechos originais estão em rodapé são de autoria do autor desta dissertação). y g , y y (Tradução do autor. Todas as traduções cujos trechos originais estão em rodapé são de autoria do autor desta dissertação). 2 I was really tired last night, but I’m better now so do tell me what you think about my book. 2.1 Espaços Mentais e Narrativa Segundo Dancygier (2011), Fauconnier (1994) definiu os espaços mentais como pacotes conceptuais estabelecidos primariamente por expressões linguísticas – que podem, também, ser visuais e gestuais – e constituem um modelo mental de como o sentido é processado cognitivamente. Em outro texto, Dancygier (2012) afirma que um Espaço Mental pode ser ativado por meio da recordação de um evento passado, retomado no curso da conversação que ocorre na situação atual. Para exemplificar, Dancygier (2011, p. 35) explica que, se um falante diz: “eu estava muito cansado noite passada, mas eu estou melhor agora, então me diz o que achou do meu livro”2, o falante evoca um espaço passado na primeira sentença, descreve seu estado atual na sentença seguinte e evoca, também, o espaço do conteúdo do livro, do conteúdo das opiniões do ouvinte em relação ao livro e do turno de fala esperado do ouvinte. Todos esses Espaços Mentais são ativados ao longo dessa parte específica da conversação. Nesse exemplo de Dancygier (2011), fica evidente que os Espaços Mentais são atribuíveis a vários conceptualizadores, e que incluem representações mentais de objetos y g , y y (Tradução do autor. Todas as traduções cujos trechos originais estão em rodapé são de autoria do autor desta dissertação). 20 concretos, movimentos conversacionais, opiniões etc. Os trabalhos em Espaços Mentais têm mostrado, conforme explica a autora, que as redes de Espaços Mentais podem representar a correlação online entre escolhas de expressão e o sentido resultante, no entanto, é preciso sinalizar que os Espaços Mentais não são um fenômeno linguístico, mas conceptuais. Além disso, a Teoria dos Espaços Mentais é uma ferramenta fundamental na identificação dos detalhes de várias formas da interação e na descoberta da correlação entre ações visíveis e sentido, fato que tem sido revelado nos resultados em estudos recentes em gesto. Acerca da concepção de Espaços Mentais no contexto narrativo, Sweetser e Stec (2016) afirmam que, na narrativa oral, ocorre o processo de compartilhamento de atenção entre os interactantes, não só por meio dos recursos linguísticos (modalidade verbal), mas também por meio do espaço gestual, dos movimentos e ações corporificadas (modalidade gestual). Sendo assim, o que é significativo para esse tipo de análise são os pontos de vista dos personagens, situados em um Espaço Narrativo (Story Space) e a interação no mundo real, situada no Espaço- base (Base Space). 3 Escolhemos essa narrativa apenas como uma ilustração para os preceitos teóricos. A narrativa de análise consta no capítulo 4. 2.1 Espaços Mentais e Narrativa Aí eu falei (2) “não, gente, pro México, acabei de sair de uma crise de asma, eu não sei se eu sou capaz... Preciso ficar aqui, eu quero fazer teatro” E tal... (3) Louca! Fonte: Dados do pesquisador 2.1 Espaços Mentais e Narrativa Fauconnier (2003), por sua vez, propõe que, à medida que o discurso se desenvolve, espaços mentais são criados para alocar informações que superam o contexto imediato. Sendo assim, os eventos passados sobre os quais se narra (ou se reencena) estão alocados no Espaço Narrativo, enquanto a interação que ocorre na situação comunicativa imediata está alocada no Espaço-base. Em relação à interação nas narrações, de forma mais específica, é esperado que o falante segmente a narração em componentes menores para falar de eventos passados e para constituir reencenações virtuais desses eventos. Esse fenômeno é crucial para que os ouvintes da narração compreendam apropriadamente o que está sendo dito, uma vez que é necessário determinar se a fala e os gestos que coocorrem com a fala estão ancorados no contexto imediato da narração (no Espaço-base) ou, alternativamente, nos eventos sobre os quais se está falando (no Espaço Narrativo). Para ilustrar essa alocação de informações no Espaço-Base e no espaço narrativo, selecionamos a narrativa da atriz Letícia Spiller3, cujas captura e transcrição podem ser observadas na Figura 1 e no Quadro 1, respectivamente, na qual ela reencena as personagens de eventos passados (seu Past Self, a apresentadora Xuxa e sua empresária da época, Marlene Mattos): 3 Escolhemos essa narrativa apenas como uma ilustração para os preceitos teóricos. A narrativa de análise consta no capítulo 4. 21 Figura 1: Narrativa de Letícia Spiller Fonte: Canal do GNT no YouTube Quadro 1: Trecho da Transcrição da narrativa de Letícia Spiller Fonte: Canal do GNT no YouTube Fonte: Canal do GNT no YouTube Quadro 1: Trecho da Transcrição da narrativa de Letícia Spiller Trecho da Narrativa de Letícia Spiller A: Letícia: Exatamente! E eu tinha paixão por teatro, né... Eu queria ser atriz, eu entrei nesse trabalho porque eu queria me virar pra pagar meus cursos sozinha (1) sem depender da minha mãe e do meu pai e aí, é... Eu fui trabalhar, eu tinha asma, né, bronquite alérgica desde criança. E nessa época ainda tinha algumas crises. E aí, eu fazia tudo pra ficar um tempo em casa, ou ficar fazendo teatro com a galera e eu nunca ia poder fazer a peça junto com eles, porque eu ia pro México junto com a Xuxa, e aí eu tava com bronquite... Fábio: Todo o Brasil queria ir pro México com a Xuxa e ela queria ficar aqui fazendo teatro... Letícia: Exatamente! Trecho da Narrativa de Letícia Spiller A: Letícia: Exatamente! E eu tinha paixão por teatro, né... Eu queria ser atriz, eu entrei nesse trabalho porque eu queria me virar pra pagar meus cursos sozinha (1) sem depender da minha mãe e do meu pai e aí, é... Eu fui trabalhar, eu tinha asma, né, bronquite alérgica desde criança. E nessa época ainda tinha algumas crises. E aí, eu fazia tudo pra ficar um tempo em casa, ou ficar fazendo teatro com a galera e eu nunca ia poder fazer a peça junto com eles, porque eu ia pro México junto com a Xuxa, e aí eu tava com bronquite... Fábio: Todo o Brasil queria ir pro México com a Xuxa e ela queria ficar aqui fazendo teatro... Letícia: Exatamente! Aí eu falei (2) “não, gente, pro México, acabei de sair de uma crise de asma, eu não sei se eu sou capaz... Preciso ficar aqui, eu quero fazer teatro” E tal (3) Louca! Fonte: Dados do pesquisador Conforme é possível observar no diagrama exposto na Figura 2, a seguir, a representação se refere à narrativa na qual Letícia Spiller (no diagrama, representada por x) conta como, quando fingia que havia sido acometida por uma crise de asma para não comparecer a uma gravação em sua época de paquita, recebeu a visita de Xuxa e de Marlene Mattos, suas chefes na época. Elaboramos, com base na representação de Fauconnier (2003, p. 36), o seguinte diagrama: 22 Figura 2: Representação dos Espaços Mentais – Narrativa Letícia Spiller x2 x3 x x1 a R N Fonte: Elaborado pelo Pesquisador. Adaptado de Fauconnier (2003, p. 36) x a R Fonte: Elaborado pelo Pesquisador. Adaptado de Fauconnier (2003, p. 36) Letícia Spiller reencena, no Espaço Narrativo (representado por N) as seguintes personagens: ela mesma no passado (Past Self) (representada por x¹), Xuxa e Marlene Mattos (representadas, respectivamente, por x² e x³). A linha reta representa a função da reencenação de x¹, x² e x³ por x, enquanto a linha pontilhada representa a interação com seus interlocutores (representados por a), no Espaço-base (representado por R)4. Dancygier (2011), por sua vez, afirma que as histórias podem, naturalmente, ser tratadas como construções linguísticas, isto é, elas se constituem como um exemplo do discurso natural, e, ao mesmo tempo em que é possível identificar escolhas linguísticas na narrativa, é possível, também, observar fatos básicos da estrutura da linguagem. 4 Em um primeiro momento, Fauconnier (2003) chamou o espaço Base de espaço de Realidade (R), e posteriormente, mudou para Base (B), por considerar que mesmo o chamado espaço de realidade é sempre uma construção cognitiva, e não a realidade em si. Trecho da Narrativa de Letícia Spiller Assim, conforme argumenta a autora, é preciso abordar as histórias em termos de sentido, o que é, segundo ela, muito desafiador, já que existem muitos recursos narrativos possíveis. A autora afirma, também, que as narrativas são constituídas por uma grande variação de fenômenos linguísticos, que são adaptados a fim cumprir as necessidades linguísticas do evento narrativo. Para exemplificar esse caso em específico, Dancygier (2011) aponta o uso do pronome “eu”, que pode ser usado para marcar a posição de narrador, o que constitui um constructo narrativo. Assim, esses usos narrativos criam tratamentos referenciais de acordo com as necessidades narrativas, considerando que o ato comunicativo aconteceu em um determinado “aqui-agora”. Considerando os múltiplos pontos de vista que podem ser marcados nas narrativas multimodais, partiremos, então, para as definições de “ponto de vista” e “perspectiva”. 4 Em um primeiro momento, Fauconnier (2003) chamou o espaço Base de espaço de Realidade (R), e posteriormente, mudou para Base (B), por considerar que mesmo o chamado espaço de realidade é sempre uma construção cognitiva, e não a realidade em si. 23 5 (i) the lamp on the table (ii) the table with the lamp on it 5 (i) the lamp on the table ( ) p (ii) the table with the lamp on it 2.2 Ponto de vista e perspectiva A capacidade de perspectivação conceptual tem sido explorada por dois autores considerados como fundadores da Linguística Cognitiva: Langacker (1987) e Talmy (2000). Esses autores focalizam as questões relativas à perspectiva a partir da noção de gramática cognitiva, o que significa reinterpretar as categorias gramaticais a partir do seu significado. O primeiro autor desenvolveu um modelo cognitivo ao qual ele denomina como Gramática Cognitiva que, por sua vez, demonstra como todo o significado parte de uma conceptualização. Talmy (2000), pensando em uma Semântica Cognitiva, focaliza nos tipos específicos de perspectivação conceptual. Ambos os autores têm identificado operações de perspectivação conceptual que são comuns à percepção visual e, também, à linguagem. De acordo com Langacker (1987), as entidades linguísticas são sistematizadas em um alto nível de organização cognitiva. Nesse sentido, o autor afirma que as expressões linguísticas dizem respeito a situações ou cenas concebidas. No entanto, o sentido de uma expressão não é suficientemente caracterizado apenas pela identificação ou descrição de uma situação em questão. Assim, nas palavras do autor, é possível, portanto, formar diferentes construções a partir de expressões relativas à mesma cena, por exemplo: (i) a lâmpada em cima da mesa e (ii) a mesa com a lâmpada em cima5. Esses exemplos ilustram o fato de que as expressões linguísticas escolhidas podem diferir no sentido de acordo com as entidades as quais elas designam, ou seja, as construções (i) e (ii) designam, respectivamente, a lâmpada, a mesa e a relação locativa entre elas. Um dos principais objetivos de Langacker (1987) diz respeito a essa designação, porém, em um sentido mais amplo. O autor se preocupa com um fenômeno no qual a designações variáveis constituem apenas um caso especial: a habilidade que os falantes possuem em construir a mesma situação básica de diferentes maneiras, a partir de imagens alternativas, por exemplo. O contraste de imagens que podem ser impostas em uma cena, de acordo com o autor, equivalem, qualitativamente, a experiências mentais diferentes e, assim, consequentemente, a imagem corporificada por uma expressão linguística constitui um aspecto crucial do seu sentido. Desse modo, no que diz respeito às imagens empregadas para estruturar situações concebidas podem variar de acordo com determinados parâmetros. Essa variação recebe o nome 24 de Ajustes Focais (focal adjustments) (LANGACKER, 1987, p. 117) e é dividida pelo autor em três categorias principais: seleção, perspectiva e abstração. Conforme define Langacker (1987, p. 2.2 Ponto de vista e perspectiva 117), os ajustes focais de seleção determinam quais particularidades de uma cena são abordadas, enquanto a perspectiva corresponde à posição a partir da qual uma cena é observada e, por fim, a abstração pertence ao nível de especificidade com o qual a situação é retratada6. No que diz respeito à seleção, Langacker (1987) afirma que as predicações são feitas em relação a domínios cognitivos específicos. Para ilustrar o fenômeno da seleção, o autor considera a expressão (iii) o grande copo de plástico azul7 (LANGACKER, 1987, p. 117) e explica que uma caracterização semântica completa de “copo” requer inúmeras especificações – como por exemplo, o formato, o material, a função, o tamanho etc. – que invocam uma variedade de domínios cognitivos. No exemplo de Langacker (1987), cada modificador seleciona um domínio em particular em relação ao que especifica mais precisamente as propriedades da entidade designada, ou seja: no que diz respeito ao material – “de plástico”; no que diz respeito à cor – “azul”; no que diz respeito à uma escala de comparação para a extensão espacial – “grande” e, finalmente, no que diz respeito à identificação dos atos de fala dos falantes – o artigo “o”. O autor explica, portanto, que uma indicação dos domínios que um predicado seleciona é um aspecto fundamental de sua descrição. Ainda no que se se refere à seleção, uma entidade designada pela predicação – o que Langacker (1987) denomina como “perfil” – é maximamente proeminente e pode ser conceptualizado como um tipo de ponto focal. Assim, o escopo da predicação – o que o autor denomina como “base” – é descritível como o contexto necessário para a caracterização do “perfil”. Nesse sentido, o autor utiliza como exemplo o predicado “tio” que é definido a partir de um domínio abstrato que consiste na concepção de uma rede de relações de parentesco. A partir do indivíduo, essa rede se estende arbitrariamente em qualquer direção na caracterização de “tio”, ou seja, os parentes relacionados e uma pessoa designada constituem o escopo da predicação e fornece o contexto com o qual a entidade perfilada é identificada e caracterizada. Langacker (1987) sistematiza a estrutura gramatical e semântica da perspectiva levada em consideração em uma cena a partir das noções de alinhamento de Figura/fundo, ponto de vista e dêixis. O autor afirma que a organização de Figura/fundo8 é um aspecto fundamental da cognição. 6 Focal adjusments of selection determine which facets of a scene are being dealt with. Perspective relates to the position from which a scene is viewed […]. Finally, abstraction pertains to the level of specificity at which a situation is portrayed. 7 8 Esse conceito é proveniente da Psicologia Gestalt 7 the big blue plastic cup 8 7 the big blue plastic cup 8 Esse conceito é proveniente da Psicologia Gestalt 2.2 Ponto de vista e perspectiva A partir dos pressupostos teóricos da gramática cognitiva, Langacker (1987) afirma 6 Focal adjusments of selection determine which facets of a scene are being dealt with. Perspective relates to the position from which a scene is viewed […]. Finally, abstraction pertains to the level of specificity at which a situation is portrayed. 7 th bi bl l ti 25 que a prevalência da organização Figura/fundo nas estruturas conceptuais são fundamentais na estrutura semântica e gramatical. Ainda, a organização de Figura/fundo não é, em geral, determinada de maneira automática por uma dada cena porque é possível estruturar a mesma cena com opções alternativas de Figura, porém, vários fatores contribuem para a naturalidade e a verossimilhança de uma cena em particular. Conforme explica Langacker (1987, p. 120): “uma região relativamente compacta que contrasta agudamente com os seus arredores demonstra uma forte tendência em ser selecionada como a Figura” (LANGACKER, 1987, p. 120).9 A fim de exemplificar, o autor argumenta que, considerando um ponto branco inserido em um fundo preto, o ponto branco é quase sempre selecionado como a Figura. A segunda noção que sistematiza a perspectiva é a de ponto de vista. Langacker (1987) afirma que a importância do ponto de vista é evidente para que se compreenda as concepções fundamentadas na experiência visual. O autor elucida que é possível olhar para um objeto físico a partir de diferentes ângulos (frente/trás, abaixo/acima etc.) e o que se observa varia de acordo com esses ângulos. Sendo assim, ao observar uma cena complexa com múltiplos participantes, é possível assumir diferentes posições em relação à cena, o que modifica a proximidade com a saliência percebida. O autor inclui, em sua definição de ponto de vista, duas noções mais específicas: ponto de vantagem e orientação. Um ponto de vantagem, segundo Langacker (1987), é a posição a partir da qual uma cena é observada. Ou seja, se levarmos em consideração uma pessoa que caminha na calçada, olhando para uma casa em específico, o ponto de vantagem em relação à casa, gradualmente, muda (LANGACKER, 1987, p. 123)10. A partir de um ponto de vantagem, diferentes orientações são possíveis para a cena observada. Já no que diz respeito à orientação, Langacker (1987) afirma que ela corresponde ao alinhamento em relação aos eixos do campo visual. O autor conclui que as predicações linguísticas relacionadas à orientação podem se basear ou não na orientação real de uma cena no campo visual. 9 A relatively compact region that contrats sharply with its sorroundings shows a strong tendency to be selected as the figure. g 10 As I walk along the sidewalk looking at a house my vantage point with respect to the house gradually shifts. 2.2 Ponto de vista e perspectiva A fim de exemplificar, o autor considera as duas possibilidades de descrever uma cena na qual uma pipa está voando acima de uma casa: (i) A pipa está acima da casa e (ii) A casa está abaixo da pipa (LANGACKER, 1987, p 123). Langacker (1987) afirma que a primeira sentença, em (i), pressupõe a matriz dimensional convencionada que se calcula em relação à superfície da terra, o que reflete a orientação do campo de visão quando se assume a 9 A relatively compact region that contrats sharply with its sorroundings shows a strong tendency to be selected as the figure. 10 As I walk along the sidewalk looking at a house my vantage point with respect to the house selected as the figure. 10 As I walk along the sidewalk looking at a house my vantage point with respect to the house gradually shifts. 26 posição de visão canônica. Já a segunda sentença, segundo o autor, ignora o sistema de coordenação convencional. Ainda no que se refere à perspectiva, a terceira noção abordada por Langacker (1987) é a de dêixis. Uma expressão dêitica, de acordo com o autor, pode ser definida como aquela que inclui referências a um elemento do Ground dentro de um escopo de predicação, considerando que o termo Ground indica o evento de fala, seus participantes e suas configurações. Dos vários tipos de expressões dêiticas elencadas por Langacker (1987), focalizaremos aquelas que, especificamente, designam um elemento do Ground. Langacker (1987) parte dos pronomes “eu” e “você”, que designam os participantes do evento de fala e, em seguida, cita os termos “aqui” e “agora”, que se referem ao espaço e o tempo da fala. Os pronomes da terceira pessoa e advérbios como “lá”, são expressões dêiticas definidas negativamente. Por exemplo: os pronomes “ele” e “ela” são caracterizados como pessoas que não fazem parte do evento de fala. Todas essas expressões dêiticas, conforme explica o autor, envolvem a concepção de um elemento do Ground como a entidade designada pela expressão em questão. Para finalizar, Langacker (1987) afirma que a abstração é o tipo final de Ajuste Focal a se considerar. O termo “abstrato” e seus derivados, de acordo com o autor, podem significar várias coisas diferentes: em um uso da palavra, a abstração é equivalente ao que o autor denomina como “seleção” e envolve a omissão da consideração de certos domínios ou propriedades. 2.2 Ponto de vista e perspectiva Em outro uso, é possível categorizar uma entidade como abstrata de acordo com o modo como ela se afasta da realidade física imediata, ou seja, algo pode ser abstrato porque é imaginário (como a palavra “unicórnio”, por exemplo); porque ocupa um domínio não-físico (o número “sete”, por exemplo); ou porque não é diretamente revelado pela experiência sensória imediata (como os “átomos”, por exemplo). O foco do autor é, no entanto, a abstração de um esquema relativo às suas instanciações. Langacker (1987) afirma que a noção de esquematicidade diz respeito ao nível de especificidade de detalhes com os quais, por exemplo, algo é caracterizado. A fim de exemplificar, o autor considera a seguinte situação: se alguém afirma que uma pessoa é alta, essa caracterização é esquemática e abstrata em relação a uma especificação mais precisa que compreende que essa pessoa possui mais de um metro e noventa. Embora a esquematicidade e seleção não digam respeito ao mesmo fenômeno, Langacker (1987) afirma que a distinção não é absoluta, uma vez que elaborar um esquema, geralmente, introduz, um domínio que não seria esperado sem a elaboração em um nível mais esquemático. Por exemplo: segundo o autor, o conceito de “polígono” introduz a noção de vários lados e cria um potencial para a especificação do seu número de lados, a fim de designar um quadrado, um pentágono, 27 hexágono etc., mas os conceitos mais esquemáticos como “coisa” e “figura geométrica” são muito abstratos para serem designados por esse parâmetro. 2.2 Ponto de vista e perspectiva A fim de sintetizar o quadro teórico oferecido por Langacker (1987), elaboramos o esquema presente no Quadro 2, a seguir: Quadro 2: Conceitos básicos da Gramática Cognitiva (Langacker, 1987) Ajustes Focais Seleção Ajustes focais de seleção que determina o aspecto da entidade sobre o qual se trata § Domínio conceptual: conhecimento inserido em sistema baseado na experiência § Perfilamento: organização de perfil/base conceptualizada pelo enunciado linguístico Perspectiva Posição com a qual um evento é observado que afeta na proeminência relativa dos participantes § Ponto de vista: perspectiva e orientação de uma cena que afeta como ela é construída Abstração Grau de especificidade com o qual um evento é retratado § Esquematicidade Fonte: Elaborado com base em Langacker (1987) Quadro 2: Conceitos básicos da Gramática Cognitiva (Langacker, 1987) Ajustes Focais Seleção Ajustes focais de seleção que determina o aspecto da entidade sobre o qual se trata § Domínio conceptual: conhecimento inserido em sistema baseado na experiência § Perfilamento: organização de perfil/base conceptualizada pelo enunciado linguístico Perspectiva Posição com a qual um evento é observado que afeta na proeminência relativa dos participantes § Ponto de vista: perspectiva e orientação de uma cena que afeta como ela é construída Abstração Grau de especificidade com o qual um evento é retratado § Esquematicidade Fonte: Elaborado com base em Langacker (1987) Quadro 2: Conceitos básicos da Gramática Cognitiva (Langacker, 1987) Ajustes focais de seleção que determina o aspecto da entidade sobre o qual se trata Ajustes focais de seleção que determina o aspecto da entidade sobre o qual se trata § Domínio conceptual: conhecimento inserido em sistema baseado na experiência § Perfilamento: organização de perfil/base conceptualizada pelo enunciado linguístico Ajustes Focais Posição com a qual um evento é observado que afeta na proeminência relativa dos participantes Fonte: Elaborado com base em Langacker (1987) Partindo, agora, para o quadro teórico oferecido por Talmy (2000), trataremos dos fenômenos com os quais as línguas podem posicionar uma parte de uma situação referente no primeiro plano de atenção a partir da menção explícita dessa parte, enquanto posiciona o restante da cena no fundo da atenção, a partir da omissão da menção. Com o objetivo de explicar a complexidade da cognição, Talmy (2000) lança um olhar para a estrutura conceptual da 28 mente. Assim, ele sugere que essa estrutura é construída por dois sistemas: o sistema conceptual e o sistema de estruturação conceptual. 11 distal, medial, or proximal 12 Windowing of attention , , p 12 Windowing of attention 11 distal, medial, or proximal 12 2.2 Ponto de vista e perspectiva Esse sistema de estruturação conceptual compreende, ainda, vários sistemas esquemáticos, tais como: i) o sistema configuracional, ii) sistema de perspectiva, iii) sistema de atenção e iv) sistema de dinâmica de forças. Neste trabalho, abordaremos os sistemas de perspectiva e de atenção. No que diz respeito ao sistema de perspectiva, Talmy (2000) explica que esse sistema esquemático consiste na perspectiva que um indivíduo pode ter em relação a uma entidade. Ainda, o autor afirma que, enquanto o sistema esquemático é presumidamente neutro às modalidades sensórias, o sistema de perspectiva é imediatamente caracterizado em termos visuais. Talmy (2000) divide o sistema de perspectiva em categorias esquemáticas, tais como: a localização perspectiva, a distância perspectiva, o modo perspectivo e o direcionamento da visão. Em relação à localização perspectiva, Talmy (2000) explica que as formas gramaticais, assim como as formas lexicais, podem especificar a localização que um ponto de perspectiva ocupa em uma cena referida. O autor argumenta que a dêixis, em sua forma básica, define a posição de um ponto de perspectiva na localização atual do falante. Isso é ilustrado, por exemplo, na escolha dos verbos “ir” ou “vir”, em um determinado contexto enunciativo. Em relação à distância perspectiva, Talmy (2000) afirma que ela é a segunda categoria esquemática que pode ser especificada por formas de classe fechada. A maioria das noções dessa categoria dizem respeito ao ponto de perspectiva e podem ser distais, mediais ou proximais em relação à sua distância de uma entidade em questão11. Já a terceira categoria esquemática pertencente ao ponto de perspectiva que é denominado por Talmy (2000) como modo de perspectiva e se refere à possibilidade de um ponto de perspectiva estar ou estacionado ou se movendo. Por fim, a categoria esquemática denominada como direcionamento da visão é baseada na possibilidade conceptual de se observar algo em uma direção particular a partir de um dado ponto de perspectiva. No que diz respeito ao sistema de atenção, Talmy aborda o que ele denomina como “janela de atenção”12. Talmy (2000) afirma que as formas linguísticas podem direcionar a distribuição de atenção de uma pessoa em uma cena, a partir de um certo tipo de padrão, a partir do posicionamento de uma ou mais janelas de atenção ao longo da cena, o que justifica a terminologia. 2.2 Ponto de vista e perspectiva Apesar do fato de apenas uma certa parte de uma referida cena ser explicitamente 29 especificada quando está em janela, é compreendido como parte da natureza desse processo que, dado um contexto apropriado, o destinatário da referida cena seria capaz de fazer inferências a respeito do que foi omitido. Talmy (2000) afirma que, geralmente, a mesma cena referida pode ser colocada em janela de diversas maneiras, ou seja, diferentes padrões de janelas selecionadas podem ser posicionados na cena. Segundo o autor, esse fenômeno constitui uma manifestação das propriedades linguísticas fundamentais da alternatividade conceptual que, para Talmy (2000), é um princípio que pertence aos sistemas de estruturação conceptual de uma língua, ou seja, um falante pode selecionar uma ou outra conceptualização como aquela que representa o complexo ideacional que o falante deseja comunicar. Levando em consideração as proposições de Langacker (1987) e de Talmy (2000), é possível observar que as operações de perspectivação conceptual são centrais na cognição e na linguagem. As classificações dos autores mencionados se fundamentam em capacidades cognitivas gerais e direcionam a atenção para diferentes aspectos da experiência humana como um fator de variação das operações de perspectivação conceptual. 2.3 Ponto de Vista e Narrativa: inter-relação entre fala e gestos Para a Linguística Cognitiva, o ponto de vista é bem mais pervasivo na cognição e linguagem humanas do que se reconhece, de modo que se concebe, ainda que nenhum produto da percepção, cognição e expressão linguísticas é independente da expressão de ponto de vista, uma vez que este constitui-se como um mecanismo importante e crucial da comunicação humana. Em outras palavras, em conformidade com as proposições de Sweetser (2012), a cognição e a comunicação humanas são permeadas pelo ponto de vista, já que nunca experienciamos o mundo sem a conceptualização baseada na experiência humana. A Linguística Cognitiva parte do pressuposto de que a semântica é, primariamente, cognitiva e não proveniente da relação direta entre a língua e o mundo. Esse princípio, como aponta Verhagen (2010), é manifestado nas pesquisas que tratam do sentido e da organização gramatical e que usam as noções de perspectiva e ponto de vista. Um grande incentivo para que esse tipo de pesquisa ocorra parte da consciência de que quanto mais os problemas linguísticos podem ser resolvidos por meio dessas noções, mais exitoso heuristicamente, nas palavras do autor, será o princípio fundamental. É nesse sentido que se destaca, dentro do âmbito da Linguística Cognitiva, a necessidade da investigação empírica. O termo que tem sido usado nas pesquisas da área para denominar as diferentes maneiras de se olhar para uma situação em particular é “construal”. 30 De acordo com Langacker (2008), no nível mais básico, o construal diz respeito às características do sentido das expressões linguísticas, como consequência do fato de que as línguas oferecem diversas maneiras de categorizar situações, seus participantes, suas características e as relações que se estabelecem entre eles. Nesse sentido, quando se leva em consideração essas questões, o autor explica como o conceito de construal pode ser aplicado: “um falante que observa, precisamente, a distribuição espacial de certas estrelas pode descrevê- las de vários modos: constelação, conjunto de estrelas, manchas de luz no céu”13. O autor explica que essas expressões são semanticamente distintas, pois demonstram construals alternativos da cena do falante, sendo que cada uma delas é compatível com as propriedades dadas. Retomando às proposições de Verhagen (2010), ele explica, ainda, que o mais importante, do ponto de vista linguístico, é o fato de que a língua disponibiliza meios para instanciar diferentes tipos de construal. 14 Conforme explica Ferrari (2016), o “termo ‘frame’ designa um sistema estruturado de conhecimento, armazenado na memória de longo prazo e organizado a partir da esquematização da experiência” (FERRARI, 2016, p. 50). distinct fashions: as a constellation, as a cluster of stars, as specks of light in the sky. 14 Conforme explica Ferrari (2016), o “termo ‘frame’ designa um sistema estruturado de conhecimento, armazenado na memória de longo prazo e organizado a partir da esquematização da experiência” (FERRARI, 2016, p. 50). 15 D d F i (2016) “L k ff (1987) d l it d M d l C iti ( p ) 15 De acordo com Ferrai (2016), “Lakoff (1987) desenvolveu o conceito de Modelo Cognitivo Idealizado (MCI), definindo-o como um conjunto complexo de frames distintos” (FERRARI, 2016, p. 51). ( , , p ) 15 De acordo com Ferrai (2016), “Lakoff (1987) desenvolveu o conceito de Modelo Cognitivo 13 A speaker who accurately observes the spatial distribution of certain stars can describe them in many distinct fashions: as a constellation, as a cluster of stars, as specks of light in the sky. 14 13 A speaker who accurately observes the spatial distribution of certain stars can describe them in many distinct fashions: as a constellation as a cluster of stars as specks of light in the sky De acordo com Ferrai (2016), Lakoff (1987) desenvolveu o conceito de Modelo Cognitivo Idealizado (MCI), definindo-o como um conjunto complexo de frames distintos” (FERRARI, 2016, p. 51). 2.3 Ponto de Vista e Narrativa: inter-relação entre fala e gestos Conforme observamos no exemplo de Langacker (2008), as descrições distintas de um mesmo fenômeno diferem em relação aos frames de conhecimento (frames of knowledge) no que diz respeito ao modo como a situação dada é caracterizada: uma distribuição particular de estrelas só é considerada como uma constelação em um frame de conhecimento, compartilhado culturalmente, a respeito da estrutura do céu. Sendo assim, ainda de acordo com o autor, um tipo de construal envolvido nesses exemplos envolvem, crucialmente, os frames14 de conhecimento ou os Modelos Cognitivos Idealizados15. Já em relação ao ponto de vista, ele seria, por si só, universal, segundo Sweetser (2012). Apesar de existirem diversas maneiras de categorizar e representar linguisticamente os pontos de vista, a experiência humana – e também a anatomia humana, já que, por exemplo, os seres humanos não são capazes de ver em trezentos e sessenta graus – limitam as maneiras pelas quais acessamos construals relativos a perspectivas. Ainda de acordo com a autora, as estruturas linguísticas das quais nos valemos na interação são moldadas pelos pontos de vista de uma descrição pré-experiencial do mundo, o que demonstra que uma única mente pode acessar múltiplos e diferentes pontos de vista em uma mesma cena. Sem essa flexibilidade cognitiva, conforme afirma a autora, os humanos não poderiam cooperar e se comunicar do modo como 13 A speaker who accurately observes the spatial distribution of certain stars can describe them in many distinct fashions: as a constellation, as a cluster of stars, as specks of light in the sky. 31 o fazemos. Por essa razão, o ponto de vista é um fenômeno de interesse especial para quase todos os estudiosos da cognição e comunicação. Ainda no que diz respeito aos múltiplos pontos de vista, Sweetser (2012) afirma que nós não somente somos capazes de manter múltiplos pontos de vista, como somos incapazes de manter um único ponto de vista de espaço ou de estrutura cognitiva quando outros humanos estão presentes. Sendo assim, uma situação que envolva múltiplos participantes é, necessariamente, estruturada por complexos e múltiplos pontos de vista. Nesse sentido, conforme Parrill (2012), quando se trata dos estudos em ponto de vista, é preciso considerar o papel deles na linguagem multimodal, levando em consideração, especificamente, as modalidades verbal e gestual. 2.3 Ponto de Vista e Narrativa: inter-relação entre fala e gestos De acordo com Parrill (2012), definir o termo “ponto de vista” apenas como a perspectiva de um conceptualizador sobre um evento ou uma cena, tal como ocorre na literatura clássica sobre o assunto, pode não ser suficiente em termos cognitivos, quando não se define o que é perspectiva. Ainda de acordo com a autora, muitos pesquisadores definem perspectiva como ponto de vista, de modo que ambos os termos sejam usados de forma indistinta, o que nos leva à importância de definir o constructo do ponto de vista. Parrill (2012) afirma que ele captura o fato de que os humanos representam eventos a partir da consciência de algum experienciador. Sendo assim, os humanos são capazes de projetar, por meio dos pontos de vista, suas perspectivas visuais, pensamentos e sentimentos. Dessa maneira, o fato de o ponto de vista tratar da perspectiva física de uma entidade, um evento ou uma cena faz com que a maioria dos pesquisadores cheguem ao consenso de que o ponto de vista, em sua forma básica, é espacial. A fim de compreender como o ponto de vista pode ser identificado no comportamento comunicativo, levando em consideração uma descrição mais sistemática, Parrill (2012) divide o fenômeno em três dimensões: o ponto de vista conceptual, que diz respeito à localização física real ou imaginada por um conceptualizador; o ponto de vista linguístico, que trata das manifestações linguísticas dessa localização; e o ponto de vista gestual, que se refere aos sinais espaciais dessa localização visíveis nos gestos. O ponto de vista conceptual, segundo Parrill (2012), pode se referir à representação mental baseada na percepção visual de uma localização física real ou imaginada. Sendo assim, as simulações de eventos podem variar de acordo com o ponto de vista: um indivíduo pode simular um evento a partir do ponto de vista de um ator na cena (ponto de vista da personagem) ou a partir de um ponto de vista mais distanciado (ponto de vista do observador). Além disso, o ponto de vista conceptual pode ser entendido como os modos pelos quais um usuário da língua 32 simula um evento, considerando que as simulações podem incluir imagens mentais, programas motores e representações de estados mentais, tanto do próprio conceptualizador como os de outras personagens da narração. Já o ponto de vista linguístico é marcado pelo que a autora nomeia como “instâncias linguísticas”. 2.3 Ponto de Vista e Narrativa: inter-relação entre fala e gestos Em termos teóricos, o ponto de vista linguístico reflete, parcialmente ou esquematicamente, o ponto de vista conceptual. De acordo com Parrill (2012), muito já foi escrito sobre as instâncias linguísticas que marcam o ponto de vista em textos escritos. Em geral, esses estudos sugerem que dispositivos linguísticos específicos funcionam como instruções que devem ser seguidas na simulação de um evento durante a compreensão ou produção da narração. A autora cita como exemplos as seguintes instâncias: i) a escolha de pronome: de acordo com ela, o uso de pronomes da segunda pessoa tendem a induzir leitores e ouvintes a adotarem um ponto de vista interno na narrativa, enquanto pronomes da terceira pessoa tendem a induzir leitores e ouvintes a adotarem um ponto de vista externo; ii) a frequência de menção a um referente no discurso em particular, que demonstra o locus do ponto de vista; iii) o uso dos modais, dos contrafactuais, dos pronomes definidos, dos verbos recíprocos e reflexivos etc. No que se refere ao ponto de vista gestual, Parrill (2012) afirma que, apesar de igual prevalência na interação, pouca atenção foi dedicada à sua emergência. Segundo a autora (2000), o ponto de vista gestual é de igual importância na compreensão do ponto de vista conceptual, pois ele sugere que o conceptualizador interage de acordo com a perspectiva de dentro ou de fora da cena: de acordo com a definição de McNeill (1995), os gestos nos quais o falante usa suas mãos como se ele fosse uma personagem da narrativa são categorizados como “gestos do ponto de vista da personagem” (por exemplo, simular o movimento dos braços durante uma corrida enquanto descreve um personagem correndo), ao passo que os gestos nos quais o falante traça a trajetória de uma personagem ou indica a sua localização, como se o falante estivesse observando a personagem a uma certa distância, são categorizados como “gestos do ponto de vista do observador”, conforme explicaremos a seguir. De acordo com McNeill (1995), o ponto de vista é onipresente nos gestos. Isso se deve ao fato de que o corpo do falante funciona como um centro dêitico, de modo que muitos gestos codificam o ponto de vista mesmo que eles não sejam uma parte ativa da conceptualização do falante. 2.3 Ponto de Vista e Narrativa: inter-relação entre fala e gestos Conforme explica Parrill (2009), um falante que produz um gesto para fora do seu corpo enquanto descreve a trajetória de pegar um objeto, pode nunca ter mencionado o ponto de origem do objeto que, por sua vez, está implícito no gesto realizado. Além disso, a autora afirma que, no que diz respeito ao uso dos gestos relacionados ao centro do corpo como origem, existe 33 uma alternância entre uma representação de terceira pessoa, mais externa em relação ao ponto de vista da narrativa; e uma representação de primeira pessoa, mais interna em relação ao ponto de vista da narrativa. Segundo Parrill (2009), essa alternância, que tem sido estudada por diversos pesquisadores, como Bryant e Tversky (1999), assim como outros pesquisadores da Língua de Sinais Americana, por exemplo, está presente na linguagem narrativa. Na linguagem narrativa, segundo McNeill (1995), os gestos que representam o ponto de vista externo à narrativa são chamados de gestos do ponto de vista do observador (O-VPT)16, enquanto os gestos que representam o ponto de vista interno à narrativa são chamados de gestos do ponto de vista da personagem (C-VPT)17. As representações externas e internas dos pontos de vista da narrativa recebem diversas terminologias dos pesquisadores (c. f. Quadro 3). Conforme explicamos anteriormente, os temos “ponto de vista” e “perspectiva” são, geralmente, usados, indistintamente. Nesse sentido, o ponto de vista é “o sentimento de distância da narrativa”18 (MCNEILL, 1995, p. 118), enquanto a perspectiva é “o lugar no qual o observador está”19 (MCNEILL, 1995, p. 192). Desse modo, o ponto de vista, expresso no gesto, pode ser ou da personagem ou do observador, podendo ser identificado de acordo com o modo como o corpo do narrador está incorporado na representação. Por outro lado, a perspectiva pode estar fora ou dentro da cena, conforme o observador estiver fora ou dentro da cena narrada. No trabalho de Kita e Özyürek (2003) que mencionamos no Quadro 3 a seguir, os gestos espontâneos são entendidos como aqueles que acompanham a fala na expressão de informação. Nesse trabalho, os autores afirmam que existem discordâncias teóricas no que diz respeito ao processo pelo qual a coordenação informacional é alcançada. Segundo eles, algumas teorias propõem que a informação codificada nos gestos não é influenciada pelas informações expressas verbalmente, enquanto outras afirmam que os gestos codificam apenas o que está compreendido na fala. p , j , p 17 C-VPT vem de character viewpoint, ou seja, ponto de vista da personagem 18 16 O-VPT vem de observer viewpoint, ou seja, ponto de vista do observador p j p p g 18 The feeling of distance from the narrative. 19 19 Where the observer stands 2.3 Ponto de Vista e Narrativa: inter-relação entre fala e gestos O estudo dos autores (2003) investiga, portanto, a coordenação informacional, levando em consideração a comparação de narrações produzidas em diferentes línguas. Os resultados do estudo apontam que os gestos usados para expressar os eventos de movimento foram influenciados pelo modo como os eventos de movimento foram expressos pela fala e pela informação espacial no estímulo que nunca foi verbalizada. Eles concluem, 34 portanto, que os gestos são gerados a partir de um processo espacial e motor e interagem online com o processo de produção da fala. No trabalho de Emmorey e colaboradores (2000), eles afirmam que descrever a localização de uma instância física em uma cena, tipicamente, requer uma perspectiva. No que diz respeito à descrição de múltiplas instâncias físicas, os autores afirmam que é preciso assumir uma perspectiva na qual o ponto de vista está dentro ou fora da cena, podendo ainda, apresentar- se como uma mescla de ambos os pontos de vista. O estudo dos autores traça um paralelo com a Língua de Sinais Americana (ASL), que usa dois formatos espaciais: o espaço do observador e o espaço diagramático. Aos participantes, falantes de inglês e ASL, foi solicitado que descrevessem um dos dois mapas utilizados no experimento. Para os falantes de ASL, os resultados apontaram que as descrições tendem a ocorrer no espaço diagramático, enquanto para os falantes de inglês, as descrições ocorrem em todo o espaço gestual, que os autores denominam de espaço 3D. No estudo de Bryant e Tversky (1999), também mencionado no Quadro 3 a seguir, os autores demonstram que o conhecimento espacial é expresso por meio de descrições. Além disso, a experiência direta dos indivíduos os induz a tomarem a perspectiva de uma personagem cercada de objetos. Ainda nesse trabalho, os autores utilizam modelos e diagramas para expressar a mesma informação: a partir dos modelos apresentados, os participantes tenderam a adotar a perspectiva da personagem (a análise do quadro teórico espacial) e a partir dos diagramas, os participantes tomaram uma perspectiva externa (partindo da análise da computação intrínseca). Quadro 3: Termos Utilizados na Descrição de Ponto de Vista e Perspectiva Fonte Definição Termo externo Termo interno McNeill (1995) Perspectiva: o lugar no qual o observador está Externo: movimento transversal. O corpo não é a origem. Podem ocorrer gestos O- VPT, mas não gestos C-VPT Interno: movimento sagital. O corpo é a origem. 2.3 Ponto de Vista e Narrativa: inter-relação entre fala e gestos Sempre C- VPT, podendo ser O-VPT 35 Fonte Definição Termo externo Termo interno Kita; Özyürek (2003) Perspectiva: não há definição Evento externo: da perspectiva do estímulo do observador (movimento transversal) Evento interno: da perspectiva do protagonista/ator (movimento sagital) Emmorey; Tversky; Taylor, (2000) Perspectiva: não há definição, mas “ponto de vista” é utilizado. O ponto de vista está fora da cena. Espaço diagramático: o sinalizador da ASL está fora do espaço. Autores notam semelhança com O- VPT O ponto de vista está dentro da cena. Espaço do observador: o sinalizador da ASL está dentro do espaço. Autores notam semelhança com C-VPT Bryant; Tversky (1999) Perspectiva: não há definição, mas “ponto de vista” é utilizado. Externo: intrínseco, o narrador não faz parte da cena Interno: egocêntrico, o narrador faz parte da cena McNeill (1995) Ponto de Vista: o sentimento de distância da narrativa O-VPT: o corpo do narrador é excluído, o narrador está fora da história (podendo estar dentro ou fora da perspectiva) C-VPT: o corpo do narrador é incluído, o narrador está presente na história (e precisa estar dentro da perspectiva) Fonte: Adaptado de Parrill (2009) Fonte: Adaptado de Parrill (2009) Fonte: Adaptado de Parrill (2009) A justificativa para a elaboração desse quadro, de acordo com a autora, se situa na necessidade de se estabelecer de que modo se compreende o que o narrador está representando, uma vez que a distinção entre os gestos do personagem (C-VPT) e os gestos do observador (O- VPT) pode ser difícil de ser identificada. Para Parrill (2009), grosso modo, os gestos C-VPT 36 indicam que o narrador está dentro do que ela denomina como “mundo da história”, enquanto os gestos O-VPT indicam que ele está de fora do “mundo da história”. Em relação ao ato mais geral de narrar, de acordo com Bressem, Ladewig e Müller (2018), esse ato é uma prática comunicativa e reconstrutiva que assume um papel extremamente relevante nas interações face a face. Segundo as autoras, ao contar uma história experienciada por eles mesmos, os falantes podem, por exemplo, ilustrar um evento, estruturar e sequenciar eventos passados, revelar dados de sua biografia e (re)afirmar a si mesmos como seres que possuem uma identidade social comum. 2.3 Ponto de Vista e Narrativa: inter-relação entre fala e gestos Além disso, as narrativas seguem técnicas de estruturação do discurso – como, por exemplo, as estratégias para iniciar a contação de uma história – assim como as estratégias de avaliação, como a criação de tensão, surpresa e envolvimento. Nesse sentido, as autoras (2008) afirmam que, para alcançarem esses objetivos comunicativos, os falantes têm acesso a diversos recursos linguísticos por meio dos quais a narração se estrutura: os recursos de estruturação e conexão marcam a estrutura global das narrativas; os conectores e partículas interrogativos participam na criação interativa de enredos; as marcas prosódicas e a estilística particular são usadas para marcar respectivas atividades de cada indivíduo. A maioria das pesquisas que focalizam a natureza das narrativas focalizam apenas os aspectos verbais. No entanto, o trabalho de Bressem e colaboradoras (2018), assim, como pretendemos nesta dissertação, lança um olhar à estrutura complexa expressa pelo corpo do falante. 2.4 Organização multimodal da narrativa McNeill (1995), que se dedicou a analisar não somente a modalidade verbal, mas, também, a modalidade gestual em narrativas, explica que os gestos que emergem na narrativa são correspondentes à estrutura da própria narração que, por sua vez, se refere à série de eventos que constroem o processo de comunicação de uma história, partindo de uma pessoa para outra(s). Cada um desses eventos pode ser referido pelo narrador no instante em que ele inicia o decurso narrativo, construído em múltiplos níveis que se caracterizam em termos de: espaço e tempo; perspectiva; distância entre o narrador e os eventos narrados; e, por fim, integração do que é sequencial com o que não é. O autor afirma, ainda, que a linguagem narrativa não se trata de uma questão bidimensional, com eixos interseccionais sintagmáticos e paradigmáticos. Na verdade, a linguagem narrativa possui uma estrutura tridimensional (uma dimensão é imagética – que é visual e cinética, assim como holística e analítica). 37 A sequência de eventos que abrange a história é apenas uma das sequências de eventos que abrangem a narrativa completa. O tipo de narrativa que faz parte dos estudos de McNeill (1995) é composto por três níveis narrativos: o nível narrativo propriamente dito, o nível metanarrativo e o nível paranarrativo. Segundo McNeill (1995), o nível narrativo diz respeito aos eventos do mundo da narrativa. O que define essas características das sentenças contidas nesse nível é que o ouvinte considerará que o que está sendo narrado é um “simulacro” da sequência de fatos ocorridos no mundo real. Essa limitação temporal é, de acordo com a definição de Labov e Waletsky (1967), nas palavras de McNeill (1995), o que define o nível narrativo. Além disso, é preciso que o nível narrativo seja destacado no curso da conversação, para que o ouvinte entenda que a ordem sequencial dos fatos narrados nesse nível faz parte da história propriamente dita – a história no mundo real. De acordo com os exemplos de McNeill (1995), a narração de filmes e desenhos, invariavelmente, possui uma sequência narrativa na qual os eventos se sucedem em uma certa ordem. Sendo assim, o nível narrativo está no núcleo da preocupação do narrador ao recontar o evento. No que diz respeito aos gestos, o autor afirma que eles possuem um papel crucial na marcação dessas sentenças que fazem parte desse simulacro do mundo real. 2.4 Organização multimodal da narrativa Ainda de acordo com McNeill (1995), os narradores não contam somente o enredo da história. Eles fazem, também, referências explícitas à estrutura da história, à medida em que ela se constrói por meio da narrativa. As sentenças que apresentam a história sobre a história são interligadas ao nível narrativo e constituem o nível metanarrativo. Diferentemente das sentenças narrativas, as sentenças meta-narrativas não se limitam pela ordem dos eventos do mundo real ou fictício (lembrando que podemos narrar uma obra de ficção, por exemplo). É por essa razão que é crucial marcar as sentenças nos dois níveis: um nível constitui o simulacro do mundo real, outro não. Qualquer referência aos eventos metanarrativos requer a habilidade de manipular a história como uma unidade e de comentar a história como um evento. Por fim, para McNeill (1995), os narradores também fazem referências à sua própria experiência ao observar os eventos da narração, o que introduz, então, o nível paranarrativo. Nesse nível, o narrador sai do seu lugar de narrador oficial e fala por si, por sua própria personalidade e a ênfase está, portanto, na relação do falante com o ouvinte. Além disso, o autor, a partir de suas análises, afirma que o decurso narrativo passa por momentos pontuais que ele chama de sequências de evento. O conceito de uma sequência de evento permite analisar, individualmente, o discurso em andamento do ponto de vista de cada sentença, uma vez que o narrador não preenche o papel de narrador durante toda a narrativa (ele é um primeiro observador, testemunhando um evento específico). Qualquer sentença pode 38 incluir uma referência a uma dessas linhas de evento e essa referência coloca a sentença no nível narrativo correspondente. As sequências de evento que McNeill (1995) identifica na narração são as seguintes: 1. Em primeiro lugar, os eventos da história acontecem. Referências a essa sequência de evento dominam o nível narrativo. 2. A história se torna “disponível” por meio do texto visual encenado pelos gestos. Referências a essa sequência de evento aparecem no nível metanarrativo. 3. A pessoa que presenciou os eventos forma uma representação na memória do texto, que pode ser mais ou menos completa em relação à sequência de eventos. Essa sequência de evento ocupa, também, o nível metanarrativo. 4. Finalmente, há a sequência de recontar os eventos a um ouvinte: a narrativa interpessoal. Esse tipo de referência interpessoal está no nível paranarrativo. 2.4 Organização multimodal da narrativa De acordo com o autor, essas linhas de evento podem formar, igualmente, o sujeito da narrativa que o ouvinte escuta. Não se trata apenas dos eventos contados na narrativa, mas, também, dos eventos de presenciar os eventos da narrativa e depois de descrevê-los. Sendo assim, a sequência de eventos da narrativa (1) está alocada no nível narrativo do discurso. O texto visual (2) e a linha de observação (3) formam o nível metanarrativo do discurso (a parte da narração que é sobre narrar). A sequência de evento interpessoal (4) forma o nível paranarrativo do discurso (a parte da história na qual o narrador sai da história e assume o papel de falante). Isso posto, o autor reafirma que a ordem da história segue a ordem dos eventos, a não ser que especifiquemos quais eventos narrativos estão sendo referidos e o nível do discurso no qual essa referência é feita. Na fala, raramente há uma distinção clara entre a referência às ações de uma personagem e referências às ações do próprio narrador, pois os mesmos dispositivos linguísticos realizam ambas as ações. No que diz respeito aos gestos, no entanto, é possível traçar uma distinção entre os níveis narrativos. 39 Figura 3: As sequências de evento e os níveis narrativos Paranarrativo Metanarrativo Narrativo Narrativa interpessoal Representação Observação TextoVisual História Fonte: Adaptado de Cassell e McNeill (1990, p. 59) Fonte: Adaptado de Cassell e McNeill (1990, p. 59) De acordo com Cassell e McNeill (1990), os narradores fazem referência a todos os três níveis narrativos e também às cinco linhas de evento durante uma história típica. Eles afirmam que adultos também demonstram um padrão típico de gesticulação correlacionado às referências aos diferentes tipos de eventos. Esse padrão específico de gestos, que não se tratam de gestos emblemáticos, indica o nível narrativo em que as sentenças se alocam. ( ) l i (A) Gestos no nível narrativo Cassell e McNeill (1990) afirmam que o tipo mais comum de gestos produzidos, em geral, nas narrativas são os gestos icônicos, uma vez que eles representam alguma característica da ação ou evento descritos e são reconhecíveis por sua forma e conteúdo. Sendo assim, as sentenças narrativas são acompanhadas por gestos icônicos, diferentemente das sentenças meta e para-narrativas. (B) Gestos no nível metanarrativo Narradores, conforme explicam os autores, dizem respeito ao ato de narrar fazendo referências explícitas à estrutura da história que está sendo contada ou à estrutura do evento que está sendo narrado. Essas referências ocorrem acompanhadas dos gestos metafóricos, cujo conceito não possui forma física. Esse tipo de gesto, ainda, aparece nas referências à estrutura da história na qual a narração pode ser objetivada e comentada na modalidade verbal e apresentada como um objeto na modalidade gestual. Os gestos dêiticos, nesse nível, 40 espacializam ou localizam no espaço físico na frente do narrador, aspectos da história que está sendo narrada. Outro tipo de gesto que os autores encontraram no nível metanarrativo e outras sentenças cuja função é meta-pragmática foram os gestos rítmicos. Esses, por sua vez, possuem um movimento que não muda em forma o conteúdo do discurso que o acompanha. Os gestos rítmicos, além de ocorrerem no nível metanarrativo, podem acompanhar a reparação de discurso e o discurso indireto. (C) Gestos no nível paranarrativo No terceiro nível narrativo, no qual os narradores falam por si fora da situação narrativa e adotam o papel de participante em uma situação socialmente definida de falantes-ouvintes, eles realizam um número menor de gestos de um tipo restrito, isto é, gestos icônicos são, virtualmente, ausentes, assim como os gestos metafóricos. Os gestos dêiticos, de acordo com as análises de Cassell e McNeill (1990), foram encontrados para apontar para os participantes do evento que, no caso da narração, são o(s) falante(s) e ouvinte(s). Goodwin e Goodwin (1992) observaram que o olhar possui um papel muito importante na estruturação da participação de falantes e ouvintes na narração de eventos. Além disso, Cassell e McNeill (1990) encontraram um padrão gestual distinto que corresponde a cada nível narrativo: para o nível narrativo, gestos icônicos; para o nível metanarrativo, gestos rítmicos, metafóricos e os de apontar para o espaço vazio; para o nível paranarrativo, gestos de apontar em direção ao interlocutor. 2.5 Reencenações de eventos passados De acordo com Sidnell (2006), na narração, os participantes contam e, recorrentemente, reencenam eventos passados. Nessas reencenações, os gestos, o olhar e a fala podem funcionar de maneira coordenada, a fim de criar um sistema de atividades interativas que dizem respeito à sustentação do enquadramento dos participantes da conversação. Ou seja: o direcionamento do olhar possui um papel fundamental na organização de reencenações, de modo que, continuamente, a face e o olhar do narrador realizam a encenação de um evento em questão e atuam na construção e compartilhamento do sentido, o que atribui ao direcionamento do olhar a função de sustentar complexos pontos de vista narrativos. 41 O olhar do falante é frequentemente utilizado para selecionar, entre os múltiplos participantes da conversação, uma pessoa em particular para a qual o momento da fala é endereçado. No entanto, quando consideramos as reencenações, momentos nos quais eventos do passado são retratados em uma narrativa, por exemplo, os falantes, regularmente, deixam de direcionar o olhar para os co-participantes. Esse redirecionamento, conforme explica Sidnell (2006), é intrinsecamente relacionado a um momento específico da fala, indicando o progresso da narração, quando, por exemplo, o falante deixa de direcionar seu olhar para os interlocutores e olha para cima no momento do curso da interação. Sendo assim, as reencenações envolvem o uso coordenado dos gestos, do olhar e da fala, construindo, dessa maneira, cursos de ação da interação direta com os coparticipantes da conversação, e, também, cursos de ação da narração de eventos passados. Sidnell (2006) explica que a fala na interação entre pessoas presentes em uma mesma cena interativa é, inerentemente, multimodal e organizada simultaneamente em diferentes níveis: as falas são organizadas em um sistema de turnos de fala que constituem um instrumento para as ações que, por sua vez, formam sequências coerentes de fala. A autora também argumenta que a ação de analisar alguns casos de discurso indireto, como as reencenações, por exemplo, é muito produtiva, uma vez que essa metodologia viabiliza uma análise focalizada em aspectos linguísticos dos enunciados originais, como por exemplo, o léxico, a gramática e a prosódia e, pensando em termos multimodais, a orientação corporal e o posicionamento dos participantes da interação, levando em consideração o direcionamento do seu olhar e os gestos produzidos na sua fala. 3 O PAPEL DOS GESTOS MANUAIS E A DA DIREÇÃO DO OLHAR NA INTERAÇÃO Neste capítulo, apresentamos um panorama sobre os Estudos de Gesto. O objetivo desse panorama é demonstrar que os estudos sobre gestos, que remontam à época da retórica antiga, com os trabalhos de Quintiliano, partiram do momento em que se deixou de pensar nos gestos espontâneos como movimentos que deveriam ser controlados. Passou-se então a pensar nos gestos espontâneos a partir de suas funções comunicacionais, e nesse sentido, Kendon (1980, 1986, 2004) propõe sua abordagem linguística dos gestos, destacando que eles não apenas possuem uma função comunicacional, mas também se estruturam como a língua – a partir da delimitação de unidades gestuais. Daí surgem os Estudos de Gestos na contemporaneidade, como os conhecemos hoje, conforme as proposições teóricas e metodológicas de autores como Kendon (2004), McNeill (1979, 1995, 2005), Müller (2004, 2013, 2018) e Cienki (2008, 2017). Em seguida, tratamos do uso simultâneo e visível de articuladores relativamente independentes, como, por exemplo, as duas mãos, a postura e orientação do torso, a orientação da cabeça, as expressões faciais e a direção do olhar, bem como o papel desses articuladores na comunicação linguística, especialmente na manutenção e na mudança do ponto de vista. 2.5 Reencenações de eventos passados A autora afirma também que o caráter visível das reencenações abre uma janela para os estudos que focalizam nas mudanças (shifts) de posição corporal e de perspectiva no curso da interação. Além disso, um único evento pode ser representado em um momento a partir da perspectiva de alguém que o testemunhou e, em outro momento, a partir da perspectiva de alguém que participou dele. Por exemplo, uma cena de uma pessoa dirigindo um carro pode ser reencenada das seguintes maneiras: i) o falante pode representar o volante a partir dos gestos e ii) pode representar, com as mãos, a trajetória do carro em questão. Considerando essa dinâmica, quando se fala nessa mudança de posição, fala-se dessa mudança entre essas duas perspectivas que ocorre dentro de um turno de fala. Conforme mencionamos anteriormente, a autora afirma que as reencenações envolvem o uso coordenado dos gestos, do direcionamento do olhar e da fala. Assim, a investigação sobre esse fenômeno possibilita o surgimento de pesquisas que dizem respeito não só à prática da reencenação, mas faz com que os 42 pesquisadores possam lançar um olhar para os modos com os quais essas diferentes modalidades funcionam em concordância na criação de momentos interativos coerentes. Sidnell (2006) conclui que a produção e recepção de reencenações constituem unidades menores de uma narração maior. No que diz respeito ao que está sendo reencenado, ela afirma que não se trata apenas da associação entre fala e gesto, mas também, trata-se de um fenômeno que compreende a corporificação física de uma personagem pelo falante, a fim de recriar um determinado evento para receptores. Ela afirma, ainda, que existem evidências que sugerem que as reencenações evocam uma manifestação intensificada para a recepção e atenção, ou seja, em determinadas situações, conforme afirma Sidnell (2006), o formato reencenado é selecionado pelo falante como um esforço para se assegurar de que os coparticipantes se engajarão em uma atitude de atenção. A autora explica, por fim, que enquanto o falante narra aos seus receptores o que aconteceu em um evento passado, as reencenações, que também são utilizadas para o atingir efeitos de humor e drama, oferecem representações virtuais dos eventos relevantes, isto é, num momento específico da narração, as reencenações pretendem demonstrar não só o que foi testemunhado pelo falante, mas sim, como um evento aconteceu. 43 3.1.1 Kendon: ações visíveis como enunciados Kendon (1980) focaliza a coordenação dos gestos com a fala e os papéis que os gestos desempenham na constituição do sentido na interação. Ele afirma que a gesticulação e a fala constituem-se como aspectos interrelacionados do enunciado. Nos seus estudos subsequentes, Kendon (1986) enfatiza a materialidade corporal que produz signos, que caracteriza seus estudos sobre gestos manuais durante a conversação. Sendo assim, ele explora as práticas nas quais o falante-gesticulador alinha a fala com os gestos, o que requer uma atenção constante às propriedades físicas (que, por sua vez, são temporais e espaciais) de ambas as modalidades (KENDON, 2004). Para Kendon (2004), os seres humanos, ao interagirem, informam, continuamente um ao outro, sobre as suas intenções, interesses, sentimentos e ideias, por meio de ações corporais visíveis. De acordo com o autor, é através da orientação do corpo e especialmente do olhar, que a informação sobre a direção e a natureza da atenção de uma pessoa são estabelecidas. Além disso, o modo como as pessoas organizam seus corpos, orientam-nos e posicionam-nos em relação a outras pessoas, como também a outros componentes do ambiente de interação, fornecem informações importantes sobre o modo como elas estão engajadas com as outras, além de expressar suas intenções e atitudes. Quando essas atividades são realizadas com a manipulação, reorganização e modificação de determinados objetos, por exemplo, é possível observar os objetivos e interesses de um indivíduo. Assim, quando se atenta para as atividades que o autor denomina como expressivas, lança-se o olhar para as configurações de ação na face e no corpo, que parecem demonstrar sentimentos e emoções, assim como passa-se a observar as ações que cumprem um papel essencial na realização de momentos de interação tais como: o cumprimento, a demonstração de gratidão ou afeição, o desafio, a ameaça, a submissão, entre outros. Outro de tipo de ação que protagonizou os estudos de Kendon (2004) corresponde àquelas que são compreendidas como parte do processo de discurso, como parte do ato de enunciar algo para alguém de modo explícito. Segundo o autor, as pessoas podem se referir a algo por meio dos gestos de apontar, utilizando a(s) mão(s) em uma ação complexa, organizada para apresentar algo, indicar seu tamanho ou formato etc. Ademais, as pessoas podem demonstrar, a partir de ações visíveis, que elas estão fazendo um pedido, fazendo uma pergunta, negando alguma coisa e, nas palavras de Kendon (2004), muitas outras coisas. 3.1 Breve histórico sobre os Estudos de Gesto na atualidade Quando se passou a pensar na comunicação não-verbal como um subdomínio das ciências linguísticas, focava-se, sobretudo, nos aspectos do comportamento em situações sociais nas quais o objeto de pesquisa era tratado como inconsciente e, de algum modo, revelador no que dizia respeito aos sentimentos e atitudes de um determinado indivíduo. Nesse contexto, os estudos de gesto passaram a protagonizar os estudos linguísticos na virada cognitivista, momento que se refere ao despertar dos Estudos de Gesto, no qual surgiram sérias discussões a respeito da origem da linguagem e a respeito do redescobrimento do caráter linguístico das Línguas de Sinais. Nesse sentido, Cienki (2016) afirma que as pesquisas em gesto, inerentemente, levam o pesquisador a gravar e analisar os dados gerados a partir de vídeo gravações e, ainda, esse tipo de análise requer muito cuidado no que diz respeito ao modelo de transcrição escolhida pois é preciso respeitar os fatores que fazem parte da interação. 44 20 Any ensemble of action that counts for others as an attempt by the actor to “give” informatio 3.1.1 Kendon: ações visíveis como enunciados Ainda de acordo com ele, existem ações visíveis que são utilizadas na realização de expressões que, do ponto de vista funcional, são similares (ou idênticas) a determinadas expressões da língua falada. O uso 45 dessas ações é denominado por Kendon (2004) como “uso de enunciados” das ações visíveis que constituem o domínio dos gestos. Kendon (2004), portanto, denomina como “gestos” as ações visíveis que são utilizadas como um enunciado ou como parte de um enunciado. Os enunciados, para o autor, referem-se “a qualquer conjunto de ações que são consideradas como uma tentativa de dar informação de algum tipo”20 (KENDON, 2004, p. 7). E por “dar informação”, baseado em Goffman (1963), Kendon (2004) entende que, quando as pessoas estão presentes em uma situação interacional, elas sempre oferecem informação sobre suas intenções, sobre seu status como seres sociais etc. No que diz respeito aos gestos, mais precisamente, Kendon (2004) argumenta que eles constituem uma denominação para as ações que possuem traços da manifestação de expressividade deliberada. Por conseguinte, conforme afirma Kendon (2004), uma unidade natural da ação gestual é iniciada quando a mão inicia a excursão a partir da posição de descanso e termina quando a mão retorna à posição de descanso. Nesse contexto, ele propõe que, tipicamente, o ato da gesticulação ou excursão gestual é constituído pelas seguintes fases: a) a preparação, fase na qual a mão se movimenta, configurada em um formato específico, da posição de descanso para o stroke; b) o stroke (traduziremos como núcleo gestual), fase que compreende o principal movimento expressivo reconhecido como “o gesto” e, por fim, c) a retração, fase na qual a mão retorna do núcleo gestual para a posição de descanso. Conforme ilustrado por Cienki e Mittelberg (2013), na Figura 4 a seguir. Figura 4: Excursão Gestual Figura 4: Excursão Gestual Fonte: Cienki e Mittelberg (2013, p. 233) Fonte: Cienki e Mittelberg (2013, p. 233) 46 Nos estudos que empreendeu desde 1980 até os dias de hoje, a partir de gravações em vídeo de interações diárias de falantes americanos, ingleses e, principalmente, italianos, Kendon tem adotado uma perspectiva funcional no que diz respeito aos gestos, examinando, atentamente, suas propriedades de formação, sua integração com a fala, suas propriedades semióticas e seu papel na interação. Ainda de acordo com o que afirma o autor (2004), os gestos contribuem para o sentido à luz do que ele denomina como “interação semântica”. 3.1.1 Kendon: ações visíveis como enunciados Assim, ele diferencia as funções referencial, pragmática e interativa dos gestos. Os gestos referenciais contribuem para conteúdo proposicional dos enunciados e fazem parte do que está sendo dito sobre algo. Os gestos pragmáticos, por sua vez, contribuem para o sentido não-referencial e são divididos em modais e performativos. Finalmente, os gestos interativos são aqueles que indicam ou endereçam a um enunciado ou outros aspectos regulatórios do processo de interação. Esta constitui a principal diferença entre Kendon e McNeill, sobre o qual trataremos na próxima seção: enquanto Kendon começa a investigar o papel do gesto na interação e suas diferentes funções inguísticas, McNeill se ocupou de demonstrar como os gestos refletem processamentos cognitivos do falante. Para McNeill o interlocutor não é importante, pois seu foco é a mente do falante. No que diz respeito, especificamente, à interação semântica, Kendon (2004) explica que os enunciados são como objetos construídos pelo material correspondente à fala e aos gestos na interação. Desse modo, o autor explica como as funções gestuais podem contribuir para a referência ao sentido ou para a expressão do enunciado. Segundo o autor, existem gestos que são usados paralelamente às palavras e expressões que são equivalentes a eles. Esses gestos enunciam o mesmo conteúdo enunciado pelas palavras e, levando em consideração a relação semântica entre gestos e palavras, Kendon (2004) explica que existe uma completa redundância. No exemplo representado na Figura 10, que foi retirado das gravações do autor de amigos jogando cartas, um falante argumenta que um dos interactantes ganhou duas vezes. Nesse momento, ele levanta a mão com o formato de “dois”, utilizando dois dedos, e mantém a mão nessa posição. 47 Figura 5: Gestos Utilizados com Expressões Verbais Correspondentes Fonte: Kendon (2004, p. 176) Fonte: Kendon (2004, p. 176) Fonte: Kendon (2004, p. 176) Ainda de acordo com Kendon (2004), os gestos podem ser utilizados em associação com uma expressão verbal que não necessariamente combina com a gesticulação. Em determinadas situações, esse tipo de gesto pode servir para especificar a dimensão de um referente que pode ser manifestado diretamente em palavras. Além disso, os gestos podem ser utilizados para indicar um significado mais geral do que o expresso pelo falante em palavras. No exemplo fornecido pelo autor, representado pela Figura 6, um falante explica para seu filho que ele precisava muito comprar um par de sapatos, mas não o fez ainda porque os sapatos são muito caros. 3.1.1 Kendon: ações visíveis como enunciados Quando ele diz “muito caros”, ele realiza um gesto no qual a mão direita produz um movimento descendente, com a palma vertical, duas vezes, em direção à sua mão esquerda que, por sua vez, está supinada. Esse gesto possui um significado no idioma napolitano: refere- se a uma surpresa negativa e, também, a algo financeiro, como, por exemplo, algo com o preço muito alto. Dessa maneira, o falante está dizendo que, quando ele descobriu o preço dos sapatos, ele teve uma surpresa negativa. 48 Figura 6: Gestos que Acompanham uma Expressão Verbal Não-correspondente Figura 6: Gestos que Acompanham uma Expressão Verbal Não-correspondente Fonte: Kendon (2004, p. 182) Fonte: Kendon (2004, p. 182) Kendon (2004) também afirma que é normal que um falante produza um gesto para enunciar um verbo ou uma locução verbal nos quais a ação do gesto é interpretável como um padrão de movimento extraído do padrão de ação performado, ou seja, nesses casos, o gesto, chamado de especificador semântico, não corresponde a um equivalente cinético do verbo lexical, mas sim, a uma encenação que demonstra uma forma de realização da ação específica. Os gestos oferecem, também, uma informação referencial que faz com que o enunciado tenha um sentido muito mais específico. No exemplo fornecido pelo autor, conforme representado pela Figura 7, um falante descreve como os soldados americanos, quando saíam da sua base perto de uma vila, costumavam deixar coisas úteis para o povo da vila. O falante explica que eles costumavam jogar laranjas e chicletes para as crianças locais. Quando ele fala “jogava laranjas e chicletes”, ele levanta sua mão do seu colo rapidamente duas vezes, direcionando o movimento para a direção de seus ombros, abrindo sua mão ligeiramente, de modo que sugere a ação de arremessar coisas, sem mencionar, por assim dizer, a precisão desse movimento. 49 Figura 7: Gestos como Especificadores Semânticos Figura 7: Gestos como Especificadores Semânticos Fonte: Kendon (2004, p. 186) Fonte: Kendon (2004, p. 186) Ainda de acordo com Kendon (2004), os gestos podem ser utilizados para elaborar a representação de um objeto de algum tipo. Nesse caso, os gestos são compreendidos pelo autor como um “exemplificador”, um “ilustrador” ou uma amostra, isto é, quando se discute sobre um determinado objeto, se o exemplar está disponível na cena interativa, o falante pode pegá- lo e apresentá-lo enquanto fala sobre ele. 3.1.1 Kendon: ações visíveis como enunciados Quando não for o caso, no entanto, uma versão do exemplar do objeto pode ser criada. No exemplo fornecido por Kendon (2004), representado na Figura 8, ele apresenta uma falante que descreve como as refeições são servidas em uma determinada situação. Quando ela menciona as tortas de limão que lhe eram servidas, ela posiciona ambas as mãos na frente de seu corpo a fim de mostrar um círculo horizontal. Nesse exemplo, as mãos se tornam uma amostra que ilustra o objeto sendo referenciado. 50 Figura 8: Gestos como uma Amostra g Fonte: Kendon (2004, p. 192) Fonte: Kendon (2004, p. 192) Fonte: Kendon (2004, p. 192) Outros gestos, de acordo com Kendon (2004), podem ser utilizados em um enunciado para fazer referência à estrutura ou ao formato de algo sendo descrito. Quando os falantes oferecem descrições de objetos, os gestos são utilizados como componentes dessas descrições. No seu exemplo, representado na Figura 9, o autor cita uma gravação na qual um grupo de garotas estão descrevendo seus quartos, seus brinquedos e seus animais de estimação. Uma delas descreve seu aquário e, ao dizer, que nele existem duas pedras grandes, ela realiza um gesto no qual ambas as mãos, com as palmas pronadas, se afastam uma da outra, em um movimento horizontal para representar a distância em que as pedras estão posicionadas em seu aquário. Esse movimento emprega, portanto, a estabilização da localização de um objeto. 51 Figura 9: Gestos que Representam Propriedades de um Objeto e Relações Espaciais Figura 9: Gestos que Representam Propriedades de um Objeto e Relações Espaciais Fonte: Kendon (2004, p. 193) Fonte: Kendon (2004, p. 193) No que diz respeito à referência dêitica, Kendon (2004) afirma que os gestos podem ser utilizados para criar objetos de referência para expressões dêiticas. Os falantes, nesse caso, criam ou descrevem um formato, ou criam um objeto, através dos gestos, desenhando-os. Essa descrição ou criação gestual torna-se o objeto de referência dêitica. No exemplo fornecido pelo autor, representado na Figura 10, a seguir, ele descreve um falante que explica como era o formato do bolo que era enviado para seu pai todos os anos, no Natal. Nesse momento, o falante se inclina em direção à mesa em sua frente e move suas mãos simetricamente, em um gesto que desenha um objeto quadrado grande que se situa sobre a mesa, ou seja, ele cria o bolo para que todos na cena enunciativa possam ver. Os receptores desse enunciado veem a imagem criada pelo falante, de modo que ela seja, efetivamente, compreendida. Nesse caso, conforme explica o autor, o desenho realizado pelo gesto ocorreu no discurso que o precedeu (o bolo de Natal), o que torna nítido o modo com o qual se interpreta o gesto. 52 Figura 10: Gestos como Objetos de Referência Dêitica Figura 10: Gestos como Objetos de Referência Dêitica Fonte: Kendon (2004, p. 148) Fonte: Kendon (2004, p. 148) Fonte: Kendon (2004, p. 148) Em linhas gerais, os gestos, de acordo com Kendon (2004), estão situados no espaço, e a produção desses gestos está relacionada a estrutura interacional da configuração espacial. O formato da mão, a orientação da palma, a trajetória e o padrão de movimento de um gesto podem ser explicados não somente pela referência ao conteúdo verbal. Portanto, as características dos gestos e os efeitos que eles proporcionam na interação são, também, circunstanciados por sua organização local e espacial. Em outro texto, Kendon (1988a) faz uma distinção entre gestos de diferentes tipos – essa distinção foi denominada, mais tarde, por McNeill (1995), como o “continuum de Kendon” –, conforme pode ser visualizado na Figura 11, a seguir. Figura 11: Continuum de Kendon (1988a) Fonte: Traduzido de McNeill (2005, p. 7) Figura 11: Continuum de Kendon (1988a) Figura 11: Continuum de Kendon (1988a) Fonte: Traduzido de McNeill (2005, p. 7) Para Kendon (1988a), a gesticulação corresponde aos movimentos que incorporam um significado relacionado à fala que o acompanha. Os gestos desse tipo são, de acordo com McNeill (2005), o tipo de gesto mais frequente no uso cotidiano, uma vez que compreendem 53 muitas variantes e usos. São considerados como parte da sentença, pois ocupam um espaço gramatical, completando a estrutura da sentença. Os emblemas, de acordo com Kendon (1988a), são os sinais convencionalizados, como, por exemplo, o dedo polegar estendido para “OK”. Por fim, a pantomima, grosso modo, diz respeito a um gesto, ou sequência de gestos que expressam uma linha narrativa, produzida sem a fala. Em um extremo do continuum, os sinais constituem palavras lexicais nas Línguas de Sinais, que possuem estruturas linguísticas próprias, incluindo padrões gramaticais, morfológicos etc. Nos termos das mudanças recíprocas que ocorrem no continuum de Kendon (1988a), nota-se que o acompanhamento obrigatório da fala se reduz dos gestos para os sinais. Nota-se, também, que o grau em que os gestos demonstram as propriedades de uma língua aumenta. Em linhas gerais, os gestos são, obrigatoriamente, acompanhados pela fala. 21 Essa proposta culminou na proposição da Teoria dos Pontos de Crescimento. Verificar McNeill et. al (2008) 3.1.2 McNeill: o que os gestos revelam sobre o pensamento Com a publicação de Conceptual basis of language, McNeill (1979), que já era conhecido por suas contribuições para os processos cognitivos envolvidos na aquisição da linguagem, sugere que os falantes realizam gestos de modo que indica que as duas modalidades, verbal e gestual, são integradas desde o início da vida, formando um sistema de imagem21. Suas observações sobre a relação do gesto com a fala foram elaboradas no artigo “So you think gestures are nonverbal?” (1985) e, depois, culminaram na sua principal obra, Hand and Mind: What Gestures Reveal About Thought, (1995). Para McNeill (1995), quando as pessoas falam, elas produzem movimentos espontâneos que chamamos de gestos. Esses movimentos são, na maioria das vezes, realizados com as mãos e braços e possuem níveis de sincronia com a modalidade verbal, uma vez que eles, juntamente com a fala, ocorrem em uma sincronia temporal muito próxima e, às vezes, possuem significados idênticos e, ainda, expressam esses significados de modo completamente distinto. Nesse sentido, conforme o autor explica, o que mais o interessa, no que diz respeito à implicação dos gestos, é a possibilidade de analisar duas formas de expressão, o discurso e a ação, a partir do mesmo sistema teórico. Ainda segundo McNeill (1995), a comparação entre gestos e fala produz um efeito de compreensão da linguagem e do pensamento que pode ser comparado ao efeito de 21 Essa proposta culminou na proposição da Teoria dos Pontos de Crescimento. Verificar McNeill et. al (2008) 54 triangulação22 na visão: muitos detalhes que, antes poderiam estar escondidos, elevam-se em uma nova dimensão. Desse modo, levar em consideração a produção de gestos, segundo o autor, ao invés de propor análises compostas por módulos semi-isolados de fala ou de gestos, por exemplo, encoraja os pesquisadores a analisarem um indivíduo como uma entidade teórica, considerando seu pensamento, sua fala, seus desejos, sentimentos e atitudes como uma unidade. Em vista do que afirma McNeill (1992) em relação à entidade teórica, verifica-se que seu objetivo é oferecer um quadro conceptual que inclua tanto os gestos como a linguagem verbal. Segundo ele, esse enquadramento teórico mais abrangente demonstra como os gestos e a fala são vinculados e, ao mesmo tempo, diferentes. 22 O termo triangulação, também atribuído às Ciências Sociais, conforme explicam Cox e Hassard (2005), surgiu das ciências militares e, decorrente da navegação e da topografia, é frequentemente entendida como um método para fixar uma posição. 3.1.2 McNeill: o que os gestos revelam sobre o pensamento Além disso, o autor afirma que o quadro conceptual propõe explicar como a fala – que se constitui linearmente no tempo – se relaciona ao tipo de pensamento – que se constitui instantâneo, imagético e global – que vemos apresentados nos gestos simultâneos. Assim, ele afirma que os gestos exibem imagens mentais que nem sempre são expressas na fala, de modo que os gestos e a fala precisam cooperar, a fim de expressar o sentido proposto pelo indivíduo. Para McNeill (1995), uma concepção de fala e gestos como um sistema integrado é acentuadamente diferente da noção de “linguagem corporal” que, segundo ele, é um produto de uma análise tradicional que compreende a língua falada exclusivamente como uma compressão de um sistema de sons associado a uma gramática. Em suma, grande parte do trabalho do autor é dedicada a demonstrar que os falantes utilizam os gestos para a expressão conceptual, isto é, as imagens sugeridas pelos gestos se referem aos conceitos do falante, indicando que ele, além de pensar em palavras, também o faz imageticamente. As primeiras observações do autor a respeito da integração gesto-fala foram responsáveis por oferecer um quadro teórico robusto para os Estudos de Gesto e os assimilaram nas perguntas de pesquisa dentro do campo da teoria psicolinguística. Hand and Mind tornou- se, assim, um divisor de águas para a Linguística, para a Psicolinguística e para a Linguística Cognitiva. No que diz respeito à definição, McNeill (1995) afirma que os gestos são as imagens intrínsecas da linguagem, ou seja, gesto e linguagem são inseparáveis porque, nas palavras do autor, os gestos orquestram o discurso. Sendo assim, o quadro teórico proposto pelo autor trata, especificamente, dos gestos que exibem imagens. Eles, de acordo com o autor são utilizados deliberadamente pelas pessoas ao demonstrarem seus pensamentos e os modos pelos quais elas 22 O termo triangulação, também atribuído às Ciências Sociais, conforme explicam Cox e Hassard (2005), surgiu das ciências militares e, decorrente da navegação e da topografia, é frequentemente entendida como um método para fixar uma posição. 55 compreendem os eventos do mundo. Desse modo, o autor classifica os gestos da seguinte maneira: gestos icônicos; gestos metafóricos; gestos rítmicos; gestos coesivos e gestos dêiticos. Os gestos icônicos, de acordo com o autor, são aqueles que mantém uma relação próxima com o conteúdo semântico da fala. 23 Aqui, e em todas as figuras subsequentes dessa seção, optamos por representar a produção gestual do mesmo modo que McNeill (1995) o faz: abaixo da imagem, é possível visualizar a parte da fala que coocorre com gesto cujo stroke está marcado em colchetes. 24 and he [bends it way back] 25 Iconic: hand appears to grip something and pull it from the upper front space back and down near to the shoulder. 24 and he [bends it way back] 3.1.2 McNeill: o que os gestos revelam sobre o pensamento A fim de exemplificar essa classificação gestual, o autor apresenta a seguinte situação: ao descrever uma cena de uma história em quadrinhos, na qual uma personagem entorta uma árvore para trás, em direção ao chão, o falante parece pegar algo com as mãos e puxar para trás (Figura 12). Esse gesto, além de revelar a memória imagética do falante ao ler a história em quadrinhos, revela também o ponto de vista que ele adota em relação ao acontecimento. Isso significa que o falante poderia ter encenado o evento a partir da perspectiva do agente (a personagem que entortou a árvore), ou a partir da perspectiva da própria árvore. Esse exemplo, de acordo com McNeill (1995), ilustra a conexão estabelecida entre a fala e o gesto: a fala demonstra como o que é representado através dos gestos pode ser incorporado em uma imagem completa do processo de pensamento de uma pessoa. Figura 12: Gestos Icônicos “E ele entorta [para trás]23”24 Gesto icônico: a mão parece pegar algo e jogar para trás do ombro.25 Figura 12: Gestos Icônicos “E ele entorta [para trás]23”24 Gesto icônico: a mão parece pegar algo e jogar para trás do ombro.25 Gesto icônico: a mão parece pegar algo e jogar para trás do ombro.25 Gesto icônico: a mão parece pegar algo e jogar para trás do ombro.2 Fonte: McNeill (1995, p. 12) 25 Iconic: hand appears to grip something and pull it from the upper front space back and down near to the shoulder. 56 Os gestos metafóricos, para McNeill (1995), são similares aos gestos icônicos no aspecto imagético. No entanto, nesse caso, o conteúdo imagético apresentado se trata mais de uma ideia abstrata do que de um evento ou objeto concretos. Conforme o autor explica, o gesto apresenta uma imagem do invisível, “a imagem de uma abstração” (MCNEILL, 1995, p. 14). O gesto representa uma metáfora de um conceito que, em termos cinéticos e visuais, possui uma imagem semelhante ao conceito. O exemplo do autor, conforme Figura 13, diz respeito a um falante que declara que o que ele acabou de assistir – e, também, o que ele está prestes a recontar – se trata de um desenho animado. A Figura 13 corresponde à ilustração do gesto metafórico que instancia a metáfora do conduto: a ideia do gênero apresentado (um desenho animado) como um container cujas barreiras são delimitadas por ambas as mãos. 26 It [was a Sylves]ter and Tweety cartoon 27 Metaphoric: Hands rise up and offer listener an “object” 3.1.2 McNeill: o que os gestos revelam sobre o pensamento McNeill (1995) explica que um desenho animado específico corresponde a uma entidade concreta. No entanto, no exemplo apresentado, o falante se refere ao gênero “desenho animado” que, por sua vez, se trata de um conceito abstrato. A entidade abstrata ganha, então, a imagem de uma forma concreta obtida pelo gesto. É nesse sentido, segundo o autor, que encontramos a metáfora: o conceito de um gênero de um certo tipo (Tópico) é apresentado como um objeto físico dotado de limites e espacialmente localizável. Figura 13: Gestos Metafóricos “Era o desenho [de Piu-Piu] e Frajola”26 Figura 13: Gestos Metafóricos “Era o desenho [de Piu-Piu] e Frajola”26 57 Gesto Metafórico: As mãos realizam um gesto ascendente e oferecem ao ouvinte um to”.27 Gesto Metafórico: As mãos realizam um gesto ascendente e oferecem ao ouvinte um to”.27 “objeto”.27 28 Nesse caso, em específico, os colchetes não marcam um stroke, mas sim o momento em que a mão realiza um gesto descendente. 30 Beat: hand rises short way up from lap and drops back down 29 When[ever she] looks at him he tries to make monkey noises Fonte: McNeill (1995, p. 14) Fonte: McNeill (1995, p. 14) A terceira categoria proposta por McNeill (1995) são os gestos rítmicos que, em inglês são chamados de Beats. A nomenclatura original desse tipo de gesto tem origem na sua semelhança com o a marcação rítmica (beating) do tempo musical. Embora autores com McClave (1991) afirmem que a sincronia não é absolutamente perfeita, nos gestos dessa categoria, as mãos se movem de acordo com a pulsação rítmica da fala. Conforme explica o autor, os gestos rítmicos, diferentemente dos icônicos e dos metafóricos tendem a possuir a mesma forma, independentemente do conteúdo. Sendo assim, ele classifica o típico gesto rítmico como um simples toque dos dedos ou da mão em um movimento que pode ser descendente ou para frente e para trás. Esse movimento tende a ser curto e rápido e o espaço no qual ele tende a acontecer se encontra na periferia do espaço gestual que, nos termos do autor, são, por exemplo, o colo e o braço da cadeira. O que distingue os gestos rítmicos das outras categorias, para McNeill (1995) é o fato de esse tipo de gestos possuírem duas fases de movimento (conceito que abordaremos na próxima seção): para dentro/para fora, para cima/para baixo etc. No exemplo de McNeill (1992), conforme Figura 14, o gesto rítmico é acompanhado da referência do tema de um episódio: o enunciado não se refere a um evento em específico, mas caracteriza uma série de eventos nos quais o gesto rítmico marca a expressão “toda hora”. Figura 14: Gestos Rítmicos 27 Metaphoric: Hands rise up and offer listener an “object” Figura 14: Gestos Rítmicos Figura 14: Gestos Rítmicos Figura 14: Gestos Rítmicos 27 Metaphoric: Hands rise up and offer listener an “object” 58 “[Toda hora] que ela olhava para ele, ele tentava imitar o barulho de um macaco”28 29 “[Toda hora] que ela olhava para ele, ele tentava imitar o barulho de um macaco”28 29 “[Toda hora] que ela olhava para ele, ele tentava imitar o barulho de um macaco”28 29 Gesto Rítmico: A mão realiza um curto movimento ascendente até o colo e depois pequenos movimentos descendentes.30 Fonte: McNeill (1995, p.16) Gesto Rítmico: A mão realiza um curto movimento ascendente até o colo e depois pequenos movimentos descendentes.30 Fonte: McNeill (1995, p.16) Fonte: McNeill (1995, p.16) Fonte: McNeill (1995, p.16) Os gestos coesivos, na concepção de McNeill (1995), servem para unir partes da fala que são tematicamente relacionadas e temporariamente separadas. Segundo o autor, no que diz respeito à função, ao passo que os gestos rítmicos marcam descontinuidade na fala, os gestos coesivos enfatizam continuidades. Já no que diz respeito à forma, o autor os considera como “ecléticos”, uma vez que eles podem consistir de gestos icônicos, metafóricos, gestos de apontar e, até mesmo, de gestos rítmicos. Além disso, a coesão gestual depende da repetição da mesma forma gestual, do mesmo movimento ou do mesmo locus no espaço gestual, isto é, os gestos repetidos demonstram a recorrência e a continuidade de um tema. Com o objetivo de exemplificar o modo pelo qual a repetição sinaliza a continuidade, McNeill (1995) apresenta a seguinte situação, conforme Figura 15: um falante, ao descrever um episódio do desenho animado, em um primeiro momento, realiza um gesto cruzado para representar a interseção de cabos aéreos, e, depois, interrompe a si mesmo para realizar um movimento para trás e para frente para representar um bonde (b), para, finalmente, voltar a realizar o gesto cruzado (c). Segundo o autor, essa interrupção ocorreu porque o falante percebeu que seria melhor explicar primeiro como os bondes, os veículos que funcionam através dos cabos aéreos, funcionam. A parte da fala que está alocada em (b), não faz parte do enredo e, segundo o julgamento do falante, não se trata de uma informação óbvia, o que requer uma explicação. Desse modo, o segundo gesto cruzado (c) funciona como um modo de direcionar os ouvintes para o tema principal da narrativa. 28 Nesse caso, em específico, os colchetes não marcam um stroke, mas sim o momento em que a mão realiza um gesto descendente. 30 Beat: hand rises short way up from lap and drops back down 59 Figura 15: Gestos Coesivos (a) [“a estação de cabos que conectam os teleféricos... um]31 Gesto icônico: ambas as mãos palmas para baixo formando um gesto cruzado 32 t t l fé i ]31 (a) [“a estação de cabos que conectam os teleféricos... um]31 (a) [“a estação de cabos que conectam os teleféricos... um]31 (a) [“a estação de cabos que conectam os teleféricos... 31 [the network of wires that hooks up the cable cars… um] 32 35 [right and there’s a whole network of these wires] 36 36 Iconic: both hands resume crisscross 34 Iconic: right hand moves back and forth at side of head 3 31 [the network of wires that hooks up the cable cars… um] 32 Iconic: both hands, palms facing down, come together at the tips and form a crisscross 33 [you know the trolley system] 34 Iconic: right hand moves back and forth at side of head 35 [right and there’s a whole network of these wires] 36 Iconic: both hands resume crisscross p g 33 [you know the trolley system] Fonte: McNeill (1995, p.16) um]31 Gesto icônico: ambas as mãos, palmas para baixo, formando um gesto cruzado.32 (b) [você sabe (como funciona) o sistema de bondes]33 Gesto icônico: a mão direita se move para frente e para atrás do lado da cabeça.34 (b) [você sabe (como funciona) o sistema de bondes] Gesto icônico: a mão direita se move para frente e para atrás do lado da cabeça.34 (c) [e existe uma rede inteira daqueles cabos]35 Gesto icônico: ambas as mãos retornam ao gesto cruzado.36 (c) [e existe uma rede inteira daqueles cabos]35 Gesto icônico: ambas as mãos retornam ao gesto cruzado.36 Fonte: McNeill (1995, p. 17) Os gestos dêiticos, a última categoria estabelecida por McNeill (1995), são também classificados por ele como os gestos de apontar. Esse tipo de gesto tem a função de indicar objetos e eventos no mundo concreto, mas, também possui um papel fundamental quando não existe, objetivamente, uma instância física para onde se apontar. O autor explica que, na interação, a maioria dos gestos de apontar são do tipo abstrato. Na Figura 16, o exemplo de McNeill (1992) descreve uma interação entre dois estudantes não familiarizados. O falante produz um gesto de apontar abstrato com a parte da fala [de onde você]. O espaço para o qual ele aponta não é o espaço onde o falante e o ouvinte estão, mas um espaço que aloca um referente introduzido. 60 Figura 16: Gestos Dêiticos “[De onde você] veio?”37 Aponta para o espaço entre ele e o interlocutor.38 Fonte: McNeill (1995, p. 18) Figura 16: Gestos Dêiticos Figura 16: Gestos Dêiticos “[De onde você] veio?”37 [ ] Aponta para o espaço entre ele e o interlocutor.38 Fonte: McNeill (1995, p. 18) Aponta para o espaço entre ele e o interlocutor.38 Aponta para o espaço entre ele e o interlocutor.38 Fonte: McNeill (1995, p. 18) Fonte: McNeill (1995, p. 18) Em resumo, os gestos, para McNeill (1995), não se constituem como o produto de um plano verbal segmental e linear, pois, apesar de possuírem uma relação intrínseca com a fala, em termos de forma, são fundamentalmente diferentes. 38 Points to space between self and interlocutor. 37 [where did you] come from before? 38 3.1.3 Müller: os gestos e seu potencial para a linguagem No que diz respeito às contribuições recentes nos Estudos de Gestos, Müller (2018) oferece um panorama a respeito das maneiras por meio das quais se faz possível explorar sistematicamente a relação entre gestos e sinais, reconstruindo a história das discussões referidas na área, focalizando a perspectiva dos autores supracitados. A autora formula, portanto, uma posição teórica que enfatiza a necessidade de se olhar para as questões da multimodalidade a partir de um ponto de vista sistemático e intercultural, considerando o uso da linguagem nas modalidades verbal e gestual. 61 Para a autora, a publicação de McNeill (1985), foi, de fato, um momento seminal nos Estudos de Gesto. Na perspectiva de Müller (2018), McNeill (1985) desafiou o pressuposto, que era dominante na época, de que os gestos não faziam parte da linguagem propriamente dita: os gestos eram considerados como parte da comunicação não-verbal. Ainda para Müller (2018), o trabalho de McNeill (1985, 1987, 1989) foi o pontapé para o surgimento de uma perspectiva psicológica e linguística sobre os gestos, mostrando que eles são um objeto de estudo de muita produtividade tanto para os psicólogos como os linguistas. Müller (2018) argumenta que, no modelo psicológico de gesto de McNeill (1995), gesto e fala se constituem como dois lados da linguagem e cada um reflete, fundamentalmente, diferentes formas de pensamento – o imagético e o proposicional. Müller (2018) traz à tona, também, as contribuições de Kendon (1980d) para os Estudos de Gesto. De acordo com ela, o autor demonstrou que gesto e fala são dois aspectos do processo de enunciação (conforme destacamos na seção anterior). Nesse sentido, Kendon (1980d) atesta que as unidades gestuais (gesticulation units) são temporariamente alinhadas às unidades de fala (speech units) e devem ser consideradas como “uma manifestação alternativa do processo pelo qual ‘ideias’ são inscritas em padrões de comportamento, (...) como se o processo de enunciação possuísse dois canais de emitir comportamento: a fala e o movimento corporal”39 (KENDON, 1980d apud MÜLLER, 2018, p. 3). A autora argumenta que a concepção de Kendon para “gestos” inclui todas as formas e funções cinéticas, desde a gesticulação espontânea até os emblemas que, por sua vez, se diferem dos gestos porque possuem uma relação fixa entre sentido e forma, isto é, traduzindo em termos linguísticos, eles são lexicalizados. 39 An alternate manifestation of the process by which ‘ideas’ are encoded into patterns of behavior which can be apprehended by others as reportive of those ideas. It is as if the process of utterance has two channels of output into behavior: one by way of speech, the other by way of bodily movement. 3.1.3 Müller: os gestos e seu potencial para a linguagem No que diz respeito à sua definição do conceito de gesto, Müller (2018) afirma que, do ponto de vista da forma baseada no uso e na interação, os gestos são movimentos corporais cujo sentido está pautado nas experiências corporificadas que são, também, dinâmicas e intersubjetivas. Essa afirmação confirma que a autora não concorda com a pressuposição de que os gestos são equivalentes a imagens. Nesse sentido, em conformidade com as asserções de Müller (2018), partimos do pressuposto de que os gestos se constituem como movimentos expressivos deliberados, motivados por: ações “como se” (as-if actions), encenações de movimentos ou representações de objetos, demonstrando níveis de convencionalização que são expressos nos termos “espontâneo”, “recorrente” e “emblemático”. 39 An alternate manifestation of the process by which ‘ideas’ are encoded into patterns of behavior which can be apprehended by others as reportive of those ideas. It is as if the process of utterance has two channels of output into behavior: one by way of speech, the other by way of bodily movement. 62 Essas asserções dizem respeito a importantes contribuições de Müller (2013, 2014), para os Estudos de Gesto. De acordo com a autora, os gestos não são motivados por ações ou representações, porque eles constituem as representações, sendo assim, Müller (2014) estabelece os quatro modos de representação gestual: 1. Encenar: As mãos se movem de modo que representam uma ação, podendo envolver a manipulação de um evento. 1. Encenar: As mãos se movem de modo que representam uma ação, podendo envolver a manipulação de um evento. Figura 17: Modo de Representação Gestual 1 – Encenar Fonte: Müller (2014, p. 1691) Fonte: Müller (2014, p. 1691) Fonte: Müller (2014, p. 1691) 2. Corporificar: As mãos representam um objeto. 2. Corporificar: As mãos representam um objeto. 2. Corporificar: As mãos representam um objeto. Figura 18: Modo de Representação Gestual 2 – Corporificar Figura 18: Modo de Representação Gestual 2 – Corporificar Fonte: Müller (2014, p. 1691) Fonte: Müller (2014, p. 1691) 63 3. Moldar: As mãos são posicionadas de modo a representar o formato do objeto a partir do contorno desenhado pelas mãos. 3. Moldar: As mãos são posicionadas de modo a representar o formato do objeto a partir do contorno desenhado pelas mãos. 3. Moldar: As mãos são posicionadas de modo a representar o formato do objeto a partir do contorno desenhado pelas mãos. Figura 19: Modo de Representação Gestual 3 – Moldar Figura 19: Modo de Representação Gestual 3 – Moldar Fonte: Müller (2014, p. 1691) Fonte: Müller (2014, p. 1691) Fonte: Müller (2014, p. 1691) 4. Desenhar: A mão ou as mãos se movem e representam, a partir das pontas dos dedos, um traço da forma do objeto. 4. Desenhar: A mão ou as mãos se movem e representam, a partir das pontas dos dedos, um traço da forma do objeto. Figura 20: Modo de Representação Gestual 4 – Desenhar 40 When the hand becomes a body sculpture of a picture or a window or a piece of paper. Figura 21: Desenvolvimento histórico dos gestos para os sinais Fonte: Adaptado de Müller (2018, p. 14) Fonte: Adaptado de Müller (2018, p. 14) Figura 20: Modo de Representação Gestual 4 – Desenhar Fonte: Müller (2014, p. 1691) Fonte: Müller (2014, p. 1691) Fonte: Müller (2014, p. 1691) 64 Além disso, a relação entre gestos e sinais é entendida por Müller (2018) de um modo sistemático, ou seja, ela defende que o termo “gesto” é mais amplo do que a noção de enunciado como ação visível, defendida por Kendon (2014), uma vez que essa noção implica que a motivação dos gestos se baseia somente nas ações do corpo, excluindo os gestos que se constituem como encenações de movimentos e exclui o que autora denomina como gestos híbridos, nos quais alguns aspectos do movimento gestual podem ser usados para expressar algum aspecto dos sentido desejado na interação, independentemente do tipo de gesto. Além disso, a definição de Kendon (2014), ainda de acordo com Müller (2018), exclui os gestos que se constituem, semioticamente, como representações de objetos, como, por exemplo “quando a mão se torna uma escultura de um corpo em uma foto, uma janela, ou um pedaço de papel”40 (MÜLLER, 2018, p. 13). Quando se trata do espectro de expressões gestuais, a autora compreende que formas gestuais não-estabilizadas e totalmente convencionalizadas podem ser empregadas por qualquer usuário da linguagem. Para Müller (2018), essas formas não são separadas abruptamente como categorias distintas. Nesse sentido, a autora propõe um continuum que parte da forma improvisada individualmente a formas que são amplamente convencionalizadas. Em relação ao continuum de Kendon, conforme estabelecido por McNeill (2005), a autora (2008) adiciona os gestos recorrentes, forma gestual híbrida e intermediária, que se aloca entre os gestos espontâneos e os emblemáticos, no que diz respeito à convencionalização. A Figura 21, traduzida de Müller (2018), inspirada no continuum de Kendon (MCNEILL, 2005), sistematiza a dinâmica histórica baseada no grau de convencionalização como um processo de constantes mudanças que é também dinâmico e sucessivo. Nesse sentido, gestos idiossincráticos (singular gestures) são considerados como aqueles que não são convencionalizados, que demonstram uma relação variável entre forma e sentido e que não são composicionais. O diagrama, ainda, ilustra a convencionalização como um processo gradual. 65 41 Verificar Lakoff e Johnson (1980) Fonte: Adaptado de Müller (2018, p. 14) Müller (2013), a partir dos trabalhos de Bühler (1982) sobre a teoria psicológica da linguagem e a teoria da expressão, propõe que os gestos possuem um potencial para a linguagem. Para a autora (2013), os gestos podem expressar estados interiores e sentimentos, podem regular o comportamento de outras pessoas e também podem representar objetos e eventos no mundo. A abordagem funcional para os gestos, de acordo com a autora, se relaciona com os três aspectos básicos de qualquer encontro comunicativo: o falante, o ouvinte e o mundo sobre o qual se fala. Levando em consideração essa sistematização, a autora categoriza os gestos a partir de sua função dominante: representação, expressão ou apelo. Quando se trata da perspectiva linguística, conforme explica Müller (2013), a função representacional é fundamental uma vez que ela explica o potencial linguístico dos gestos, demonstrando a integração funcional desses com a estrutura da parte verbal do enunciado. Segundo Müller (2013), o fato de os gestos poderem representar outras entidades que não eles mesmos deve-se à condição anatômica humana, de que as mãos possuem a flexibilidade para multiplicar formatos, para se mover em uma ampla variedade de modos, e, também, para ocupar vários lugares em um espaço amplo. Os movimentos de mão são altamente articulados e constituem-se como os instrumentos mais importantes para que os humanos lidem com o mundo. A função representacional dos gestos, de acordo com Müller (2013), divide-se em duas categorias: i) a função representacional que representa ações, objetos ou eventos concretos – os gestos representacionais e ii) a função representacional que se refere a ações, objetos e eventos abstratos – os gestos metafóricos. No 66 exemplo apresentado por Müller (2013), representado na Figura 22 a seguir, vemos um gesto que representa um desempacotamento: ambas as mãos reencenam repetitivamente o movimento de desempacotar. Figura 22: Função Representacional 1: Ações, Objetos e Eventos concretos Figura 22: Função Representacional 1: Ações, Objetos e Eventos concretos Fonte: Müller (2013, p. 207) Fonte: Müller (2013, p. 207) Já na Figura 23 a seguir, vemos a representação de uma mulher que encena um gesto muito parecido com o do exemplo anterior: ela encena, também, o movimento de desempacotar, porém, nesse caso, trata-se do desempacotamento de segredos. No alemão, figurativamente, essa expressão diz respeito ao ato de contar segredos. Nesse exemplo, vemos um caso de um gesto metafórico. 67 Figura 26: Função Expressiva 3: Raiva ou Ira Fonte: Müller (2013, p. 211) Fonte: Müller (2013, p. 211) No que se refere à função de apelo, Müller afirma que eles são utilizados para regular o comportamento de outras pessoas. Eles podem ser direcionados a um público maior, conforme a Figura 27 – na qual é ilustrado um indivíduo utilizando ambas as mãos, com as palmas pronadas, executando um movimento descendente, para acalmar a plateia – mas, é possível observá-los, também, em uma escala menor, nas interações face a face, quando os utilizam para solicitar que um interlocutor faça silêncio, conforme a Figura 28 – na qual é ilustrada uma falante que posiciona o dedo indicador entre os lábios, para expressar “silêncio”. Figura 23: Função representacional 2: Gestos Metafóricos Fonte: Müller (2013, p. 208) Fonte: Müller (2013, p. 208) Na função gestual de expressar emoções, de acordo com a autora (2013), encontramos gestos como o de levantar as mãos para expressar alegria ou triunfo, esconder o rosto para expressar tristeza ou sofrimento, ou mover o punho para baixo para representar raiva. De acordo com a autora, essa categoria gestual está relacionada a uma determinada expressão facial, a uma orientação do olhar em particular (sobre o qual trataremos na próxima seção) e a uma postura corporal. Ela afirma, ainda, que os gestos que atendem a essa função correspondem a expressões do corpo como um todo. A fim de exemplificar, Müller (2013) argumenta que, em situações de extrema felicidade ou triunfo, as pessoas tendem a levantar seus braços, em um movimento ascendente, conforme ilustrado na Figura 24, a seguir. A proeminência de movimentos ascendentes se relaciona, segundo a autora, à metáfora conceptual41 FELIZ É PARA CIMA, o que demonstra as experiências corporificadas que indicam a origem experiencial das metáforas conceptuais e a utilização de expressões verbais que se relacionam à alegria e incorporam essa ideia, a exemplo da expressão “estou para cima”, para representar alegria. Ao expressar tristeza ou sofrimento cobrindo o rosto ou os olhos com a mão, o corpo acompanha: a cabeça está inclinada para baixo, assim como os ombros, conforme ilustrado na Figura 25. Aqui, segundo a autora, encontramos outra metáfora conceptual, TRISTE É PARA BAIXO, e, mais uma vez, é possível verificar como as metáforas se relacionam a experiência corporificadas. Por fim, para expressar ira ou raiva, a autora afirma que as pessoas do hemisfério 68 ocidental tendem a usar seus punhos para realizar movimentos descendentes de maneira enérgica, conforme ilustrado na Figura 26. Figura 24: Função Expressiva 1: Alegria ou Triunfo Figura 24: Função Expressiva 1: Alegria ou Triunfo Figura 24: Função Expressiva 1: Alegria ou Triunfo Fonte: Müller (2013, p. 211) Figura 25: Função Expressiva 2: Tristeza ou Sofrimento Fonte: Müller (2013, p. 211) Fonte: Müller (2013, p. 211) Figura 25: Função Expressiva 2: Tristeza ou Sofrimento Fonte: Müller (2013, p. 211) Fonte: Müller (2013, p. 211) 69 Figura 26: Função Expressiva 3: Raiva ou Ira Fonte: Müller (2013, p. 213) 42 It opens up pathways to a linguistic understanding of gestures: the understanding of gestures as linguistic derives in part from their functions in language, and this can be realized by researchers if they do a close analysis of gestural forms. Figura 28: Função Apelativa 2: Interação Face a Face Figura 28: Função Apelativa 2: Interação Face a Face Fonte: Müller (2013, p. 213) Fonte: Müller (2013, p. 213) Em suma, Müller (2013) apresenta os gestos como um meio de expressão e afirma, ainda, que eles preconizam uma perspectiva corporificada e funcional o que permite que se compreenda os gestos a partir de uma perspectiva linguística. Nas palavras da autora, “a compreensão dos gestos de maneira linguística se origina em parte de suas funções na linguagem e isso pode ser percebido pelos pesquisadores se eles empreenderem uma análise profunda das formas gestuais”42 (MÜLLER, 2013, p. 214). Figura 27: Função Apelativa 1: Público Amplo Figura 27: Função Apelativa 1: Público Amplo Figura 27: Função Apelativa 1: Público Amplo Fonte: Müller (2013, p. 213) Fonte: Müller (2013, p. 213) Fonte: Müller (2013, p. 213) 70 3.1.4 Cienki: análise de metáfora nos gestos No que diz respeito à integração dos Estudos de Gesto à Linguística Cognitiva, Cienki (2016) afirma que ambas as áreas passaram a estreitar sua relação. O reconhecimento mútuo de ambas as áreas torna essa relação muito produtiva, conforme explica o autor, mas, também de acordo com ele, não se trata de uma simples admiração: levando em consideração a necessidade de lançar um olhar empírico para os gestos, o princípio baseado no uso da Linguística Cognitiva, sobretudo no que se refere ao uso de dados de vídeos gravados de interações amplia o escopo das análises, oferecendo uma visão muito mais ampla do fenômeno estudado pela área do conhecimento que se convencionou chamar “Estudos de Gesto”. O mesmo autor, que dedicou boa parte de seus estudos à metáfora, um dos conceitos basilares da Linguística Cognitiva, explica que existem diversas maneiras com as quais se pode abordar o estudo em metáfora e, consequentemente, existem diversas maneiras para se abordar 42 It opens up pathways to a linguistic understanding of gestures: the understanding of gestures as linguistic derives in part from their functions in language, and this can be realized by researchers if they do a close analysis of gestural forms. 71 os Estudos de Gesto (CIENKI, 2010). A metáfora é vista nos trabalhos de Cienki (2005, 2013, 2017) a partir de uma perspectiva conceptual e semiótica e, também, é relacionada aos modos de pensar imagético e idiossincrático que pode ser refletido nos gestos. Ainda segundo o autor (2016) as análises semióticas focalizam o uso metafórico de diferentes formas de estruturas icônicas nos gestos. Ele afirma também que os gestos, por um lado, são estudados como uma janela que permite o acesso aos processos de pensamento do falante e gesticulador, ou seja, argumenta-se que os gestos podem revelar o pensamento metafórico, que, por sua vez, pode ocorrer com ou sem o acompanhamento da fala. Por outro lado, também conforme explica o autor, os gestos são entendidos como uma ferramenta utilizada pelo ouvinte e visualizador para que se construa o sentido pretendido pelo falante. 43 Draw the line é uma expressão idiomática utilizada na língua inglesa que diz respeito a impor limites, isto é, o ato de “desenhar a linha” significa estabelecer um limite. 44 a way of seeing one thing in terms of something different 3.1.4 Cienki: análise de metáfora nos gestos A fim de explicar como funciona essa relação entre os gestos e a metáfora, Cienki (2017) exemplifica essa relação da seguinte maneira: considerando a situação em que uma pessoa fala a respeito de uma decisão que pode ser tomada em relação ao que é moralmente certo ou errado, ela diz “you have to draw your line and figure out on which side of it you fall”, em uma tradução literal, teríamos “você precisa desenhar sua linha43 e descobrir de que lado você cai”. A partir do exemplo dado por Cienki (2017), é possível entender que a frase “desenhar sua linha” pode se referir à demarcação de dois espaços fisicamente separados, os quais, no contexto de uma oposição binária, que, nesse caso, se refere ao que é moralmente correto e errado, podem ser compreendidos como uma separação metafórica das categorias morais (correto e errado) em termos de uma divisão espacial, produzida a partir de uma relação arbitrária entre as palavras e a ação. No entanto, segundo Cienki (2017), quando se leva em consideração as situações nas quais os gestos representam conceitos, eles o fazem iconicamente, ou seja, as imagens icônicas que os gestos formam funcionam como o que o autor denomina como as “bases da metáfora”. A Figura 29 representa o gesto produzido no exemplo apresentado por Cienki (2017). 43 Draw the line é uma expressão idiomática utilizada na língua inglesa que diz respeito a impor limites, isto é, o ato de “desenhar a linha” significa estabelecer um limite. 72 Figura 29: Gesto “desenhar a linha” (draw the line) 44 a way of seeing one thing in terms of something different Figura 29: Gesto “desenhar a linha” (draw the line) Fonte: Cienki (2017, p. 2) Fonte: Cienki (2017, p. 2) Enquanto o falante enuncia a frase “você precisa desenhar sua linha”, ele produz o seguinte gesto: ele posiciona o dedo indicador da sua mão direita para fora do corpo e o move em um gesto descendente. O formato da mão no movimento, realizado com o dedo indicador, de acordo com o autor, fornece a visualização de um tracejado na forma de uma linha vertical. Para Cienki (2017) esse exemplo sistematiza como as pesquisas sobre metáfora nos gestos difere das pesquisas que analisam as metáforas na fala. Do ponto de vista teórico, o autor argumenta, a partir da descrição de Burke (1945), que a metáfora é “uma maneira de ver uma coisa em termos de algo diferente44” (BURKE, 1945 apud CIENKI, 2017, p. 2). Quando se trata das pesquisas em formas visíveis de expressão, a exemplo das pesquisas sobre gestos, a argumentação de Burke possui um significado especial, como propõe Cienki (2017): quando vemos gestos realizados pelos falantes em um momento de interação, é possível que vejamos alguns aspectos das ideias que são expressas por meio dos formatos e movimentos das mãos. Desse modo, os pesquisadores de gesto e, especialmente, da emergência de metáforas nos gestos, compreendem, segundo Cienki (2017), as palavras de Burke (1945) não somente em termos do significado físico da palavra “ver”, mas em termos do significado metafórico ENTENDER É VER. Em um dos trabalhos mais recentes de Cienki (2017) são propostas as diretrizes para a identificação de metáfora nos gestos – Metaphor Identification Guidelines for Gesture (MIG- G). Nesse trabalho, em relação aos gestos metafóricos, Cienki (2017) afirma que eles são estudados como a expressão de metáforas conceptuais, uma vez que encenam o Domínio-fonte 44 a way of seeing one thing in terms of something different 73 das metáforas. Além disso, os estudos em LC concebem a metáfora como um fenômeno cognitivo e, nesse sentido, a noção do pensamento, por mais abstrata que seja, está baseada na experiência corpórea. Uma das grandes preocupações do autor consiste em, justamente, identificar como os gestos se relacionam com as expressões metafóricas e como os gestos destacam a natureza da metaforicidade. Ainda conforme Cienki (2017), é possível encontrar metáforas (ou expressões metafóricas) em vários aspectos do comportamento humano, não somente na linguagem. Figura 29: Gesto “desenhar a linha” (draw the line) De acordo com o autor, estudos apontam que gestos espontâneos realizados durante a “produção de linguagem”, especialmente os gestos de mão e antebraço, podem constituir expressões metafóricas. Levando em consideração as orientações para a identificação de metáfora nos gestos (Metaphor Identification Guidelines for Gestures – MIG-G) (CIENKI, 2016), é preciso seguir os seguintes passos: 1) identificar o núcleo gestual (gesture stroke), nesse passo, é preciso assistir à amostra selecionada sem o som e em câmera lenta; 2) descrever as quatro formas de cada núcleo gestual: formato da mão, orientação da palma, movimento e localização do gesto no espaço; 3) identificar se o gesto possui função referencial ou pragmática; 4) identificar os modos de representação icônica: a- enacting (encenar): quando as mãos representam a ação funcional de uma entidade; b- embodying (incorporar): quando as mãos dão forma a uma entidade em si; c- holding (segurar): quando as mãos simulam o contorno da superfície de uma entidade; d- tracing (traçar): quando as mãos desenham uma entidade; 5) identificar o referente físico do gesto, nesse passo, é preciso verificar se a entidade referida pelo gesto é concreta ou abstrata; 6) identificar o tópico contextual referido pelos gestos. Embora o objetivo deste trabalho não seja relacionado à metáfora, as diretrizes para a identificação de metáforas nos gestos permitem que observemos que esse modelo de análise nos oferece uma visão detalhada de suas representações, da saliência de sua produção e do esforço com o qual o gesto foi produzido. Além disso, o autor se baseou no MGA (Methods of Gesture Analysis: “From form to meaning”) para formular o MIG-G, mesmo conjunto de métodos que gerou o Sistema Linguístico de Anotação Gestual, sobre o qual trataremos no Capítulo 4. 3.2 Gestos e outros articuladores independentes: o papel do olhar na interação Conforme explicam Sweetser e Stec (2016), diferentemente da sequência de sons da língua falada, os gestos que coocorrem com a fala promovem o uso simultâneo e visível de 74 articuladores relativamente independentes, como, por exemplo, as duas mãos, a postura e orientação do torso, a orientação da cabeça, as expressões faciais e o olhar. Esses articuladores são fundamentais na comunicação linguística, especialmente na manutenção e mudança do ponto de vista. Embora se entenda que os gestos manuais são os mais estudados, bem como que os termos “gestos” e “gestos manuais” são muitas vezes utilizados como sinônimos, as autoras argumentam que a definição de “gesto” que elas utilizam é mais abrangente, uma vez que inclui todos os articuladores multimodais que são, conforme as autoras, externamente observáveis e centralmente envolvidos não só na comunicação, mas também na ação e atenção cotidianas. No entanto, dada a padronização de “gesto manual” na literatura mais ampla, como “gesto”, descreveremos os demais articuladores, como direção do olhar (eye-gaze), movimento de cabeça, movimento de torso etc. A proposição de articuladores independentes apresentada por Sweetser e Stec (2016) parte das proposições de Dudis (2004). O autor afirma que uma distinção pode ser feita entre as duas zonas corporais (os gestos manuais e as expressões faciais) em termos de seu papel em potencial na criação linguística de expressões visuais na Língua de Sinais Americana (ASL). Os articuladores manuais, de acordo com Dudis (2004), juntamente com a expressão facial do sinalizador, podem contribuir para a criação de um elemento visível de uma determinada expressão linguística, enquanto outras partes do corpo, como o torso, por exemplo, contribuem para a criação de outros elementos. Ainda, conforme explicaremos melhor no capítulo dedicado às reencenações, as zonas de particionamento do corpo podem estar alocadas em diferentes Espaços Mentais (FAUCONNIER, 1980). A fim de traçar um panorama, as pesquisas sobre o papel da interação no olhar, segundo Kidwell (2014), surgiram a partir da curiosidade de pesquisadores de diversas áreas pelas características e condições do corpo na interação natural. Ainda segundo a autora, o direcionamento do olhar é, para as pessoas (incluindo crianças muito novas), um recurso fundamental na iniciação ou inibição da interação. Além disso, o olhar, integrado ao movimento corporal e postura, expressa graus de engajamento em uma interação, levando em consideração os articuladores supracitados (a cabeça, o torso e os gestos manuais), particularmente quando um indivíduo está envolvido em atividades múltiplas e simultâneas. (2) A recipient should be gazing at the speaker when the speaker is gazing at the hearer. 45 (1) A speaker should obtain the gaze of [her] recipient during the course of a turn at-talk. 3.2 Gestos e outros articuladores independentes: o papel do olhar na interação O comportamento do olhar humano, conforme explica Rossano (2012), constitui um sistema altamente evoluído, de forma que, por uma questão fisiológica, os seres humanos são capazes de detectar a direção de olhar de outros indivíduos a uma determinada distância, representando uma função comunicativa, o que os torna únicos entre as espécies primatas. No 75 que diz respeito aos experimentos que comprovam a singularidade do olhar humano, Rossano (2012) cita os trabalhos de Tomasello (2007) e de Gibson e Pick (1963). No primeiro, a partir de evidências empíricas, foi demonstrado que, enquanto as crianças humanas confiam no olhar de adultos para moldar suas experiências, os filhotes de macaco confiam na direção da cabeça, sugerindo que a presença da esclera (a parte branca do olho), conforme ilustrado na Figura 30, evoluiu com a interação cooperativa humana, para facilitar a percepção sobre o que os adultos estão olhando. Já o trabalho de Gibson e Pick (1963) estabeleceu que humanos podem julgar, devido a uma condição fisiológica, a direção do olhar de outros até mesmo por meio da visão periférica. Figura 30: Comparação entre o Olhar Humano e o Olhar de um Chimpanzé Figura 30: Comparação entre o Olhar Humano e o Olhar de um Chimpanzé Fonte: Rossano (2012 p. 22) Fonte: Rossano (2012 p. 22) Partindo, mais precisamente, para os papéis que são atribuídos ao olhar, Rossano (2012), explica que um grande número de trabalhos se dedicou a analisar a relação entre o olhar e os papéis de participação, tais como falantes e ouvintes, considerando o framework de participação, pois a noção de que o olhar se relaciona com o papel de participação é comum na literatura: muitos autores sugerem que, nas interações dialógicas, as pessoas tendem a olhar mais para os participantes quando estão falando do que quando estão escutando. Ainda tratando da interação, Rossano (2012) cita duas regras45 que dizem respeito ao comportamento do olhar na conversação, propostas por Goodwin (1980, 1981), a partir da 45 (1) A speaker should obtain the gaze of [her] recipient during the course of a turn at-talk. 76 análise de cada caso dos seus dados: “1) um falante deve obter o olhar de seu receptor durante o curso de turnos de fala e 2) um receptor deve direcionar seu olhar ao falante quando este direcionar seu olhar ao ouvinte” (ROSSANO, 2012, p. 312). Em outras palavras, se o receptor olha, na maior parte do tempo, o falante o encontrará olhando de volta, toda vez que olhar para o receptor. Se o receptor não estiver olhando para o falante, o falante possui recursos para solicitar o olhar do receptor. Rossano (2012) afirma que Goodwin, ao propor essas regras, declara que o comportamento visual dos participantes da interação é mais interrelacionado do que independente e sugere uma ordem normativa no que diz respeito aos turnos de fala. Nesse sentido, buscamos provar com as nossas análises de dados do português brasileiro, que o olhar se constitui como um articulador independente em relação a outros articuladores como os gestos manuais, por exemplo. Ainda de acordo como Rossano (2012), existem trabalhos que focalizam outro aspecto do olhar. Muitos pesquisadores trataram da função reguladora do olhar, comprovando que o olhar funciona como um indicador para demonstrar que um falante A terminou de falar, podendo permitir que o turno de fala seja alterado. 46 Nomes atribuídos às narrativas. Originalmente, License, Snow and Cats. Figura 30: Comparação entre o Olhar Humano e o Olhar de um Chimpanzé Nesse sentido, ele cita o trabalho de Bavelas, Coates e Johnson (2002), no qual as autoras comprovam que os ouvintes tendem a responder quando o falante direciona o olhar para eles e que os falantes tendem a direcionar o olhar para fora da linha falante-ouvinte assim que um indivíduo termina de responder algo que foi perguntado. Assim, ambos, falantes e ouvintes, utilizam o olhar para coordenar suas ações. Bavelas, Coates e Johnson (2002) propuseram compreender os modos com os quais os ouvintes, ao produzirem determinadas respostas ao estímulo da fala. A partir de análises do diálogo gravado de pessoas não conhecidas, eles destacam dois aspectos importantes para o seu trabalho. O primeiro é que os participantes podem se valer tanto da modalidade oral quanto da gestual para expressarem o sentido desejado na comunicação. Nesse sentido, eles propõem o conceito de atos visíveis de sentido, que consistem, primordialmente, dos gestos manuais e expressões faciais. Eles consideram, com base em McNeill (1995), que essa gama de atos não- verbais não serve como um canal separado das palavras para a expressão de sentido, pelo contrário, os falantes integram suas palavras com os atos visíveis, complementar e coordenadamente. O outro aspecto seminal para o seu trabalho se baseia na noção de reciprocidade. Segundo Bavelas, Coates e Johnson (2002), o diálogo face a face possui um alto nível de reciprocidade que, de acordo com eles, está relacionado à probabilidade e à latência de resposta da outra pessoa. 77 3.3 As funções do olhar em narrativas: o estabelecimento de diferentes pontos de vista O trabalho de Sweetser e Stec (2016), que serve como base para o nosso, foi elaborado a partir de um corpus de vídeos semiespontâneos coletado por Stec em 2012. Os participantes desses vídeos são falantes nativos do inglês americano e, a fim de participar do experimento, levaram um amigo ao local da gravação e, posteriormente, as pesquisadoras solicitaram que eles contassem uma narrativa pessoal. Aqui, discutiremos três narrativas analisadas pelas autoras. A fim de apresentar um panorama a respeito da distribuição dos gestos em narrativas, assim como estabelecer a correlação entre diferentes aspectos dos gestos, o espaço gestual e os espaços mentais, discutiremos a seguir, três narrativas analisadas por Sweetser e Stec (2016): Certidão, Neve e Gatos46, ilustradas na Figura 31, dividida em a, b e c. Em Certidão, ilustrada em a, a narradora, que está localizado no lado esquerdo do quadro, conta a história de quando foi à prefeitura de uma cidade pequena com outra mulher e precisou requerer uma certidão de casamento de um funcionário. Ao contar a história, ela encena seu eu-passado e o funcionário, simultaneamente e, enquanto isso, ela também interage com seu interlocutor no Espaço-base. Em Neve, ilustrada em b, a mulher na esquerda e o homem na direita são casados e o homem está recontando a história de quando passaram por uma viagem de carro perigosa em uma nevasca. O homem, simultaneamente, reencena o seu eu-passado e também o de sua esposa. Já em Gatos, ilustrada em c, a narradora, localizada à direita do quadro, descreve como seu namorado gosta de colocar um cesto de roupa suja em cima do seu gato para ver o que acontece. Ela encena o seu eu-passado, o namorado e até mesmo o gato, além de interagir com seu interlocutor no espaço base. p Memory space: Espaço da Memória (Veriricar Sweetser e Stec, 2012) Storty: Espaço Narrativo 47 Speaker-hearer line: Linha falante-ouvinte 47 Speaker-hearer line: Linha falante-ouvinte Memory space: Espaço da Memória (Veriricar Sweetser e Stec, 2012) Storty: Espaço Narrativo Figura 31: Ilustração das narrativas “Certidão”, “Neve” e “Gatos” Figura 31: Ilustração das narrativas “Certidão”, “Neve” e “Gatos” Fonte: Sweetser e Stec (2016, p. 242) Fonte: Sweetser e Stec (2016, p. 242) Fonte: Sweetser e Stec (2016, p. 242) 78 As autoras afirmam que, nessas narrativas, é possível observar que o olhar possui um papel fundamental, que permite que o narrador se engaje, simultaneamente, em mais de um nível de construção de sentido. A narrativa Certidão, por exemplo, envolve um grande número de gestos manuais, sendo que o olhar facilita as transições entre o espaço narrativo e o espaço base. A co-narração de Neve, enquanto o casal revive a experiência passada, oferece uma perspectiva do uso do olhar como uma forma de manter a experiência compartilhada no presente, mesmo quando se trata de uma narração do passado. Já a narrativa Gatos envolve dois amigos que interagem, ilustrando os modos como a metainteração ocorre ao longo do conteúdo da narrativa. As três narrações, por conseguinte, demonstram o funcionamento de representações vividas do ponto de vista de personagens, com alterações na posição e segmentação do corpo para representar múltiplas personagens. De acordo com Sweetser e Stec (2016), o fato de uma história ser narrada no tempo passado se constitui como uma evidência de que os eventos narrados não tratam do aqui-agora do espaço-base, mas sim, do espaço narrativo, cujo conteúdo, em relação ao espaço-base, é passado. No caso dos gestos, conforme afirmam as autoras, a questão é um pouco mais complexa: se levarmos em consideração, por exemplo, uma pessoa que conta uma história, no tempo passado, produz um gesto que represente o ato de correr, mas levanta a mão, com a palma direcionada ao seu interlocutor, interrompendo-o apenas no espaço base, essa interrupção se mantém fora do espaço narrativo. Sendo assim, Sweetser e Stec (2016) afirmam que a segmentação espacial oferece uma evidência crucial para a investigação acerca da marcação de espaços na narração: assim como acontece na emergência de gestos discursivos, os narradores tendem a utilizar a área do espaço base , diretamente entre o falante e o ouvinte, como o locus da interação do mundo real entre eles e, do mesmo modo, tendem a escolher uma área, no espaço físico, de um dos lados da linha falante-ouvinte, para representar o Espaço Mental do conteúdo da narrativa – o espaço narrativo. A representação dessa atividade, considerando as narrativas discutidas pelas autoras, está representada na Figura 32. Figura 31: Ilustração das narrativas “Certidão”, “Neve” e “Gatos” 79 Figura 32: Segmentação Corporal nas narrativas Certidão(a), Neve (b) e Gatos (c) Figura 32: Segmentação Corporal nas narrativas Certidão(a), Neve (b) e Gatos (c) Fonte: Sweetser e Stec (2006, p. 243)47 Fonte: Sweetser e Stec (2006, p. 243)47 Os gestos que representam os eventos no espaço narrativo ocorrem na parte física do espaço gestual dedicado à história, enquanto os gestos que emergem da interação entre os interlocutores no “aqui-agora” ocorrem, Espaço-base, ao longo da linha falante-ouvinte. Sweetser e Stec (2016) afirmam que a modalidade gestual utiliza mais articuladores independentes do que a fala, o que o torna, também, menos sequencial: diferentes articuladores gestuais de um narrador podem, simultaneamente, encenar diferentes partes de uma cena ou diferentes personagens de uma cena. Nesse sentido, conforme afirmam as autoras, os termos “ponto de vista do personagem” e “ponto de vista do observador” têm sido usados para caracterizar o contraste entre i) os gestos icônicos que representam as ações do corpo de uma personagem através do corpo do falante e ii) os gestos realizados pelo corpo do falante para representar objetos ou aspectos gerais da cena observada. Essa segmentação corporal é uma característica da ação corporal comunicativa, reconhecida tanto pelas Línguas de Sinais como nos Estudos de Gesto. Conforme demonstram Sweetser e Stec (2016), o olhar de um falante, assim como a direção da cabeça e as expressões faciais podem representar ações relativas a uma personagem da narrativa, enquanto as mãos do falante podem representar as ações de outra personagem. Se levarmos em consideração que essas encenações são acompanhadas por enunciados atribuídos a duas personagens da narrativa e tendem a ocorrer na área física destinada ao espaço narrativo, isso não causa um problema na comunicação, uma vez que nem o narrador e nem o falante 80 encontram empecilhos para interpretar que esses enunciados dizem respeito ou à personagem ou ao falante, pois os pontos de vista de cada um estão demarcados por diferentes articuladores corporais. O olhar de um narrador, ainda de acordo com as autoras (2016), pode representar tanto o olhar de uma personagem como o olhar do próprio falante na interação. Figura 32: Segmentação Corporal nas narrativas Certidão(a), Neve (b) e Gatos (c) Esse fenômeno de alternância entre o espaço narrativo e o espaço base nos gestos que coocorrem com a fala pode ser melhor visualizado na Figura 33, que dizem respeito, respectivamente à transcrição e as fotos da narrativa Certidão, na qual uma narradora conta para as pesquisadoras a história de que, na sua certidão de nascimento, que foi rasurada, consta, erroneamente, que ela é homem o que confunde um funcionário da prefeitura e, consequentemente, a impediu de adquirir a certidão de casamento. O olhar da narradora representa, alternativamente, i) o olhar cético do funcionário, olhando para o eu-passado dela e para o documento imaginado - conforme ilustrado nas imagens (inserir os números) e ii) o olhar do seu eu-passado, que era direcionado para o funcionário - conforme ilustrado na Figura 33. Figura 33: Narrativa “Certidão” Fonte: Sweetser e Stec (2016, p. 246) Fonte: Sweetser e Stec (2016, p. 246) No decorrer dessa sequência, as mãos da narradora representam a mão do funcionário da prefeitura segurando o documento, mesmo que sua face e olhar estejam representando seu eu-passado, que não está segurando o documento. Na Figura 33, as imagens 1 e 2 correspondem ao momento em que a narradora encena o funcionário manipulando seu documento e a narradora está olhando para seu interlocutor. Na imagem 3, a narradora representa o funcionário devolvendo o documento para o seu eu-passado. Nesse caso, segundo as autoras, é possível observar dois pontos de vista de personagem distintos, o eu-passado da narradora e o funcionário da prefeitura, representados simultaneamente pelo corpo segmentado da narradora. 81 Ainda de acordo com Sweetser e Stec (2016), o Espaço-base da interação está sempre presente na narração oral. É por esse motivo que os ouvintes não se sentem ofendidos, nas palavras das autoras, ao terem a atenção dividida quando o corpo e o olhar do narrador representam vividamente as personagens da narração. Esse comportamento é tão enraizado na comunicação humana que os falantes tendem a procurar o olhar dos interlocutores para assegurar que a atenção apropriada está sendo dada às sequências reencenadas. O olhar narrativo pode permanecer representando apenas o olhar do falante na interação em curso, mesmo que outras partes do corpo do falante não estejam, efetivamente, representando os seus movimentos, mas sim, as ações de uma das personagens da narração. As autoras destacam uma forte relação entre o olhar e a expressão facial. Figura 32: Segmentação Corporal nas narrativas Certidão(a), Neve (b) e Gatos (c) Na narrativa “Gatos”, na qual a narradora conta a história de como seu namorado gosta de brincar com seu gato, as mãos da narradora se situam no espaço narrativo, preparadas para a expressão do conteúdo narrativo, enquanto o olhar e a direção da cabeça já estão direcionados para a história. Essa atividade está representada na Figura 34: a cabeça da narradora se move em direção ao seu interlocutor ao longo do espaço base na interação presente na linha falante-ouvinte, conforme representado na imagem 2. No final da interação com seu interlocutor, a narradora marca o retorno ao conteúdo da narrativa linguisticamente, quando enuncia a palavra “então”, enquanto o olhar retorna ao espaço da narrativa, conforme representado na imagem 4. Figura 34: Narrativa “Gatos” Fonte: Sweetser e Stec (2016, p. 251) Figura 34: Narrativa “Gatos” Fonte: Sweetser e Stec (2016, p. 251) Por fim, conforme explicam as autoras, uma forma de integração de pontos de vista é alcançada pela combinação da encenação corporal da personagem com o olhar e fala narrativos do espaço base. As autoras afirmam, ainda, que em qualquer momento da narrativa, o narrador pode interagir com os interlocutores no espaço base e retornar para a performance da narrativa no espaço narrativo. Se o narrador precisa se direcionar para os ouvintes no meio da narrativa, ele tende a direcionar sua cabeça e olhar para esse ouvinte por um determinado período de 82 tempo, mesmo que os gestos, por exemplo, estejam alocados no espaço narrativo. Essa atividade, de certa maneira, interrompe o curso narrativo e o enredo. No entanto, as autoras afirmam que essa interrupção pode estar presente apenas na modalidade gestual, o que funciona como um dispositivo de verificação visual em relação aos interlocutores, sem interromper, verbalmente, o curso da narração. Esse fenômeno pode ser visualizado na Figura 35, que diz respeito à narrativa “Neve”, na qual o narrador conta a história de como ele e sua esposa dirigiam perigosamente em uma nevasca. Figura 35: Narrativa “Neve” Fonte: Sweetser e Stec (2016, p. 252) Figura 35: Narrativa “Neve” 48 A trilha 3 diz respeito à transcrição que se refere ao GAT 2 (SELTING, et. al. 2009) e faz parte do bloco 2 do LASG (BRESSEM; MÜLLER; LADEWIG, 2013) Figura 35: Narrativa “Neve” Fonte: Sweetser e Stec (2016, p. 252) No caso específico dessa narrativa, o narrador está encenando uma viagem que ele e sua esposa, que se encontra do lado dele, fizeram juntos. No momento da encenação, o casal está alocado em uma posição espacial muito semelhante à posição espacial do evento narrado. Enquanto o narrador encena ambas as partes da conversação durante a viagem, ele, muito brevemente, vira sua cabeça e encontra o olhar de sua esposa, antes e depois de mencionar seu enunciado passado, “narrador: oh, vamos voltar e [minha mulher] vai e diz não não não continue indo continue indo”. O texto de Sweetser e Stec (2016) que discutimos na seção anterior, teve como objetivo apresentar pelo menos algumas maneiras com as quais o olhar contribui para a manutenção de múltiplos pontos de vista na estrutura gestual de narrativas do inglês americano. As autoras afirmam que os estudos sobre o olhar são significativos principalmente porque evidenciam o modo como os Espaços Mentais são criados a partir do Espaço-base. 83 49 GNT é um canal de televisão por assinatura que faz parte dos Canais Globo. Criado em 1991, com o nome de Globosat News Television, nessa época, seu foco era transmitir notícias. A partir de 2003, uma reformulação passou a orientar seu foco para os assuntos de interesse do universo familiar, com uma programação que oferece entretenimento e informação, abordando, com leveza, os mais diversos temas, como comportamento, gastronomia, moda, sexo, séries, documentários e filmes. 4 PROCEDIMENTOS METODOLÓGICOS Neste capítulo, descrevemos, de forma panorâmica, a narrativa analisada e abordamos os procedimentos de coleta e análise dos dados. Em seguida, trataremos de questões específicas que dizem respeito à análise de dados multimodais nas narrativas em questão, levando em consideração os dois articuladores multimodais: os gestos manuais e direção do olhar. O primeiro passo da nossa análise, foi verificar as escolhas linguísticas de cada ocorrência com base nos pressupostos de Dancygier (2011), em cada trecho narrativo (trilha 1). A partir de então, partimos para a análise multimodal das ocorrências, com respaldo na identificação e descrição dos núcleos (strokes) gestuais, conforme definição de Kendon (2004), na trilha 2, e, posteriormente, identificamos a forma do gesto, levando em consideração os parâmetros que dizem respeito ao bloco 1 do Sistema Linguístico de Anotação Gestual (LASG) (BRESSEM; LADEWIG; MÜLLER, 2013), nas trilhas 4, 5, 6, 7, 8 9, 1048. Já no que diz respeito à marcação de Espaços mentais pelos gestos, utilizamos os parâmetros de Cassell e McNeill (1990) que relacionam os gestos com o segmento da narração em que determinada interação se aloca, seja ele parte de uma interação no aqui-agora (Ground) ou parte de uma interação no “mundo” da narrativa (Espaço Narrativo), na trilha 11. Já no que diz respeito à marcação de Espaços Mentais pela direção do olhar, utilizamos os parâmetros de Sweetser e Stec (2016), que afirmam que o olhar de um narrador pode representar tanto o olhar de uma personagem (no Espaço Narrativo) como o olhar do próprio narrador na interação (no Ground), na trilha 16. Nesse contexto, analisamos, também a direção do olhar, na trilha 14, e a direção do tronco, na trilha 15. Em termos metodológicos, somente nos casos em que os gestos estão alocados no Espaço Narrativo, analisamos, com base em McNeill (1995), se eles se configuram como gestos do ponto de vista do observador (O-VPT) ou como gestos do ponto de vista da personagem (C- VPT), na trilha 12. Ainda nesses casos, analisamos os gestos quanto à sua função dominante no interior da narrativa: representação, expressão ou apelo, conforme Müller (2013), na trilha 13. Somente nos casos em que a direção do olhar marcou o Espaço Narrativo, analisamos, também, a sua função no interior da narrativa, de acordo com os pressupostos de Sweetser e Stec (2016): i) encenação da personagem e ii) narração propriamente dita, na trilha 17. 84 p g 50 De acordo com o site da GNT, o programa de entrevistas “Que História É Essa, Porchat?” foi criado por Fábio Porchat, produzido pelo Porta dos Fundos e exibido desde 6 de agosto de 2019 no canal de televisão por assinatura. O talk show venceu o Prêmio APCA de Melhor Programa de TV, em 2019. 49 GNT é um canal de televisão por assinatura que faz parte dos Canais Globo. Criado em 1991, com o nome de Globosat News Television, nessa época, seu foco era transmitir notícias. A partir de 2003, uma reformulação passou a orientar seu foco para os assuntos de interesse do universo familiar, com uma programação que oferece entretenimento e informação, abordando, com leveza, os mais diversos temas, como comportamento, gastronomia, moda, sexo, séries, documentários e filmes. 50 De acordo com o site da GNT, o programa de entrevistas “Que História É Essa, Porchat?” foi criado por Fábio Porchat, produzido pelo Porta dos Fundos e exibido desde 6 de agosto de 2019 no canal de televisão por assinatura. O talk show venceu o Prêmio APCA de Melhor Programa de TV, em 2019. 51 Laboratório de Estudos em Linguagem e Cognição (LeCogLing - DGP/CNPq), coordenado pela Prof.ª Dr.ª Maíra Avelar. 4.1 Procedimentos da Coleta de Dados O objetivo do presente trabalho é investigar, a partir de dados multimodais, o comportamento dos gestos e da direção do olhar em narrativas multimodais do português brasileiro, em contextos narrativos. Para tanto, utilizamos excertos do programa do canal GNT49, um dos canais de assinatura do grupo Globo, disponibilizados no YouTube, “Que História É Essa, Porchat?”50, apresentado pelo ator e humorista Fábio Porchat. Nesse programa, o apresentador convida três personalidades da televisão e da música, para que eles compartilhem histórias engraçadas e surpreendentes. A escolha do programa se justifica pela dinâmica de narração de eventos passados, e, também, por sua configuração espacial: o convidado interage com o apresentador, com os outros dois convidados, com a plateia e, ocasionalmente, com o público de casa, acessado pela câmera. Coletamos, do canal do Youtube da atração, uma narrativa na qual a atriz Marisa Orth relata a experiência de que, quando solteira, saiu para jantar com um rapaz “muito bem recomendado” por suas amigas e, surpreendentemente, descobriu que se tratava de um homem casado. O episódio foi ao ar no dia 12 de novembro de 2019 e contou com a presença da cantora Ivete Sangalo e da atriz e humorista Samanta Schmütz. p , g , , , , 50 De acordo com o site da GNT, o programa de entrevistas “Que História É Essa, Porchat?” foi criado por Fábio Porchat, produzido pelo Porta dos Fundos e exibido desde 6 de agosto de 2019 no canal de televisão por assinatura. O talk show venceu o Prêmio APCA de Melhor Programa de TV, em 2019. 85 Figura 36: Narrativa de Marisa Orth Figura 36: Narrativa de Marisa Orth Fonte: Youtube do canal GNT Figura 36: Narrativa de Marisa Orth Fonte: Youtube do canal GNT Fonte: Youtube do canal GNT Fonte: Youtube do canal GNT 4.2 Procedimentos Metodológicos de Análise 52 It addresses necessary aspects for a description of gestures’ forms and for a reconstruction of their meanings and functions with and without speech and explicates underlying theoretical and methodological assumptions. 4.2 Procedimentos Metodológicos de Análise A fim de realizar as análises das amostras selecionadas, utilizamos o ELAN (SLOETJES; WITTENBURG, 2008), software profissional de anotações complexas que permite a análise de arquivos audiovisuais. Esse programa, que vem sendo utilizado pelo Laboratório de Estudos em Cognição e Linguagem – LeCogLing51, desde sua fundação, é um software muito útil para análises linguísticas uma vez que o usuário pode adicionar inúmeros tipos de anotação textual seja para arquivos de áudio e também para mídias audiovisuais, como no nosso caso que trabalhamos com dados multimodais. De acordo com os desenvolvedores do software, uma anotação pode ser uma sentença, uma palavra, um comentário, uma tradução ou uma descrição de qualquer aspecto observado na mídia selecionada através de trilhas que são editáveis e podem ser preenchidas, conforme é possível observar na Figura 37. 86 Figura 37: Captura de tela do ELAN Figura 37: Captura de tela do ELAN Fonte: Dados do Pesquisador Fonte: Dados do Pesquisador De acordo com Bressem, Ladewig e Müller (2013), diversos tipos de transcrição e anotação são utilizados no campo dos Estudos de Gesto. Nesse sentido, levando em consideração uma perspectiva baseada na abordagem da linguística cognitiva e nas análises gestuais, as autoras propõem o Sistema Linguístico de Anotação Gestual – The Linguistic Annotation System for Gestures (LASG), que oferece diretrizes para a anotação dos gestos de acordo com os níveis de descrição linguística. O sistema, além de propor uma análise dos gestos a partir de uma perspectiva de uma gramática multimodal, nas palavras das autoras, “aborda aspectos necessários para uma descrição das formas gestuais e uma reconstrução de seus sentidos e funções com e sem a fala” (BRESSEM; LADEWIG; MÜLLER, 2013, p. 1099)52. A primeira parte do bloco 1 do LASG (BRESSEM; LADEWIG; MÜLLER, 2013), ainda, possui quatro parâmetros para a análise da forma de gestos baseadas em Bressem (2013): formato da mão, orientação, movimento e posição no espaço gestual. Nas Figuras a seguir, optamos por dividir as trilhas em duas fases: no primeiro momento, as trilhas que dizem respeito à narrativa de forma geral unidas às trilhas que tratam da forma e função dos gestos manuais e, no segundo momento, as trilhas que dizem respeito à direção do olhar. Essa divisão é meramente ilustrativa e didática, uma vez que no ELAN (SLOETJES; WITTENBURG, 2008), todas as trilhas fazem parte de uma mesma trilha-mãe. Figura 38: Trilhas de Análise do Articulador Multimodal “Gestos” Fonte: Dados do Pesquisador Fonte: Dados do Pesquisador Fonte: Dados do Pesquisador Figura 39: Trilhas de Análise do Articulador Multimodal “Direção do Olhar” Fonte: Dados do Pesquisador Figura 39: Trilhas de Análise do Articulador Multimodal “Direção do Olhar” Fonte: Dados do Pesquisador Nas próximas seções, abordaremos os parâmetros utilizados em cada uma das trilhas de análise. 4.2 Procedimentos Metodológicos de Análise 52 It addresses necessary aspects for a description of gestures’ forms and for a reconstruction of their meanings and functions with and without speech and explicates underlying theoretical and methodological assumptions. 87 Figura 38: Trilhas de Análise do Articulador Multimodal “Gestos” 4.2.1 Trilha 1: Trecho Narrativo Para que conseguíssemos analisar a narrativa de maneira mais apurada, optamos por utilizar uma divisão que respeitasse os seus elementos, sendo assim, seguindo a lógica clássica 88 do enredo (GANCHO, 2006), que concebe a narrativa em quatro partes, sendo elas, a exposição (ou introdução ou apresentação), a complicação (ou desenvolvimento), o clímax e o desfecho (desenlace ou conclusão), dividimos a narrativa em questão em três blocos: i) a exposição (00m34s), que diz respeito à introdução ou apresentação dos fatos iniciais da narrativa e das personagens; ii) o clímax (00m53s), que se refere ao conflito, o momento culminante da história, isto é, o momento de maior tensão e iii) o desfecho (00m34s), que corresponde à conclusão do conflito. A exposição da narrativa inicia no instante em que a atriz pergunta ao apresentador “posso contar minha história?” e introduz a sua narrativa. Já no clímax, ela apresenta o conflito que surgiu a partir de uma informação obtida entre ela, como personagem, e o homem com quem saiu, em um diálogo entre eles. Finalmente, no desfecho ela apresenta a mudança de atitude dela mesma, como personagem, que constitui a conclusão do conflito. Levando em consideração que os excertos que analisamos são curtos, optamos por compreender a complicação da narrativa (GANCHO, 2006) como parte do clímax. 4.2.2 Trilha 2: Stroke (Núcleo) Conforme explicamos no capítulo teórico, para Kendon (2004), uma unidade da ação gestual é iniciada quando a mão inicia a excursão, a partir da posição de descanso, e termina quando a mão retorna à posição de descanso. Desse modo, o autor propõe que, tipicamente, o ato da gesticulação ou excursão gestual é constituída pelas seguintes fases: i) a preparação, fase na qual a mão sai, configurada em um formato específico, da posição de descanso para o núcleo; ii) o stroke (aqui, traduziremos como núcleo gestual), fase que compreende o principal movimento expressivo reconhecido como “o gesto” e, por fim, iii) a retração, fase na qual a mão retorna do núcleo gestual para a posição de descanso. Sendo assim, nessa trilha, marcamos o momento no qual o gesto manual se inicia e, em seguida, avaliaremos se trata-se de um núcleo gestual simples ou de uma frase gestual conforme explicamos no Quadro 4, a seguir: Quadro 4: Tipos de Núcleo Gestual Núcleo simples Quando o gesto é composto por apenas um formato de mão, orientação da palma e uma direção de movimento entre a preparação e a retração. Quadro 4: Tipos de Núcleo Gestual Quando o gesto é composto por apenas um formato de mão, orientação da palma e uma direção de movimento entre a preparação e a retração. 89 Frase Gestual Quando um ou mais dos parâmetros “formato da mão”, “orientação da palma” e “direção do movimento” é alterado entre a preparação e a retração ou quando o núcleo gestual simples é repetido durante as fases de preparação e retração. Fonte: Elaborado pelo autor. Frase Gestual Quando um ou mais dos parâmetros “formato da mão”, “orientação da palma” e “direção do movimento” é alterado entre a preparação e a retração ou quando o núcleo gestual simples é repetido durante as fases de preparação e retração. Fonte: Elaborado pelo autor. Quando um ou mais dos parâmetros “formato da mão”, “orientação da palma” e “direção do movimento” é alterado entre a preparação e a retração ou quando o núcleo gestual simples é repetido durante as fases de preparação e retração. Fonte: Elaborado pelo autor. Nessa trilha de análise, preenchemos a porção de texto proferida no instante em que marcamos o núcleo gestual. 4.2.2 Trilha 2: Stroke (Núcleo) Considerando o contexto referencial, Cienki (2017) indica que ela é importante para a identificação do tópico enunciado pelo falante, servindo como evidência para a interpretação do que é relacionado ao próprio discurso e para qual dos interlocutores o enunciado é orientado. 4.2.3 Trilha 3: Transcrição O Sistema Linguístico de Anotação Gestual (LASG), conforme explicam Bressem, Müller e Ladewig (2013), no que diz respeito à anotação da fala, sugere o sistema GAT 2 (Gesprächsanalytisches Transkriptionssystem) (SELTING et al., 2009), em português, Sistema de Transcrição para Análise da Conversa que, de acordo com Schröder e colaboradores (2016) “é um sistema de transcrição para notação da fala e prosódia da fala-em-interação cotidiana autêntica. a transcrição das ocorrências analisadas” (SCHRÖDER et al, 2016, p. 8). Nesse sentido, nessa trilha, em cada ocorrência, transcrevemos, com base no GAT 2 (SELTING et al., 2009) a fala que coocorre com os gestos analisados, conforme é possível observar no Capítulo 5 e nos Anexos B, D, E e F. Esse é o único procedimento de análise que não faz parte do bloco 153 do LASG (BRESSEM; MÜLLER, LADEWIG, 2013). 53 Esse parâmetro faz parte do Bloco 2 do LASG (BRESSEM; MÜLLER; LADEWIG, 2013), que diz respeito à Análise Sequencial. p p respeito à Análise Sequencial. 4.2.4 Trilha 4: Formato das Mãos Conforme explicam Bressem, Müller e Ladwig (2013), para iniciar a análise dos articuladores envolvidos no gesto manual, é preciso partir do formato das mãos. Conforme 90 explicam as autoras, LASG está baseado em uma abordagem semiótica para os gestos, que compreende uma separação entre forma, sentido e função gestual. Nesse sentido, segundo Bressem (2013), para a descrição do parâmetro “formato da mão”, inspirado no sistema de codificação para a língua de sinais alemã, são utilizadas quatro categorias básicas: mão aberta, punho, dedos individuais e combinação de dedos, conforme ilustramos no Quadro 5. Quadro 5: Formato das Mãos Mão aberta A palma é o aspecto mais proeminente da configuração da mão Punho A mão está configurada como um punho Dedos individuais Dedos individuais determinam a configuração da mão. Combinação de dedos A combinação de dedos por si só, ou acompanhados da palma dominam o formato da mão. Fonte: Adaptado de Bressem (2013, p. 1085). 91 4.2.5 Trilha 5: Orientação das Palmas 4.2.5 Trilha 5: Orientação das Palmas O parâmetro “orientação”, conforme explica Bressem (2013), depende da orientação da palma e do espaço gestual. Desse modo, esse procedimento é dividido em duas partes, nos quais, em primeiro lugar, identificamos a orientação da palma e, em seguida, a orientação da mão em relação ao espaço gestual no qual o gesto é realizado. Sendo assim, os parâmetros de análise do primeiro procedimento são: palma para cima, palma para baixo, palma lateral, palma vertical e palma diagonal, conforme ilustrado no Quadro 6. A fim de elaborar uma trilha de análise que seja coerente com os nossos dados, para o segundo procedimento, decidimos modificar o sistema de anotação da seguinte maneira: ao invés de utilizar os parâmetros “em direção ao centro”; “para fora do centro”; “em direção ao corpo” e para fora do corpo”, decidimos utilizar apenas “em direção ao corpo”, considerando o corpo do falante e “para fora do corpo”, conforme ilustrado no Quadro 7. Quadro 6: Orientação das Palmas Palma para cima Palma para baixo Palma lateral Quadro 6: Orientação das Palmas Quadro 6: Orientação das Palmas 92 92 Palma vertical Palma diagonal Fonte: Elaborado pelo pesquisador com base em Bressem (2013). Fonte: Elaborado pelo pesquisador com base em Bressem (2013). Fonte: Elaborado pelo pesquisador com base em Bressem (2013). 93 Quadro 7: Orientação das Palmas em Relação ao Espaço Gestual Em direção ao corpo Para fora do corpo Fonte: Elaborado pelo pesquisador com base em Bressem (2013, p. 1087). Fonte: Elaborado pelo pesquisador com base em Bressem (2013, p. 1087). 4.2.6 Trilha 6: Posição do Movimento Bressem (2013) afirma que, no que diz respeito ao parâmetro “posição”, o LASG (BRESSEM; MÜLLER; LADEWIG, 2013) utiliza o conceito fundamentado por McNeill (1995) que divide o espaço gestual em setores, a partir de um sistema denominado pelo autor como “quadrados concêntricos”. No entanto, também de acordo com Bressem (2013), para que nossa análise conseguisse tratar de movimentos e posições mais detalhadas no espaço gestual, utilizamos nas nossas trilhas de análise as dimensões de Fricke (2005) que estabelece as quatro dimensões para o espaço gestual: i) 0 = próprio corpo do falante, ii) 1 = distância próxima do corpo, iii) 2 = distância média do corpo, e iv) 3 = distância longa do corpo. 94 4.2.7 Trilha 7: Tipos do Movimento 4.2.7 Trilha 7: Tipos do Movimento De acordo com Bressem (2013), os padrões de movimento são categorizados com base nas seguintes categorias: i) movimento reto, ii) movimento arqueado, iii) movimento circular, iv) movimento em espiral, v) movimento em zigzag e vi) movimento em linha S, conforme é possível observar no Quadro 8, a seguir. O LASG (BRESSEM; MÜLLER; LADEWIG, 2013), ainda, trata dos tipos de movimentos realizados com o punho e com determinadas combinações de dedo. No entanto, até o momento, os parâmetros anteriormente mencionados foram os suficientes para o tipo de análise que propomos nesta dissertação. Quadro 8: Tipos do Movimento i) movimento reto ii) movimento arqueado iiii) movimento circular iv) movimento em espiral v) movimento em zigzag vi) movimento em linha S Fonte: Elaborado pelo pesquisador com base em Bressem (2013, p.1088). Quadro 8: Tipos do Movimento Fonte: Elaborado pelo pesquisador com base em Bressem (2013, p.1088). 95 4.2.8 Trilha 8: Direção do Movimento 4.2.8 Trilha 8: Direção do Movimento Ainda de acordo com Bressem (2013), após descrever o tipo de movimento, é preciso especificá-lo, ainda, quanto à sua direção. Sendo assim, três direções principais são distinguidas: i) movimentos ao longo do eixo horizontal (direita e esquerda); ii) movimentos ao longo do eixo vertical (para cima e para baixo), conforme é possível observar na Figura 40, e iii) movimentos ao longo do eixo sagital (para fora do corpo e em direção ao corpo), conforme é possível observar na Figura 41. Figura 41: Movimentos ao longo do Eixo Sagital Figura 41: Movimentos ao longo do Eixo Sagital Fonte: Traduzido de Bressem (2013, p. 1090) Fonte: Traduzido de Bressem (2013, p. 1090) 4.2.9 Trilha 9: Qualidade do Movimento Bressem (2013), em seguida, trata do parâmetro qualidade do movimento que, por sua vez, é responsável por descrever os seguintes aspectos dos gestos: i) tamanho (reduzido ou ampliado), ii) velocidade (acelerado ou lento) e iii) fluxo do movimento. Nessa pesquisa, optamos por analisar somente o terceiro aspecto, pois consideramos que apenas o parâmetro “fluxo” pode ser revelador no nível de analisar as possíveis encenações realizadas pela gesticuladora. Figura 40: Movimento ao longo dos Eixos Horizontal e Vertical Figura 40: Movimento ao longo dos Eixos Horizontal e Vertical Fonte: Traduzido de Bressem (2013, p. 1089) Fonte: Traduzido de Bressem (2013, p. 1089) 96 4.2.11 Trilha 11: Marcação dos Espaços Mentais (Gestos) 4.2.11 Trilha 11: Marcação dos Espaços Mentais (Gestos) Em relação à marcação de Espaços Mentais pelos gestos, utilizamos os parâmetros de Cassell e McNeill (1990) que relacionam os gestos com o segmento da narração em que determinada interação se aloca, seja ele parte de uma interação no aqui-agora (Ground) ou parte de uma interação no “mundo” da narrativa (Espaço Narrativo). Esses parâmetros podem ser visualizados no Quadro 10, a seguir. Quadro 10: Marcação dos Espaços Mentais pelos Gestos Ground Os gestos manuais são relativos à interação imediata no espaço base. Em geral, são gestos dêiticos e rítmicos. EN Os gestos manuais são relativos ao espaço narrativo e dizem respeito às entidades presentes na narração. Em geral, são gestos icônicos e metafóricos. Fonte: Elaborado pelo pesquisador com base em Cassell e McNeill (1990). Quadro 10: Marcação dos Espaços Mentais pelos Gestos Ground Os gestos manuais são relativos à interação imediata no espaço base. Em geral, são gestos dêiticos e rítmicos. EN Os gestos manuais são relativos ao espaço narrativo e dizem respeito às entidades presentes na narração. Em geral, são gestos icônicos e metafóricos. Fonte: Elaborado pelo pesquisador com base em Cassell e McNeill (1990). Quadro 10: Marcação dos Espaços Mentais pelos Gestos Fonte: Elaborado pelo pesquisador com base em Cassell e McNeill (1990). 4.2.10 Trilha 10: Modo de Representação Gestual Nessa trilha, partimos para os pressupostos de Müller (2014) que afirma que os gestos não são motivados por ações ou representações, porque eles constituem as representações, sendo assim, a autora estabelece os quatro modos de representação gestual: i) Encenar: As mãos se movem de modo que representam uma ação, podendo envolver a manipulação de um evento. ii) Corporificar: As mãos representam um objeto. iii) Moldar: As mãos são posicionadas de modo a representar o formato do objeto a partir do contorno desenhado pelas mãos. iv) 97 Desenhar: A mão ou as mãos se movem e representam, a partir das pontas dos dedos, um traço da forma do objeto. Esses parâmetros podem ser visualizados no Quadro 9, a seguir. Desenhar: A mão ou as mãos se movem e representam, a partir das pontas dos dedos, um traço da forma do objeto. Esses parâmetros podem ser visualizados no Quadro 9, a seguir. Quadro 9: Modos de Representação Gestual Encenar Corporificar Moldar Desenhar Fonte: Elaborado com base em Müller (2013, p. 1691) Quadro 9: Modos de Representação Gestual Fonte: Elaborado com base em Müller (2013, p. 1691) 98 4.2.12 Trilha 12: Ponto de Vista Gestual Conforme explicamos no capítulo teórico, Parrill (2000) aponta que o ponto de vista gestual é de igual importância na compreensão do ponto de vista conceptual, pois ele sugere que o conceptualizador interage de acordo com a perspectiva de dentro ou fora da cena: de acordo com a definição de McNeill (1995) os gestos nos quais o falante usa suas mãos como se ele fosse uma personagem da narrativa são categorizados como “gestos do ponto de vista da personagem” (por exemplo, simular o movimento dos braços durante uma corrida enquanto descreve um personagem correndo) , ao passo que os gestos nos quais o falante traça a trajetória de uma personagem ou indica a sua localização, como se o falante estivesse observando o personagem de uma distância são categorizados como “gestos do ponto de vista do observador. É importante ressaltar, ainda, que essa trilha só será preenchida, caso, na trilha anterior, o espaço marcado for o narrativo (EN). 99 Quadro 11: Marcação dos Pontos de Vista Gestuais na Narrativa Quadro 11: Marcação dos Pontos de Vista Gestuais na Narrativa Quadro 11: Marcação dos Pontos de Vista Gestuais na Narrativa Personagem Quando os gestos são utilizados pelo falante como se ele fosse uma personagem da narrativa Observador Quando os gestos são utilizados pelo falante como se ele traçasse a trajetória de uma personagem ou de uma entidade, ou, ainda, indicasse a localização de uma personagem ou de uma entidade em uma narrativa Fonte: Elaborado pelo pesquisador com base em McNeill (1995) 4.2.14 Trilha 14: Direção do Olhar Com base em Sweetser e Stec (2016), na trilha denominada “Direção do Olhar”, verificamos se o olhar da narradora está direcionado para à Linha-Falante-Ouvinte, ou para outra localização no espaço gestual, podendo ser para cima, para baixo ou para a esquerda e para a direita. 4.2.13 Trilha 13: Função dos Gestos no Interior da Narrativa Conforme explicamos no capítulo teórico, Müller (2013), a partir dos trabalhos de Bühler (1982) sobre a teoria psicológica da linguagem e a teoria da expressão, propõe que os gestos possuem um potencial para a linguagem, uma vez que eles podem ser utilizados pra preencher as mesmas funções básicas da linguagem. Para ela, os gestos podem expressar estados interiores e sentimentos, podem regular o comportamento de outras pessoas e podem representar objetos e eventos no mundo. A abordagem funcional para os gestos, de acordo com a autora, se relaciona com os três aspectos básicos de qualquer encontro comunicativo: o falante, o ouvinte e o mundo sobre o qual se fala. Levando em consideração essa sistematização, a autora categoriza os gestos a partir de sua função dominante: i) representação, ii) expressão ou iii) apelo, sendo esses os parâmetros de análise, conforme ilustramos no Quadro 12. Quadro 12: Função dos Gestos no Interior da Narrativa Representação Quando os gestos representam outras entidades. i) função representacional que representa ações, objetos ou eventos concretos ii) função representacional que se refere a Quadro 12: Função dos Gestos no Interior da Narrativa Representação Quando os gestos representam outras entidades. i) função representacional que representa ações, objetos ou eventos concretos ii) função representacional que se refere a Quadro 12: Função dos Gestos no Interior da Narrativa Representação Representação 100 ações, objetos e eventos abstratos – os gestos metafóricos. Expressão Encontramos gestos como o de levantar as mãos para expressar alegria ou triunfo, esconder o rosto para expressar tristeza ou sofrimento, ou mover o punho para baixo para representar raiva. De acordo com Müller (2013), essa categoria gestual está relacionada a uma determinada expressão facial, uma orientação do olhar em particular (sobre o qual trataremos na próxima seção) e uma postura corporal. Apelo Os gestos dessa categoria são utilizados para regular o comportamento de outras pessoas. Fonte: Elaborado pelo pesquisador, com base em Müller (2013) Os gestos dessa categoria são utilizados para regular o comportamento de outras pessoas. Fonte: Elaborado pelo pesquisador, com base em Müller (2013) 4.2.16 Trilha 16: Marcação dos Espaços Mentais (Direção do Olhar) 4.2.16 Trilha 16: Marcação dos Espaços Mentais (Direção do Olhar) Sweetser e Stec (2016) afirmam que a segmentação espacial oferece uma evidência crucial para a investigação acerca da marcação de Espaços Mentais: assim como acontece na emergência de gestos discursivos, os narradores tendem a utilizar a área do Espaço-base, diretamente entre o falante e o ouvinte, como o locus da interação do mundo real entre eles e, do mesmo modo, tendem a escolher uma área, no espaço físico, de um dos lados da linha falante-ouvinte, para representar o Espaço Mental do conteúdo da narrativa – o espaço narrativo. Sendo assim, nessa trilha, identificamos se o olhar está direcionado para a SHL – a linha falante-ouvinte (speaker-hearer line), marcando o espaço base (ground), ou para o um dos lados dessa linha, marcando o espaço narrativo (EN), conforme sintetizamos no Quadro 13, a seguir. Quadro 13: Marcação de Espaços Mentais pela direção do olhar Ground O olhar está direcionado para a speaker- hearer line, demarcando a interação no espaço base (Ground) EM O olhar está direcionado para um dos lados da SHL (considerando a esquerda, direita, para cima e para baixo), demarcando a interação no espaço narrativo com os elementos da narração. Fonte: Elaborado pelo pesquisador com base em Sweetser e Stec (2016) Quadro 13: Marcação de Espaços Mentais pela direção do olhar Fonte: Elaborado pelo pesquisador com base em Sweetser e Stec (2016) 4.2.15 Trilha 15: Direção do Tronco Ainda com base em Sweetser e Stec (2016), na trilha “Direção do Tronco”, buscamos verificar a direção para a qual o tronco da narradora se direciona, podendo ser para o centro, para a esquerda ou para a direita. Esse parâmetro é fundamental, levando em consideração a configuração espacial do programa. 101 4.2.17 Trilha 17: Função da Direção do Olhar no Interior da Narrativa Conforme explicamos no capítulo teórico, Sweetser e Stec (2016) demonstram que o olhar de um falante, assim como a direção da cabeça e as expressões faciais podem representar ações relativas a uma personagem da narrativa, assim como podem fazer parte da narração propriamante dita. Se levarmos em consideração que essas encenações são acompanhadas por enunciados atribuídos a duas personagens da narrativa e tendem a ocorrer na área física destinada ao espaço narrativo, não haverá um problema na comunicação, uma vez que nem o 102 narrador nem o falante encontrarão empecilhos para interpretar que esses enunciados dizem respeito ou à personagem ou ao falante em si. Sendo assim, com base nos pressupostos das autoras, levamos em consideração duas funções principais do olhar na narrativa: i) encenação da personagem e ii) narração, conforme explicamos no Quadro 14. É importante ressaltar que essa trilha somente será preenchida quando o olhar marcar o espaço narrativo. Quadro 14: Função da Direção do Olhar no Interior da Narrativa Encenação Quando o olhar do narrador representa o olhar de uma personagem na narração. Narração Quando o olhar do narrador não expressa o olhar de uma personagem específica e não está inserido no ground. Fonte: Elaborado pelo pesquisador com base em Sweetser e Stec (2016) Quadro 14: Função da Direção do Olhar no Interior da Narrativa Fonte: Elaborado pelo pesquisador com base em Sweetser e Stec (2016) onte: Elaborado pelo pesquisador com base em Sweetser e Stec (2016) No Capítulo 5, a seguir, trataremos das análises multimodais realizadas com base nos parâmetros apresentados neste capítulo. Buscamos, com base nos pressupostos téorico- metodológicos dos autores mencionados anteriormente, lançar um olhar para os dois articuladores multimodais, os gestos e a direção do olhar, levando em consideração seu potencial para a marcação de Espaços Mentais no contexto narrativo. 103 5 ANÁLISE DOS DADOS A narrativa que escolhemos para a nossa análise foi proferida pela atriz Marisa Orth, que relata, para o apresentador Fábio Porchat, para a plateia e para os outros convidados do programa – a atriz Samantha Schmütz e a cantora Ivete Sangalo –, o momento no qual, por indicação de suas amigas, sai para jantar com um homem e descobre que ele escondia o fato de ser casado. Tal como proposto Fauconnier (2003), é possível observar na narrativa em questão que, à medida que o discurso da narradora se desenvolve, espaços mentais são criados para alocar informações que vão além do contexto imediato, alocado no Espaço-base. Levando em consideração que a história sobre a qual se narra está situada no tempo passado – fato que, automaticamente, é marcado pelo uso dos verbos no pretérito – observamos que, naturalmente, essas informações estão alocadas no Espaço Narrativo, enquanto a interação imediata da atriz com o apresentador do programa e com as outras convidadas está alocada no Ground, o Espaço- base. Na Figura 42, a seguir, podemos observar a disposição espacial das interações presentes no programa. Figura 42: Disposição Espacial da Narrativa de Marisa Orth Fonte: Dados do Pesquisador./Canal da GNT no Youtube Figura 42: Disposição Espacial da Narrativa de Marisa Orth Fonte: Dados do Pesquisador./Canal da GNT no Youtube Na Figura 43, é possível observar alguns elementos importantes que fazem parte da configuração espacial da narrativa com a qual trabalhamos: em A, temos a narradora em questão, a atriz Marisa Orth; em B, temos o apresentador do programa que, na maioria das vezes, possui o papel de mediador, uma vez que ele conhece previamente as histórias que serão 104 narradas, atribuindo conexões entre elas; em C, temos Samantha Schmütz, interlocutora que, antes de Marisa Orth proferir a narrativa que analisamos, contou uma história a respeito de um encontro que também deu errado; em D, temos Ivete Sangalo que muitas vezes interage a narradora; em E, temos a plateia; e em F, temos o espaço que configura o semicírculo, que permite que o telespectador, de casa, sinta-se parte dessa roda de conversa. Conforme mencionamos anteriormente, Marisa Orth narra a tentativa de um encontro romântico com um homem recomendado por suas amigas, cujos nomes não são mencionados. 5 ANÁLISE DOS DADOS Sendo assim, é possível observar que ela reencena, em específico, as seguintes personagens desse evento passado: seu past self, isto é, seu eu-passado, suas amigas dando determinadas recomendações para ela e o homem em questão. De acordo com essas informações, e com base na representação de Fauconnier (2003, p. 36), elaboramos o diagrama, representado pela Figura 43, a seguir: Figura 43: Representação dos espaços mentais – Narrativa Marisa Orth Figura 43: Representação dos espaços mentais – Narrativa Marisa Orth x2 x3 Figura 43: Representação dos espaços mentais – Narrativa Marisa O x x1 a R N Fonte: Dados do Pesquisador. Adaptado de Fauconnier (2003, p. 36) Fonte: Dados do Pesquisador. Adaptado de Fauconnier (2003, p. 36) Conforme é possível observar no diagrama representado na Figura 44, esse esquema diz respeito à interação que ocorre no Espaço-base e, também, às narrações e reencenações que ocorrem no Espaço Narrativo. Marisa Orth, representada por x, reencena, no Espaço Narrativo (representado por N), ela mesma no passado (Past Self) – seu eu-passado – representado por x¹), a instância que corporifica a(s) pessoa(s) que recomendou(aram) o homem para ela, representada por x² e o homem do encontro, representado por x³. As linhas retas representam a função da reencenação de x¹, x² e x³ por x, enquanto a linha pontilhada representa a interação com seus interlocutores (representados por a), no Espaço-base, representado por R. Seguindo a lógica clássica do enredo (GANCHO, 2006), que concebe a narrativa em quatro partes, sendo elas, a exposição (ou introdução ou apresentação), a complicação (ou desenvolvimento), o clímax e o desfecho (desenlace ou conclusão), dividimos a narrativa em questão em três blocos: i) a exposição (00m34s), que diz respeito à introdução ou apresentação 105 dos fatos iniciais da narrativa e das personagens; ii) o clímax (00m53s), que se refere ao conflito, o momento culminante da história, isto é, o momento de maior tensão e iii) o desfecho (00m34s), que corresponde à conclusão do conflito54. A exposição da narrativa inicia no instante em que a atriz pergunta ao apresentador “posso contar minha história?” e introduz a sua narrativa. Já no clímax, ela apresenta o conflito que surgiu a partir de uma informação obtida entre ela, como personagem, e o homem com quem saiu, em um diálogo entre eles. Finalmente, no desfecho ela apresenta a mudança de atitude dela mesma, como personagem, que constitui a conclusão do conflito. 54 Conforme explicamos no Capítulo 4, devido ao caráter curto das nossas narrativas, optamos por compreender a complicação da narrativa (GANCHO, 2006) como parte do clímax. 5 ANÁLISE DOS DADOS Nas próximas subseções, referentes à análise multimodal dos dados da narrativa em questão, apresentaremos uma descrição das ocorrências referentes às três amostras de análise: 3 (três) ocorrências do bloco “Exposição”, 5 (cinco) ocorrências do bloco “Clímax” e 3 (três) ocorrências do bloco “Desfecho”. Na transcrição, representamos os turnos de fala, considerando as iniciais dos participantes dessa interação. Em primeiro lugar, analisamos as escolhas linguísticas de cada ocorrência com base nos pressupostos de Dancygier (2011). Em seguida, partimos para a análise multimodal das ocorrências, com respaldo na identificação e descrição dos núcleos (strokes) gestuais, conforme definição de Kendon (2004) e, posteriormente, identificamos a forma do gesto, levando em consideração os parâmetros descritos no Capítulo 4, que dizem respeito ao bloco 1 do Sistema Linguístico de Anotação Gestual (LASG) (BRESSEM; LADEWIG; MÜLLER, 2013). Já no que diz respeito à marcação de Espaços mentais pelos gestos, utilizamos os parâmetros de Cassell e McNeill (1990) que relacionam os gestos com o segmento da narração em que determinada interação se aloca, seja ele parte de uma interação no aqui-agora (Ground) ou parte de uma interação no “mundo” da narrativa (Espaço Narrativo). Já no que diz respeito à marcação de Espaços Mentais pela direção do olhar, utilizamos os parâmetros de Sweetser e Stec (2016), que afirmam que o olhar de um narrador pode representar tanto o olhar de uma personagem (no Espaço Narrativo) como o olhar do próprio narrador na interação (no Ground). Somente nos casos em que os gestos marcaram o Espaço Narrativo, analisamos, com base em McNeill (1995), se eles se configuram como gestos do ponto de vista do observador (O-VPT) ou como gestos do ponto de vista da personagem (C-VPT). Ainda nesses casos, analisamos os gestos quanto à sua função dominante no interior da narrativa: representação, expressão ou apelo, conforme Müller (2013), a partir dos trabalhos de Bühler (1982) sobre a 106 teoria psicológica da linguagem e a teoria da expressão. Somente nos casos em que a direção do olhar marcou o Espaço Narrativo, analisamos, também, a sua função no interior da narrativa, de acordo com os pressupostos de Sweetser e Stec (2016): i) encenação da personagem e ii) narração propriamente dita. A fim de representar os dois articuladores multimodais com os quais trabalhamos, representamos com setas amarelas os gestos manuais e com setas vermelhas pontilhadas a direção do olhar. 5.1 Análise do bloco “Exposição” No Quadro 15, a seguir, colocamos em evidência, no bloco “Exposição”, as discussões propostas por Dancygier (2011), nas quais ela afirma que as narrativas podem ser tratadas como construções linguísticas, constituídas como um exemplo do discurso natural narrativo, no qual o falante, por meio da segmentação da narrativa, assume os papéis de narrador e assume, também, os papéis das personagens em questão, identificando as escolhas linguísticas da narrativa e lançando um olhar para a sua estrutura. A escolha das ocorrências ilustradas no Quadro 15 se justifica pelo bom enquadramento da câmera, que nos permitiu ter uma visualização completa, mesmo que de ângulos diferentes, das expressões faciais e dos gestos da narradora. Quadro 15: Escolhas linguísticas como evidências da estrutura da narrativa no bloco “Exposição” Ocorrência Transcrição Papel assumido EXPOSIÇÃO Ocorrência 1 eu tava solteira Narradora não ri AL:to Personagem: A pessoa cujo nome não é mencionado, que recomendou o homem para ela. ro 15: Escolhas linguísticas como evidências da estrutura da narrativa no bloco “Exposição” Quadro 15: Escolhas linguísticas como evidências da estrutura da narrativa no bloco ro 15: Escolhas linguísticas como evidências da estrutura da narrativa no bloco EXPOSIÇÃO Ocorrência 1 Ocorrência 1 107 EXPOSIÇÃO Ocorrência 2 EXPOSIÇÃO Ocorrência 3 faz um brinquINHO menor Personagem: A pessoa cujo nome não é mencionado, que recomendou o homem para ela. Fonte: Elaborado pelo pesquisador EXPOSIÇÃO Ocorrência 2 EXPOSIÇÃO Ocorrência 3 faz um brinquINHO menor Personagem: A pessoa cujo nome não é mencionado, que recomendou o homem para ela. Fonte: Elaborado pelo pesquisador EXPOSIÇÃO Ocorrência 2 EXPOSIÇÃO Ocorrência 3 Fonte: Elaborado pelo pesquisador EXPOSIÇÃO Ocorrência 2 Fonte: Elaborado pelo pesquisador Fonte: Elaborado pelo pesquisador Ainda de acordo com Dancygier (2011), os recursos narrativos, como, por exemplo, o uso do pronome “eu” (04 eu tava solteira) das frases imperativas (24 não ri AL:to) e interrogativas, marcam o constructo narrativo do narrador e, alternativamente, da personagem, além disso, cria tratamentos referenciais de acordo com as necessidades narrativas, respeitando o fato de que o evento narrado aconteceu em um determinado “aqui- agora”. Em conformidade com o que afirma a autora, Bressem e colaboradoras (2018) explicam que o ato de narrar constitui-se como uma prática comunicativa e reconstrutiva, que possui uma função extremamente relevante na comunicação face a face. 5.1 Análise do bloco “Exposição” Nas palavras de Bressem e colaboradoras (2018), ao narrar uma experiência pessoal, os falantes podem, por exemplo, ilustrar uma determinada situação, estruturar e sequenciar eventos passados, revelar dados de sua biografia etc. Sendo assim, esses recursos narrativos instituem técnicas de estruturação do discurso muito bem estabelecidas no corpo da narrativa. 55 Samantha Schmütz, na narrativa que contou no programa, revelou detalhes de uma história que aconteceu durante uma viagem internacional. No México, a atriz e comediante conheceu um rapaz durante uma viagem e as coisas não foram bem como planejadas. Assim que ela termina de contar sua história, a atriz Marisa Orth pede o turno de fala “Posso contar minha história? Lembro muito uma história assim” 5.1.1 Análise Multimodal da Ocorrência 1 do Bloco “Exposição” Na Figura 44 a seguir, apresentamos a representação multimodal da ocorrência 1 que consideramos representativa, uma vez que, por estar no bloco “exposição”, corresponde ao momento no qual a atriz direciona o olhar e o corpo a um dos seus interlocutores e apresenta o plano de fundo de sua narrativa, indicando os personagens envolvidos na situação comunicativa: MO para Marisa Orth, FP para Fábio Porchat, IS para Ivete Sangalo e SS para Samantha Schmütz. Ao longo do bloco “Exposição”, a atriz, conforme é possível observar no exemplo a seguir, solicita o turno de fala ao apresentador e, então, inicia a narração, indicando, 108 por meio do gesto utilizado e da marcação da direção do olhar, que sua narrativa se relaciona com a narrativa de Samanta Schümtz55. Figura 44: Representação multimodal da ocorrência 1 do bloco “Exposição” Figura 44: Representação multimodal da ocorrência 1 do bloco “Exposição” 01: MO lembro muito uma história assim 02: (gesto) 01: MO lembro muito uma história assim 02: (gesto) (gesto) 02: (gesto) 03 MO: me lembro muito uma história sim 03 MO: me lembro muito uma história sim 03 MO: me lembro muito uma história sim 03 MO: me lembro muito uma história sim 04 eu tava solteira 05 <<apontando com o dedo indicador da mão direita em 06 direção à direita>> Fonte: Elaborado pelo pesquisador 55 Samantha Schmütz, na narrativa que contou no programa, revelou detalhes de uma história que aconteceu durante uma viagem internacional. No México, a atriz e comediante conheceu um rapaz durante uma viagem e as coisas não foram bem como planejadas. Assim que ela termina de contar sua história, a atriz Marisa Orth pede o turno de fala “Posso contar minha história? Lembro muito uma história assim” 109 No que diz respeito a essa ocorrência, notamos que a frase gestual é iniciada com um gesto de apontar que ocorre com a sequência B1 “lembro muito de uma história assim”, indicando que, apesar de a interação ser composta por vários ouvintes, a narradora endereça o seu corpo com mais frequência para Samantha Schümtz, que está localizada à sua direita. No que diz respeito ao turno de fala, quando a narradora enuncia a sequência B1, notamos que o gesto de apontar acontece para indicar que sua história possui semelhanças com a narrativa contada por Samantha Schümtz, que também diz respeito a um encontro romântico. 5.1.1 Análise Multimodal da Ocorrência 1 do Bloco “Exposição” Também por isso, o tronco da narradora está direcionado para a direita. Na retração, o gesto é composto por uma mão aberta, com a palma para baixo. A mão também está direcionada para fora do corpo da falante, em direção à mesma interlocutora, conforme a seta em amarelo, assim como a direção do movimento. A direção do olhar, representada pela seta vermelha pontilhada está alocada no Ground, uma vez que essa interação acontece no Espaço-base. Trata-se, portanto de uma marcação do Ground, tanto pelo olhar – ela direciona o olhar para um interlocutor imediato, – quanto pelos gestos – o gesto em questão trata-se de um gesto no nível paranarrativo que coocorre com a sequência 03 “eu tava solteira”. Isto é, conforme afirmam Cassell e McNeill (1990), essa ocorrência faz parte do terceiro nível narrativo, denominado nível paranarrativo, pois, nesse caso, a narradora fala exercendo o papel de narradora. Embora ela assuma a posição de falante (considerando os papéis falante e ouvinte), ela, nesse trecho, fala de fora da situação narrativa. Nesse caso, também, tratam-se de gestos dêiticos, pois ela está apontando – tanto através do dedo indicador, como através da mão aberta com a palma para baixo – para um de seus ouvintes, Samantha Schmütz, que, conforme mencionamos anteriormente, havia contado uma história que também dizia respeito a uma desventura amorosa. Associando o nível paranarrativo às discussões propostas por Dancygier (2011), verificamos, no âmbito das escolhas linguísticas, o discurso indireto que representa a intervenção da narradora. Considerando que os gestos marcam o Ground é, não analisamos o ponto de vista gestual, a função dominante no interior da narrativa. Do mesmo modo, pelo fato de a direção do olhar também marcar o Ground, não analisamos sua função na narrativa. 5.1.2 Análise multimodal da ocorrência 2 do bloco “Exposição” Na Figura 45 a seguir, apresentamos a representação multimodal da ocorrência 2, que também faz parte do bloco 1 - “exposição”. Nessa ocorrência, podemos observar os eventos que, posteriormente, levarão ao clímax. 110 Figura 45: Representação multimodal da ocorrência 2 do bloco “Exposição” Figura 45: Representação multimodal da ocorrência 2 do bloco “Exposição” 24 não ri AL:to 25 <<levantando as duas mãos para cima junto com a direção 26 do olhar>> 27 não abre a [boca] 28 SS: [gente] 24 não ri AL:to 25 <<levantando as duas mãos para cima junto com a direção 26 do olhar>> 27 não abre a [boca] 28 SS: [gente] 29 MO: não deixa ele PERCEBER [que você é inteligente] 30 SS: [gente não VAle ] 31 né 29 MO: não deixa ele PERCEBER [que você é inteligente] 30 SS: [gente não VAle ] 32 IS: [esse é o cara pra você: ] 32 IS: [esse é o cara pra você: 111 33 MO: [não deixa ele perceber que você é inteligente ] Fonte: Elaborado pelo pesquisador Nessa segunda ocorrência, com base no bloco 1 do LASG (BRESSEM; LADEWIG, MÜLLER, 2013), é possível observar um gesto simples realizado pelas duas mãos abertas, cuja direção do movimento é para cima no núcleo, para baixo na retração e, ainda, na retração, uma das mãos se direciona para a esquerda. No núcleo, as palmas se direcionam lateralmente, e, na retração, para baixo. Observamos também que o gesto, cuja qualidade do movimento é acentuada, expressa a metáfora conceptual MAIS É PARA CIMA, uma vez que a expressão proferida diz respeito a falar com mais volume, acompanhada da unidade “24 não ri AL:to”, momento em que o seu volume de voz também aumenta. No núcleo, na sequência 02, o olhar está direcionado para cima, o que representa o olhar narrativo que faz parte da encenação de um personagem na narrativa, conforme Sweetser e Stec (2016), que deu um conselho para o eu-passado da narradora. Trata-se, portanto de uma marcação visual e gestual do Espaço Narrativo, no nível narrativo, já que o olhar não está direcionado para a linha falante- ouvinte (speaker-hearer line), mas sim, para cima, segundo Cassell e McNeill (1990) e Sweetser e Stec (2016). Por outro lado, o gesto, a partir do ponto de vista da personagem x² da narração, o que configura o gesto como um gesto C-VPT, de acordo com as categorizações de McNeill (1995), -nesse caso, a pessoa que fez recomendações para o eu-passado da narradora -, possui uma função expressiva, segundo as categorizações de Müller (2013). 5.1.3 Análise multimodal da ocorrência 3 do bloco “Exposição” 5.1.3 Análise multimodal da ocorrência 3 do bloco “Exposição” 5.1.3 Análise multimodal da ocorrência 3 do bloco “Exposição” Na Figura 46 a seguir, apresentamos a representação multimodal da ocorrência 3, a última corresponde ao bloco 1 - “exposição”. Essa ocorrência, em conformidade com a anterior, também compreende os eventos que levarão ao clímax da narrativa. Figura 46: Representação multimodal da ocorrência 3 do bloco “Desfecho” Figura 46: Representação multimodal da ocorrência 3 do bloco “Desfecho” Figura 46: Representação multimodal da ocorrência 3 do bloco “Desfecho” 34 faz um brinquINHO menor 35 <<levantando ambas as mãos em direção à sua própria 36 orelha e seus dedos indicadores e polegares 37 corporificam dois brincos pequenos>> 34 faz um brinquINHO menor 35 <<levantando ambas as mãos em direção à sua própria 36 orelha e seus dedos indicadores e polegares 37 corporificam dois brincos pequenos>> 34 faz um brinquINHO menor 35 <<levantando ambas as mãos em direção à sua própria 36 orelha e seus dedos indicadores e polegares 37 corporificam dois brincos pequenos>> 34 faz um brinquINHO menor 35 <<levantando ambas as mãos em direção à sua própria 36 orelha e seus dedos indicadores e polegares 37 corporificam dois brincos pequenos>> Fonte: Elaborado pelo pesquisador Figura 45: Representação multimodal da ocorrência 2 do bloco “Exposição” Nesse sentido, considerando as escolhas linguísticas conforme proposto por Dancygier (2011), nesse caso, observamos a presença do discurso, no segmento 24 não ri AL:to que, no modo imperativo, representa fielmente o discurso de uma das personagens da interação (a personagem x²) sobre a qual a narradora está falando, o que confirma o ponto de vista gestual da personagem. No momento de retração, as palmas se direcionam para baixo (conforme 05) e, posteriormente, acompanhada da direção do olhar e do tronco, a mão esquerda da narradora se direciona para a direita, correspondente à posição de Ivete Sangalo no espaço físico que, por sua vez, faz uma interrupção (conforme linha 32). Sendo assim, na realização do gesto que marca o Espaço Narrativo, notamos um deslocamento da direção do olhar, que parte do Espaço Narrativo para o Ground. O que pode ser interpretado como um recurso complexo que as narrativas multimodais possuem para a manutenção da marcação de mais de um Espaço Mental na mesma ocorrência. 112 Fonte: Elaborado pelo pesquisador No que se refere à forma dessa ocorrência, de acordo com os parâmetros do LASG ((BRESSEM; LADEWIG, MÜLLER, 2013), observamos que há uma combinação dos dedos polegar e indicador unidos a ambas as palmas laterais, representando o gesto clássico para representar medidas e quantidades pequenas, que coocorre com o segmento 34 faz um brinquINHO menor. O movimento das mãos é ascendente, mas o movimento dos dedos é constituído pela diminuição da distância entre os dedos combinados em direção ao próprio corpo da falante. E, no que diz respeito ao critério à qualidade do movimento, verificamos que se trata de um movimento fraco em relação ao seu fluxo. Além disso, em relação ao Modo de Representação Gestual, verificamos que o gesto corporifica um objeto físico da narração, que nesse caso, representa um brinco pequeno. Com respaldo nos parâmetros de Cassell e McNeill (1990), identificamos a marcação do gesto no Espaço Narrativo, considerando sua motivação icônica de representar um objeto 113 presente na narrativa a partir do modo “corporificar”. Segundo os autores, os gestos icônicos são os mais presentes no nível narrativo, uma vez que eles são reconhecidos pela forma e conteúdo e, nesse caso, é muito evidente a tentativa de configurar um aspecto do objeto sendo representado pelas mãos: o “brinquinho menor” que significa um brinco pequeno. Com base em McNeill (1995), categorizamos o gesto como O-VPT, uma vez que ele representa iconicamente o brinco a partir do ponto de vista do observador. Nesse caso, também, conforme os pressupostos de Dancygier (2011), observamos a presença do discurso direto, no segmento 34 faz um brinquINHO menor que, no modo imperativo, representa o discurso de uma das personagens da interação (a personagem x²). Já no que diz respeito à sua função dominante na narrativa, categorizamos o gesto como “representacional”, já que ele faz referência a um objeto que nessa interação é concreto. Partindo para as categorizações de Sweetser e Stec (2016), identificamos que a direção do olhar marca o Ground, uma vez que ela o direciona para um dos interlocutores, na linha Falante-Ouvinte. 5.1.4 Discussão dos dados do bloco “Exposição” A fim de instanciar os resultados do bloco “Exposição”, apresentamos, a seguir, uma discussão mais geral dos parâmetros de marcação de Espaços Mentais nas três ocorrências analisadas. No que diz respeito aos gestos manuais, encontramos uma marcação do Ground (ocorrência 1) e duas marcações do Espaço Narrativo (ocorrências 2 e 3). Já no que diz respeito à direção do olhar, encontramos duas marcações do Ground (ocorrências 1 e 3) e uma marcação do Espaço Narrativo. Conforme é possível observar no Quadro 16 a seguir, nas ocorrências 1 e 2 os gestos e a direção do olhar marcam simultaneamente o mesmo Espaço Mental (seja ele o Ground ou o Espaço Narrativo), enquanto na ocorrência 3, os articuladores marcam, independentemente, Espaços diferentes. Quadro 16: Marcação de Espaços Mentais nas ocorrências do bloco “Exposição” Ocorrência Gesto Direção do Olhar 1 Ground Ground 2 Espaço Narrativo Espaço Narrativo 3 Espaço Narrativo Ground Fonte: Elaborado pelo pesquisador Quadro 16: Marcação de Espaços Mentais nas ocorrências do bloco “Exposição” 114 No que diz respeito à categorização de McNeill (1995) a respeito do ponto de vista gestual, que pode ser do observador (O-VPT) ou da personagem (C-VPT), de forma geral, no bloco “Exposição” encontramos uma ocorrência cujo gesto marca o ponto de vista da personagem (C-VPT) (ocorrência 2) e uma ocorrência cujo gesto marca o ponto de vista do observador (O-VPT), conforme é possível observar no Quadro 17, a seguir: Quadro 17: Ponto de Vista dos Gestos Narrativos no bloco “Exposição” Ocorrência Ponto de Vista Gestual 1 Não analisamos, pois o gesto marca o Ground 2 C-VPT 3 O-VPT Fonte: Elaborado pelo pesquisador Quadro 17: Ponto de Vista dos Gestos Narrativos no bloco “Exposição” Fonte: Elaborado pelo pesquisador Partindo, agora, para as funções dos gestos alocados no Espaço Narrativo, utilizando a categorização de Müller (2013), que, a partir do trabalho de Bühler (1982), classifica os gestos a partir de sua função dominante: representação, expressão ou apelo. Os gestos que possuem a função representacional, de acordo com a autora, são aqueles que representam ações, objetos ou eventos concretos ou ações, objetos e eventos abstratos. Os gestos que possuem a função expressiva são aqueles que expressam emoções, tais como, alegria ou triunfo, tristeza ou sofrimento e raiva. Ainda de acordo com a autora (2013), os gestos que possuem a função apelativa são aqueles utilizados para regular o comportamento de outras pessoas. 5.1.4 Discussão dos dados do bloco “Exposição” Sendo assim, a partir desses critérios, analisamos os gestos que marcaram o Espaço Narrativo, sendo os presentes nas ocorrências 2 e 3, conforme é possível observar no Quadro 18, a seguir. Quadro 18: Função Gestual no Interior da Narrativa no bloco “Exposição” Ocorrência Função Gestual na Narrativa 1 Não analisamos, pois o gesto marca o Ground 2 Função Expressiva 3 Função Representativa Fonte: Elaborado pelo pesquisador Quadro 18: Função Gestual no Interior da Narrativa no bloco “Exposição” Ocorrência Função Gestual na Narrativa 1 Não analisamos, pois o gesto marca o Ground 2 Função Expressiva 3 Função Representativa Fonte: Elaborado pelo pesquisador Quadro 18: Função Gestual no Interior da Narrativa no bloco “Exposição” Fonte: Elaborado pelo pesquisador 115 Na ocorrência 2, o gesto é expressivo pois indica a expressão metafórica MAIS É PARA CIMA, conforme explicamos anteriormente. Na ocorrência 3, por sua vez, o gesto é representativo, uma vez que a narradora está encenando uma personagem que, embora regulasse o comportamento do seu eu-passado, utiliza um gesto que representa uma instância física presente na cena enunciativa, nesse caso um brinco pequeno (34 faz um brinquINHO menor) que deveria ser usado a fim de não assustar o companheiro de encontro. Em seguida, no que se refere à função da direção do olhar, conforme explicamos anteriormente, no capítulo 3, Sweetser e Stec (2016) demonstram que o olhar de um falante, assim como a direção da cabeça e as expressões faciais podem representar ações relativas a uma personagem da narrativa, assim como podem fazer parte da narração propriamente dita. Sendo assim, levamos em consideração duas funções principais do olhar na narrativa: i) encenação da personagem e ii) narração propriamente dita. Apenas na ocorrência 2, a direção do olhar marca o Espaço Narrativo, conforme é possível observar no Quadro 19, a seguir. Quadro 19: Função da Direção do Olhar no Interior da Narrativa no bloco “Exposição” Ocorrência Função da Direção do Olhar na Narrativa 1 Não analisamos, pois a direção do olhar marca o Ground 2 Função de Encenação de Personagem 3 Não analisamos, pois a direção do olhar marca o Ground Fonte: Elaborado pelo Pesquisador. 5.1.4 Discussão dos dados do bloco “Exposição” Quadro 19: Função da Direção do Olhar no Interior da Narrativa no bloco “Exposição” Ocorrência Função da Direção do Olhar na Narrativa 1 Não analisamos, pois a direção do olhar marca o Ground 2 Função de Encenação de Personagem 3 Não analisamos, pois a direção do olhar marca o Ground Fonte: Elaborado pelo Pesquisador. Quadro 19: Função da Direção do Olhar no Interior da Narrativa no bloco “Exposição” Fonte: Elaborado pelo Pesquisador. Levando em consideração a única ocorrência do bloco “Exposição” que atendeu os critérios de análise de função da direção do olhar na narrativa (ocorrência 2), encontramos a função da encenação de uma personagem, uma vez que o articulador não está marcando a sequência linear da história e diz respeito à encenação de uma personagem específica da narração: as pessoas que deram conselhos a respeito do comportamento do eu-passado da narradora. 116 116 5.2 Análise do bloco “Clímax” No Quadro 20, a seguir, colocamos em evidência, também, no bloco “Clímax”, as discussões propostas por Dancygier (2011). Do mesmo modo em relação ao bloco de análises anterior, no “Clímax”, é possível identificar, a partir das construções linguísticas, a narradora assumindo os papéis de narrador e das personagens em questão, levando em consideração as escolhas linguísticas. A seleção das ocorrências, ilustradas no Quadro 20 a seguir, se justifica, também, pelo bom enquadramento da câmera, que nos permitiu ter uma visualização completa das expressões faciais e dos gestos. Quadro 20: Escolhas linguísticas como evidências da estrutura da narrativa no bloco “Clímax” Ocorrência Transcrição Papel assumido CLÍMAX Ocorrência 1 MO:?hm?hm Personagens: O homem e o eu passado da narradora, respectivamente. CLÍMAX Ocorrência 2 RIA das piAdas DEle Personagem: A pessoa cujo nome não é mencionado, que recomendou o homem para ela. CLÍMAX coMEçou a falar dos FIlhos Narradora Quadro 20: Escolhas linguísticas como evidências da estrutura da narrativa no bloco ro 20: Escolhas linguísticas como evidências da estrutura da narrativa no bloco “Clímax” CLÍMAX Ocorrência 1 Ocorrência 1 CLÍMAX Ocorrência 2 Ocorrência 2 CLÍMAX CLÍMAX 117 Ocorrência 3 CLÍMAX Ocorrência 4 e eu feLIZ falei Narradora CLÍMAX Ocorrência 5 é garÇOM Personagem: o homem. Fonte: Elaborado pelo pesquisador Ocorrência 3 CLÍMAX Ocorrência 4 Ocorrência 4 CLÍMAX Ocorrência 5 é Fonte: Elaborado pelo pesquisador Fonte: Elaborado pelo pesquisador Do mesmo modo em que analisamos as ocorrências do bloco anterior, aqui, no bloco “Clímax”, primeiramente, lançamos um olhar para as escolhas linguísticas de cada ocorrência com base nos pressupostos de Dancygier (2011) a respeito dos recursos narrativos. Logo após, na análise multimodal das ocorrências identificamos a forma do gesto, levando em consideração os parâmetros descritos no Capítulo 4, que dizem respeito ao bloco 1 do Sistema Linguístico de Anotação Gestual (LASG) (BRESSEM; LADEWIG; MÜLLER, 2013). Ainda, com base em Cassell e McNeill (1990), categorizamos os gestos a partir da sua relação com o Ground e com o Espaço Narrativo. Já no que diz respeito à marcação de Espaços Mentais pela direção do olhar, utilizamos os parâmetros de Sweetser e Stec (2016). Ainda seguindo os nossos parâmetros de análise, nos casos em que os gestos marcaram o Espaço Narrativo, analisamos, com base em McNeill (1995), se eles se configuram como gestos do ponto de vista do observador (O-VPT) ou como gestos do ponto de vista da personagem (C-VPT). 5.2 Análise do bloco “Clímax” Analisamos, também nesses casos, os gestos quanto à sua função dominante no interior da narrativa: representação, expressão ou apelo, conforme Müller (2013). Conforme analisamos o bloco “Exposição”, somente nos casos em que a direção do olhar marcou o Espaço Narrativo, analisamos a sua função no interior da narrativa, de acordo com os 118 pressupostos de Sweetser e Stec (2016): i) encenação da personagem e ii) narração propriamente dita. pressupostos de Sweetser e Stec (2016): i) encenação da personagem e ii) narração propriamente dita. 5.2.1 Análise multimodal da ocorrência 1 do bloco “Clímax” Na Figura 47, a seguir, que representa a primeira ocorrência do bloco “Clímax”, é possível observar que a história se encaminha para o momento culminante. Figura 47: Representação multimodal da ocorrência 1 do bloco “Clímax” 80 aí fomos no restaurante 81 papo bom e tal e eu NUde 82 ?hm?hm ?hm?hm 83 não bebe 84 FP: nã [hahahaha] 85 SS: [hahahaha] 86 IS: [hahahaha] 87 MO:?hm?hm 88 <<com o dedo indicador para cima em um movimento 89 repetitivo da esquerda para a direita>> 80 aí fomos no restaurante 81 papo bom e tal e eu NUde 82 ?hm?hm ?hm?hm 83 não bebe 84 FP: nã [hahahaha] 85 SS: [hahahaha] 86 IS: [hahahaha] 87 MO:?hm?hm 88 <<com o dedo indicador para cima em um movimento 89 repetitivo da esquerda para a direita>> 119 Fonte: Elaborado pelo pesquisador/Canal da GNT no Youtube. Fonte: Elaborado pelo pesquisador/Canal da GNT no Youtube. Nessa ocorrência, foi possível observar, com base no LASG (BRESSEM; LADEWIG; MÜLLER, 2013) que a frase gestual se inicia com um gesto clássico realizado com o dedo indicador da mão direita da narradora, com a palma lateral, que representa o gesto clássico de negação, em um movimento repetitivo e arqueado para a esquerda e depois para a direita. Esse gesto coocorre com a interjeição comunicativa negativa em 87 MO:?hm?hm, que faz parte de uma reencenação que, na pergunta 83 não bebe representa o homem e, na resposta, momento no qual ocorre o gesto, representa a reencenação do eu passado da própria narradora. Já na retração, no momento em que as mãos da narradora retornam à posição de descanso, verificamos a combinação dos dedos indicador e polegar, em um movimento que diminui a distância entre eles que representa, por meio da modalidade gestual, a quantidade de bebida que ela ingere. Tanto no núcleo como na retração, o gesto marca o Espaço Narrativo, conforme Cassell e McNeill (1990) pois faz referência a um momento que faz parte do trecho narrativo e, além disso, não está direcionado para nenhum dos interlocutores, cumprindo a função representacional no interior da narrativa, conforme Müller (2013). Do mesmo modo, em relação à direção do olhar, conforme Sweetser e Stec (2016), consideramos uma marcação do Espaço Narrativo, já que, em ambos os casos, está direcionado para baixo e cumpre a função de “encenação de uma personagem”. Figura 47: Representação multimodal da ocorrência 1 do bloco “Clímax” Conforme Dancygier (2011), os recursos narrativos podem se relacionar com os gestos da seguinte maneira: a presença dos gestos em coocorrência com o discurso direto (tal como é possível observar em 87 MO:?hm?hm)confirmam o ponto de vista gestual C-VPT, conforme McNeill (1995), nesse caso, representando a personagem x¹ que é respondente ao eu-passado da narradora. 120 5.2.2 Análise multimodal da ocorrência 2 do bloco “Clímax” 5.2.2 Análise multimodal da ocorrência 2 do bloco “Clímax” Na Figura 48 a seguir, apresentamos a representação multimodal da ocorrência 2, do bloco Clímax” Figura 48: Representação multimodal da ocorrência 2 do bloco “Clímax” 91 vinha os aMIgos gays as aMIgas. 92 RIA das piAdas DEle 93 <<com as duas mãos com as palmas laterais e paralelas, 94 realiza um movimento descendente 95 ria [((incompreensível, aprox. 2 seg))] Fonte: Elaborado pelo pesquisador/Canal da GNT no Youtube. Figura 48: Representação multimodal da ocorrência 2 do bloco “Clímax” 91 vinha os aMIgos gays as aMIgas. 92 RIA das piAdas DEle 93 <<com as duas mãos com as palmas laterais e paralelas, 94 realiza um movimento descendente 95 ria [((incompreensível, aprox. 2 seg))] Fonte: Elaborado pelo pesquisador/Canal da GNT no Youtube. 91 vinha os aMIgos gays as aMIgas. 92 RIA das piAdas DEle 93 <<com as duas mãos com as palmas laterais e paralelas, 94 realiza um movimento descendente 95 ria [((incompreensível, aprox. 2 seg))] Fonte: Elaborado pelo pesquisador/Canal da GNT no Youtube. Na segunda ocorrência do “Clímax”, verificamos, com base em Bressem, Ladewig e Müller (2013) que o gesto é realizado com as duas mãos laterais com as palmas paralelas, em um movimento para baixo que marca o ritmo de fala, em um movimento acentuado. A nosso ver, esse movimento acentuado serve para confirmar o teor imperativo do segmento de faça presente no segmento 92 RIA das piAdas DEle. Além disso, percebemos também, que o volume da voz também é acentuado. O formato das mãos nesse movimento instância a ideia de “piada” (uma ideia abstrata) como um objeto marcado no espaço físico, isto é, nesse caso, de acordo com Cassell e McNeill (1990), consideramos que se trata de um gesto que marca, metaforicamente, o Espaço Narrativo, cumprindo, de acordo com Müller (2013) uma função representacional (que, nesse caso, conforme mencionamos, se configura de forma metafórica). Ainda, nesse caso, no que diz respeito à marcação produzida pela direção do olhar, identificamos, com base em Sweetser e Stec (2016) a marcação do Ground, uma vez que ele está direcionado para o interlocutor imediato da narradora. Retomando às discussões de 121 Dancygier (2011), verificamos a presença do discurso direto o que, relacionado às discussões de McNeill (1995), confirma o ponto de vista C-VPT, que representa a personagem x³, ou seja, a(s) pessoa(s) que deu(ram) conselhos para o eu-passado da narradora. 5.2.3 Análise multimodal da ocorrência 3 do bloco “Clímax” Na Figura 49 a seguir, apresentamos a representação multimodal da ocorrência 3 do bloco Clímax. Figura 49: Representação multimodal da ocorrência 3 do bloco “Clímax” 97 MO: e ah papo vem 98 coMEçou a falar dos FIlhos 99 <<com as duas palmas para cima realiza um gesto de 100 apresentar>> Fonte: Elaborado pelo pesquisador/Canal da GNT no Youtube Figura 49: Representação multimodal da ocorrência 3 do bloco “Clímax” 97 MO: e ah papo vem 98 coMEçou a falar dos FIlhos 99 <<com as duas palmas para cima realiza um gesto de 100 apresentar>> l b d l i d /C l d G b 97 MO: e ah papo vem 98 coMEçou a falar dos FIlhos 99 <<com as duas palmas para cima realiza um gesto de 100 apresentar>> Fonte: Elaborado pelo pesquisador/Canal da GNT no Youtube. Fonte: Elaborado pelo pesquisador/Canal da GNT no Youtube. Em relação à forma dessa ocorrência, de acordo com os parâmetros do LASG (BRESSEM; LADEWIG; MÜLLER, 2013), observamos que o gesto é realizado com ambas as mãos abertas com as palmas para cima, em um movimento arqueado, para fora do corpo, que coocorre com o segmento de fala 98 coMEçou a falar dos FIlhos. Nesse caso, o gesto representa a ação de “começar”, uma vez que apresenta os tópicos tratados pelo personagem do homem naquele momento da interação. Nesse sentido, Cienki (2013) afirma que os gestos que fazem parte da família gestual “mão aberta, palma para cima” (Palm-up open- hand-PUOH) são os gestos clássicos de apresentar. Nesse caso, conforme explicam Cassell e McNeill (1990), a narradora trata sobre o ato de narrar fazendo uma referência à estrutura da narrativa que está sendo dita: no momento em que ela menciona que o personagem x² 122 “começou” a falar sobre os filhos. Em outras palavras, é possível interpretar que esse gesto de apresentar, ainda de acordo com Cassell e McNeill (1990) está presente no nível metanarrativo, ou seja, trata-se de uma marcação do Ground e, adicionalmente, se relacionarmos com os pressupostos de Dancygier (2011), é possível afirmar que a presença do discurso indireto confirma esse fato. Simultaneamente aos gestos, verificamos que a direção do olhar marca o Ground, pois, de acordo com Sweetser e Stec (2016,) ela está ancorando o corpo na interação presente no aqui-agora. 5.2.4 Análise multimodal da ocorrência 4 do bloco “Clímax” Na Figura 50 a seguir, apresentamos a representação multimodal da ocorrência 4, que também faz parte do “Clímax”. Figura 50: Representação multimodal da ocorrência 4 do bloco “Clímax” 102 pior que ele adorou 103 ficou bem começou a falar dos Filhos 104 falou TEnho filhos 105 e eu feLIZ falei 106 <<ambas as palmas para cima em um movimento 107 ascendente>> Fonte: Elaborado pelo pesquisador/Canal da GNT no Youtube. 102 pior que ele adorou 103 ficou bem começou a falar dos Filhos 104 falou TEnho filhos 105 e eu feLIZ falei 106 <<ambas as palmas para cima em um movimento 107 ascendente>> Fonte: Elaborado pelo pesquisador/Canal da GNT no Youtube. Nesta ocorrência, é possível observar, com base em Bressem, Müller e Ladewig (2013), que se trata de um gesto simples realizado com as duas mãos abertas e com as palmas para cima. Esse gesto revela que a posição de descanso da maioria dos gestos realizados pela 123 narradora está localizada na altura do seu colo. No que diz respeito ao movimento, observamos que se trata de um movimento reto e acentuado, cuja direção é para cima e que, ainda, instancia a metáfora conceptual MAIS É PARA CIMA: ao dizer 105 e eu feLIZ falei, a narradora levanta ambos os braços rapidamente. Com base no que explica Müller (2013), em situações de felicidade ou triunfo, as pessoas tendem a levantar seus braços, em um movimento ascendente, cuja proeminência se relaciona, também, à metáfora conceptual FELIZ É PARA CIMA, o que demonstra as experiências corporificadas que indicam a origem experiencial das metáforas conceptuais e a utilização de expressões verbais que se relacionam à alegria e incorporam essa ideia, a exemplo da expressão “estou para cima”, utilizada no PB para representar alegria, o que justifica sua função expressiva no interior da narrativa. Nesse caso, com base em Cassell e McNeill (1990), os gestos manuais marcam, o Espaço Narrativo, a partir do ponto de vista C-VPT, já que se trata da perspectiva da personagem x² da narração, com base em McNeill (1995). Embora se observarmos, de acordo com Dancygier (2011), que o recurso narrativo presente nessa ocorrência seja uma marca da narradora (discurso indireto), consideramos que o gesto marca o Espaço Narrativo, uma vez que ele diz respeito a uma personagem que faz parte efetivamente da narrativa. No que se refere à direção do olhar, conforme Sweetser e Stec (2016), direcionado para um dos interlocutores imediatos, marca o Ground, permitindo que a narradora se engaje com os interlocutores no nível do aqui-agora. 5.2.5 Análise multimodal da ocorrência 5 do bloco “Clímax” Na Figura 51 a seguir, apresentamos a representação multimodal da ocorrência 5, que finaliza o bloco “Clímax”. 124 Figura 51: Representação multimodal da ocorrência 5 do bloco “Clímax” 129 MO: [não não] 130 os meus filhos moram coMIgo 131 e a MÃE DEles mora conosco também 132 é garÇOM 133 <<dedo indicador apontado para cima>> Fonte: Elaborado pelo pesquisador/Canal da GNT no Youtube. Figura 51: Representação multimodal da ocorrência 5 do bloco “Clímax” Figura 51: Representação multimodal da ocorrência 5 do bloco “Clímax Nesta ocorrência, chegamos ao ponto culminante da história, no qual o personagem do homem revela, a partir do segmento 03 “Não, não! É... Moram comigo, e a mãe deles mora conosco também”, que é casado. Como é possível observar na Figura 51, a narradora utiliza o espaço à direita de seu corpo para gesticular, a partir de um gesto simples, acentuado, realizado com o dedo indicador, em um movimento ascendente (também para a direita), conforme o LASG (BRESSEM; MÜLLER; LADEWIG, 2013). Esse gesto coocorre com o segmento 132 é garÇOM e, de acordo com Cassell e McNeill (1990), marca o Espaço Narrativo, uma vez que leva os interlocutores a compreenderem que, naquele momento a mão deixa de representar o gesto da narradora e passa a representar o gesto da personagem x², ou seja, o homem com o qual o eu-passado da narradora estava jantando. Ainda, de acordo com Müller (2013), esse gesto possui a função apelativa, pois é uma tentativa de chamar o garçom para evitar uma situação constrangedora, o que representa a regulação do comportamento do garçom que, a partir do pedido do homem, deveria fazer-se presente na cena. Analisando os recursos narrativos da modalidade verbal (DANCYGIER, 2011), é possível observar que a presença do discurso direto em 132 é garÇOM confirma o ponto de vista gestual C-VPT que, a partir da categorização de McNeill (1995), faz referência ao personagem x², conforme mencionamos anteriormente. O olhar, por sua vez, conforme as categorizações de Sweetser e Stec (2016), marca o Ground, uma vez que está direcionado para 125 o interlocutor imediato localizado no aqui-agora. O interlocutor imediato da narradora, nesse caso é Fábio Porchat – para quem, na maioria das ocorrências do bloco “Clímax” ela direciona o corpo e o olhar –. 5.2.5 Análise multimodal da ocorrência 5 do bloco “Clímax” Ele consegue interpretar, sem ruídos, a segmentação corporal da narradora (que divide seu corpo entre i- a personagem no Espaço Narrativo (marcação gestual) e ii- a narradora no Ground (marcação visual). 5.2.6 Discussão dos dados do bloco “Clímax” Para instanciar os resultados do bloco “Clímax”, apresentamos uma discussão mais geral dos parâmetros de marcação de Espaços Mentais nas cinco ocorrências analisadas. No que diz respeito aos gestos manuais, encontramos uma marcação do Ground (ocorrência 3) e quatro marcações do Espaço Narrativo (ocorrências 1, 2, 4 e 5). Já no que diz respeito à direção do olhar, encontramos quatro marcações do Ground (ocorrências 2, 3, 4 e 5) e uma marcação do Espaço Narrativo. Conforme é possível observar no Quadro 21 a seguir, nas ocorrências 1 e 3 os gestos e a direção do olhar marcam simultaneamente o mesmo Espaço Mental: na ocorrência 1, ambos os articuladores marcam o Espaço Narrativo, enquanto na ocorrência 3, eles marcam o Ground. Nas outras ocorrências, os articuladores, independentemente, marcam Espaços diferentes. Quadro 21: Marcação de Espaços Mentais nas ocorrências do bloco “Clímax” Ocorrência Gesto Direção do Olhar 1 Espaço Narrativo Espaço Narrativo 2 Espaço Narrativo Ground 3 Ground Ground 4 Espaço Narrativo Ground 5 Espaço Narrativo Ground Fonte: Elaborado pelo pesquisador Quadro 21: Marcação de Espaços Mentais nas ocorrências do bloco “Clímax” Fonte: Elaborado pelo pesquisador Em relação à categorização de McNeill (1995) a respeito do ponto de vista gestual, que pode ser do observador (O-VPT) ou da personagem (C-VPT), de forma geral, no bloco “Clímax” encontramos quatro ocorrências cujos gestos marcam o ponto de vista da personagem (C-VPT) (ocorrências 1,2,4 e 5) e não encontramos a marcação do ponto de vista do observador (O-VPT), conforme é possível observar no Quadro 22, a seguir: 126 Quadro 22: Ponto de Vista dos Gestos Narrativos no bloco “Clímax” Quadro 22: Ponto de Vista dos Gestos Narrativos no bloco “Clímax” Ocorrência Ponto de Vista Gestual 1 C-VPT 2 C-VPT 3 Não analisamos, pois o gesto marca o Ground 4 C-VPT 5 C-VPT Fonte: Elaborado pelo pesquisador Fonte: Elaborado pelo pesquisador Já no que diz respeito às funções dos gestos alocados no Espaço Narrativo, utilizando a categorização de Müller (2013), classificamos os gestos presentes nas ocorrências 1, 2, 4 e 5 a partir de sua função dominante: representação, expressão ou apelo, conforme é possível observar no Quadro 23, a seguir. 5.2.6 Discussão dos dados do bloco “Clímax” Quadro 23: Função Gestual no Interior da Narrativa no Bloco “Clímax” Ocorrência Função Gestual na Narrativa 1 Função Representacional 2 Função Representacional 3 Não analisamos, pois o gesto marca o Ground 4 Função Expressiva 5 Função Apelativa Fonte: Elaborado pelo pesquisador Quadro 23: Função Gestual no Interior da Narrativa no Bloco “Clímax” Fonte: Elaborado pelo pesquisador Na ocorrência 1, consideramos a função do gesto como representacional porque, no instante da sua realização, a narradora está encenando seu eu-passado no momento em que negava uma informação (nesse caso, ao ser perguntada se bebia) a partir de um gesto realizado com o dedo indicador que representa a negação. Na ocorrência 2, também consideramos a função representacional. No entanto, nesse caso, trata-se de uma representação metafórica pois a narradora representa os limites espaciais da piada (uma ideia abstrata) como um objeto concreto no espaço físico. Na ocorrência 4, classificamos o gesto levando em consideração sua função expressiva porque ele instancia as expressões metafóricas MAIS É PARA CIMA e 127 FELIZ É PARA CIMA, conforme explicamos anteriormente. Na ocorrência 5, por sua vez, trata-se de um gesto apelativo, uma vez que a narradora está encenando a personagem do homem do encontro no momento em que ele solicita a presença do garçom para se desvencilhar do estranhamento causado pelo fato de ele ter revelado que era casado. Em seguida, partindo para a função da direção do olhar, seguindo, ainda as categorizações de Sweetser e Stec (2016) que tratam das duas funções principais do olhar na narrativa: i) encenação da personagem e ii) narração propriamente dita. Apenas na ocorrência 1, a direção do olhar marca o Espaço Narrativo, conforme é possível observar no Quadro 24, a seguir. Quadro 24: Função da Direção do Olhar no Interior da Narrativa no bloco “Clímax” Ocorrência Função da Direção do Olhar na Narrativa 1 Função de Encenação de Personagem 2 Não analisamos, pois a direção do olhar marca o Ground 3 Não analisamos, pois a direção do olhar marca o Ground 4 Não analisamos, pois a direção do olhar marca o Ground 5 Não analisamos, pois a direção do olhar marca o Ground Fonte: Elaborado pelo Pesquisador. Quadro 24: Função da Direção do Olhar no Interior da Narrativa no bloco “Clímax” Fonte: Elaborado pelo Pesquisador. Fonte: Elaborado pelo Pesquisador. 5.2.6 Discussão dos dados do bloco “Clímax” Levando em consideração a única ocorrência do bloco “Clímax” que atendeu os critérios de análise de função da direção do olhar na narrativa (ocorrência 1), encontramos a função de encenação de uma personagem. Notamos, portanto, que nesse caso, a narradora utiliza o olhar na representação da expressão facial de seu eu-passado, ao recusar a bebida. 5.3 Análise do bloco “Desfecho” Novamente, com base em Dancygier (2011), identificamos as escolhas linguísticas utilizadas pela narradora a fim de assumir diferentes papéis nesse trecho narrativo. Conforme é 128 possível observar no Quadro 25, a seguir. Mais uma vez, a seleção de ocorrências do bloco “Desfecho” se justifica pelo bom enquadramento da câmera, que nos permite visualizar todos os articuladores multimodais. Quadro 25: Escolhas linguísticas como evidências da estrutura da narrativa no bloco “Desfecho” Ocorrência Transcrição Papel assumido DESFECHO Ocorrência 1 foi esse TEMpo Narradora DESFECHO Ocorrência 2 comecei aí Narradora DESFECHO Ocorrência 3 não por favor Personagens: o eu passado da narradora e o homem. Fonte: Elaborado pelo pesquisador Quadro 25: Escolhas linguísticas como evidências da estrutura da narrativa no bloco “Desfecho” DESFECHO Ocorrência 1 fo DESFECHO Ocorrência 1 Ocorrência 1 DESFECHO Ocorrência 2 DESFECHO Ocorrência 2 Ocorrência 2 DESFECHO Ocorrência 3 DESFECHO Ocorrência 3 Fonte: Elaborado pelo pesquisador Assim como nas análises dos blocos “Exposição” e “Clímax”, conforme pode-se verificar nas sessões anteriores, utilizamos no bloco “Desfecho” os mesmos parâmetros: analisamos as escolhas linguísticas de cada ocorrência com base nos pressupostos de Dancygier 129 (2011). Além disso, analisamos os gestos com base no bloco 1 do Sistema Linguístico de Anotação Gestual (LASG) (BRESSEM; LADEWIG; MÜLLER, 2013). Para a marcação de Espaços mentais pelos gestos, utilizamos os parâmetros de Cassell e McNeill (1990) que dizem respeito aos gestos que marcam o Ground e, também, aos gestos que marcam o Espaço Narrativo. No que se refere à marcação de Espaços Mentais pela direção do olhar, utilizamos os parâmetros de Sweetser e Stec (2016), que afirmam que o olhar de um narrador pode representar tanto o olhar de uma personagem (no Espaço Narrativo) como o olhar do próprio narrador na interação (no Ground). Utilizamos, também, os parâmetros que dizem respeito, especificamente, aos gestos – o ponto de vista gestual (com base em McNeill (1995)) e sua função no interior da narrativa (com base em Müller (2013)) e a direção do olhar – sua função na narrativa (com base em Sweetser e Stec (2016)) no Espaço Narrativo. 5.3.1 Análise multimodal da ocorrência 1 do bloco “Desfecho” Na Figura 52 a seguir, apresentamos a representação multimodal da ocorrência 1, que inicia o bloco “Desfecho” Figura 52: Representação multimodal da ocorrência 1 do bloco “Desfecho” 135 FP: um outro homem 136 um outro rapaz: 137 MO: foi esse TEMpo 138 <<mãos com as palmas diagonais em um movimento 139 ascendente>> Figura 52: Representação multimodal da ocorrência 1 do bloco “Desfecho” 135 FP: um outro homem 136 um outro rapaz: 137 MO: foi esse TEMpo 138 <<mãos com as palmas diagonais em um movimento 139 ascendente>> 136 um outro rapaz: 137 MO: foi esse TEMpo 130 140 sentiram a biGORna que caiu na sala 141 SS: NOSsa 142 MM: foi mais ou menos essa biGORna que eu recebi Fonte: Elaborado pelo pesquisador/Canal da GNT no Youtube. 140 sentiram a biGORna que caiu na sala 141 SS: NOSsa 142 MM: foi mais ou menos essa biGORna que eu recebi Fonte: Elaborado pelo pesquisador/Canal da GNT no Youtube. 140 sentiram a biGORna que caiu na sala 141 SS: NOSsa 142 MM: foi mais ou menos essa biGORna que eu recebi Fonte: Elaborado pelo pesquisador/Canal da GNT no Youtube. 140 sentiram a biGORna que caiu na sala 141 SS: NOSsa 142 MM: foi mais ou menos essa biGORna que eu recebi Fonte: Elaborado pelo pesquisador/Canal da GNT no Youtube. 140 sentiram a biGORna que caiu na sala 141 SS: NOSsa 142 MM: foi mais ou menos essa biGORna que eu recebi Fonte: Elaborado pelo pesquisador/Canal da GNT no Youtube. Nesta ocorrência, observamos, a partir dos parâmetros do bloco 1 do LASG (BRESSEM, MÜLLER, LADEWIG, 2013), que a sequência gestual é iniciada com um gesto realizado por ambas as mãos, com as palmas diagonais, em um movimento ascendente e reto, que coocorre com o segmento 137 MO: foi esse TEMpo. Na retração, o gesto coocorre com o segmento 140 sentiram a biGORna que caiu na sala com as mãos em um movimento descendente. Ao analisar essa sequência gestual, em que as palmas se encontram voltadas uma para a outra “como se” (as-if) (CIENKI, 2005) a narradora estivesse segurando, em um primeiro momento, a ideia de tempo, o que evidencia a metáfora IDEIAS SÃO OBJETOS (MCNEILL; CASELL; LEVY, 1993). Considerando os pressupostos de Cassell e McNeill (1990), verificamos que o Espaço Narrativo é marcado por um gesto metafórico, uma vez que torna a instância de tempo como um objeto no interior da narrativa. Ao levarmos em consideração as discussões de Dancygier (2011), é possível observar a presença do discurso indireto em 137 MO: foi esse TEMpo e também em 140 sentiram a biGORna que caiu na sala. No entanto, levando em consideração que o gesto faz referência a um objeto – metafórico nesse caso – que faz parte da história em si, a partir do ponto de vista O-VPT, já que se refere ao tempo como objeto observado da perspectiva de fora, conforme McNeill (1995). Pensando, especificamente, na função do gesto no interior da narrativa, com base em Müller (2013), verificamos que se trata da função representacional de um objeto metafórico. 140 sentiram a biGORna que caiu na sala 141 SS: NOSsa 142 MM: foi mais ou menos essa biGORna que eu recebi Fonte: Elaborado pelo pesquisador/Canal da GNT no Youtube. 131 No que se refere à direção do olhar, de acordo com Sweestser e Stec (2016), em um primeiro momento, a narradora olha para a esquerda, onde encontra-se uma de suas interlocutoras imediatas (Ivete Sangalo), e em seguida, olha para a direita, onde encontra-se outra interlocutora imediata (Samantha Schmütz) – que interage com ela no segmento 05 SS: 141 SS: NOSsa o que evidencia uma interação no Ground que diz respeito ao segmento 140 sentiram a biGORna que caiu na sala. No que se refere à direção do olhar, de acordo com Sweestser e Stec (2016), em um primeiro momento, a narradora olha para a esquerda, onde encontra-se uma de suas interlocutoras imediatas (Ivete Sangalo), e em seguida, olha para a direita, onde encontra-se outra interlocutora imediata (Samantha Schmütz) – que interage com ela no segmento 05 SS: 141 SS: NOSsa o que evidencia uma interação no Ground que diz respeito ao segmento 140 sentiram a biGORna que caiu na sala. 5.3.2 Análise multimodal da ocorrência 2 do bloco “Desfecho” Na Figura 53 a seguir, apresentamos a representação multimodal da ocorrência 2, que também faz parte do bloco “Desfecho” Figura 53: Representação multimodal da ocorrência 2 do bloco “Desfecho” 146 IS: aí você tirou o [nude ] 147 MM: aí começou [a farra] 148 aí o volume aumentou 149 estávamos num restaurante pequeno 150 eu comecei 151 já viu atraÇÃO fatal:: ((ri)) 152 comecei aí 153 <<ambas as mãos com as palmas para baixo em um movimento 154 para baixo>> Fonte: Elaborado pelo pesquisador/Canal da GNT no Youtube. Figura 53: Representação multimodal da ocorrência 2 do bloco “Desfecho” 146 IS: aí você tirou o [nude ] 147 MM: aí começou [a farra] 148 aí o volume aumentou 149 estávamos num restaurante pequeno 150 eu comecei 151 já viu atraÇÃO fatal:: ((ri)) 152 comecei aí 153 <<ambas as mãos com as palmas para baixo em um movimento 154 para baixo>> Fonte: Elaborado pelo pesquisador/Canal da GNT no Youtube. 132 Na segunda sequência gestual do “Desfecho”, de acordo com o LASG (BRESSEM, MÜLLER; LADEWIG, 2013), a frase gestual é iniciada com as duas mãos abertas com a palma para baixo, em um movimento descendente e também para a direita, em coocorrência com o segmento 04 Eu comecei, o que marca o ritmo da ação da narradora em mudar de postura logo após descobrir que estava sendo enganada. Conforme explica Cienki (2008), os gestos rítmicos marcam as palavras e as frases como significantes para o seu conteúdo discursivo e pragmático, o que, de acordo com o Cassell e McNeill (1990), faz uma referência ao Ground. Ao relacionarmos com o que propõe Dancygier (2011), verificamos a presença do discurso indireto, o que, nesse caso, confirma a marcação do Ground. Ainda nesse caso, no que diz respeito à marcação produzida pela direção do olhar, também identificamos, a partir dos pressupostos de Sweetser e Stec (2016) a marcação do Ground, porque o olhar da narradora está direcionado para Fábio Porchat, um dos seus interlocutores imediatos na interação. 5.3.3 Análise multimodal da ocorrência 3 do bloco “Desfecho” Na Figura 54 a seguir, apresentamos a representação multimodal da ocorrência 3, que finaliza o bloco “Desfecho”. Figura 54: Representação multimodal da ocorrência 3 do bloco “Desfecho” 175 não eu tô falando super baixo 176 que loucura agora 177 você vai enfiar a mão no seu BOLso direito 178 e tiRAR a alian::ça 179 ((incompreensível, aprox. 1 seg)) 180 eu falei Figura 54: Representação multimodal da ocorrência 3 do bloco “Desfecho” 175 não eu tô falando super baixo 176 que loucura agora 177 você vai enfiar a mão no seu BOLso direito 178 e tiRAR a alian::ça 179 ((incompreensível, aprox. 1 seg)) 180 eu falei Figura 54: Representação multimodal da ocorrência 3 do bloco “Desfecho” Figura 54: Representação multimodal da ocorrência 3 do bloco “Desfecho” Figura 54: Representação multimodal da ocorrência 3 do bloco “Desfe 175 não eu tô falando super baixo 176 que loucura agora 177 você vai enfiar a mão no seu BOLso direito 178 e tiRAR a alian::ça 179 ((incompreensível, aprox. 1 seg)) 180 eu falei 175 não eu tô falando super baixo 176 que loucura agora 177 você vai enfiar a mão no seu BOLso direito 178 e tiRAR a alian::ça 179 ((incompreensível, aprox. 1 seg)) 180 eu falei 133 181 agora será que eu preciso repetir a terceira vez:: 182 ele falou 183 não por favor 184 <<com as palmas verticais, realiza um movimento 185 descendente>> 186 FP: ele [tinha a aliança no bol:so] 187 MO: [TInha a aliança no bolso ] 188 IS: [filho da pleuras ] 189 FP: O::lha só 190 MO: aí foi Fonte: Elaborado pelo pesquisador/Canal da GNT no Youtube. Fonte: Elaborado pelo pesquisador/Canal da GNT no Youtube. Nessa ocorrência, chegamos ao final do bloco “Desfecho”. Nessa sequência, a narradora, ao encenar o personagem do homem, verificamos com base em Bressem, Müller e Ladewig (2013) inicia a frase gestual com ambas as mãos laterais com a palma para fora em relação ao seu corpo em um movimento ascendente no núcleo em coocorrência com o segmento 183 não por favor. Na retração, a palma da mão esquerda é direcionada para baixo, enquanto a mão direita é direcionada para um “bolso imaginário”, considerando que, no segmento 177 você vai enfiar a mão no seu BOLso direito, a narradora menciona o fato de ele estar com a aliança dentro de um bolso na reencenação do evento passado. Fundamentando-nos nos pressupostos de Cassell e McNeill (1990), categorizamos a marcação do gesto no Espaço Narrativo, já que a mão da narradora funciona, a nosso ver, como um simulacro da mão da personagem x² no momento do evento passado contado, o que configura, também, a função representacional da ação de colocar algo no bolso. Figura 54: Representação multimodal da ocorrência 3 do bloco “Desfecho” 175 não eu tô falando super baixo 176 que loucura agora 177 você vai enfiar a mão no seu BOLso direito 178 e tiRAR a alian::ça 179 ((incompreensível, aprox. 1 seg)) 180 eu falei De acordo com McNeill (1995), encontramos, portanto, um gesto cujo ponto de vista é C-VPT, ou seja, o ponto 134 de vista da personagem o que é confirmado pela modalidade verbal, na presença do discurso direto, conforme Dancygier (2011). No caso da marcação do Espaço Mental a partir da direção do olhar, conforme Sweetser e Stec (2016), verificamos que o olhar se desloca do interlocutor imediato e se direciona para baixo, também em direção ao “bolso imaginário” que representa, no Espaço Narrativo, a encenação da personagem do homem no evento. Trata-se de uma marcação gestual (CASSELL; MCNEILL, 1990), e visual (SWEETSER, STEC, 2016) do Espaço Narrativo. 5.3.4 Discussão dos dados do bloco “Desfecho” Finalmente, para instanciar os resultados do bloco “Desfecho”, apresentamos a discussão mais geral dos parâmetros de marcação de Espaços Mentais nas três ocorrências analisadas. No que diz respeito aos gestos manuais, encontramos uma marcação do Ground (ocorrência 2) e duas marcações do Espaço Narrativo (ocorrências 1 e 3). Já no que diz respeito à direção do olhar, encontramos duas marcações do Ground (ocorrências 1 e 2) e uma marcação do Espaço Narrativo (ocorrência 3). No Quadro 26, a seguir, é possível observar que, nas ocorrências 2 e 3 os gestos e a direção do olhar marcam simultaneamente o mesmo Espaço Mental: na ocorrência 2, ambos os articuladores marcam o Ground; e, na ocorrência 3, eles marcam o Espaço Narrativo. Os articuladores, ainda conforme é possível observar no Quadro, marcam, de forma independente, diferentes Espaços na ocorrência 1. Quadro 26: Marcação de Espaços Mentais nas ocorrências do bloco “Desfecho” Ocorrência Gesto Direção do Olhar 1 Espaço Narrativo Ground 2 Ground Ground 3 Espaço Narrativo Espaço Narrativo Fonte: Elaborado pelo pesquisador Quadro 26: Marcação de Espaços Mentais nas ocorrências do bloco “Desfecho” Fonte: Elaborado pelo pesquisador No que diz a respeito do ponto de vista gestual, que pode se configurar como gesto do ponto de vista do observador (O-VPT) ou do ponto de vista da personagem (C-VPT) (MCNEILL, 1995), de forma geral, no bloco “Desfecho” encontramos uma ocorrência cujo gesto marca o ponto de vista do observador (O-VPT), (ocorrência 1) e uma ocorrência cujo gesto marca o ponto de vista da personagem (ocorrência 3), conforme é possível observar no Quadro 27, a seguir: 135 Quadro 27: Ponto de Vista dos Gestos Narrativos no bloco “Desfecho” Ocorrência Ponto de Vista Gestual 1 O-VPT 2 Não analisamos, pois o gesto marca o Ground 3 C-VPT Fonte: Elaborado pelo pesquisador Quadro 27: Ponto de Vista dos Gestos Narrativos no bloco “Desfecho” Seguindo para as funções dos gestos alocados no Espaço Narrativo, ainda a partir da categorização de Müller (2013), classificamos os gestos que marcaram o Espaço Narrativo a partir de sua função dominante: representação, expressão ou apelo. No Quadro 28, a seguir, verificamos que as ocorrências 1 e 3 atenderam aos critérios de função gestual no interior da narrativa. 5.3.4 Discussão dos dados do bloco “Desfecho” Quadro 28: Função Gestual no Interior da Narrativa no Bloco “Desfecho” Ocorrência Função Gestual na Narrativa 1 Função Representacional 2 Não analisamos, pois o gesto marca o Ground 3 Função Representacional Fonte: Elaborado pelo pesquisador Quadro 28: Função Gestual no Interior da Narrativa no Bloco “Desfecho” Fonte: Elaborado pelo pesquisador Na ocorrência 1, categorizamos o gesto como representacional, pois trata-se de uma representação metafórica da ideia de tempo como um objeto concreto no espaço físico. Na ocorrência 3, por sua vez, o gesto é representacional porque a narradora está encenando uma personagem que, nesse caso, refere-se ao comportamento corporal do homem no momento em que se passa o evento, a partir de uma reencenação que envolve a segmentação corporal ao encenar uma personagem. Em seguida, em relação à função da direção do olhar, com base nas categorizações de i) encenação da personagem e ii) narração propriamente dita, propostas por Sweetser e Stec (2016), identificamos que, apenas na ocorrência 3, a direção do olhar marca o Espaço Narrativo, conforme é possível observar no Quadro 29, a seguir. 136 Quadro 29: Função da Direção do Olhar no Interior da Narrativa no bloco “Desfecho” Ocorrência Função da Direção do Olhar na Narrativa 1 Não analisamos, pois a direção do olhar marca o Ground 2 Não analisamos, pois a direção do olhar marca o Ground 3 Função de Encenação de Personagem Fonte: Elaborado pelo Pesquisador. Fonte: Elaborado pelo Pesquisador. Levando em consideração a única ocorrência do bloco “Desfecho” que atendeu os critérios de análise de função da direção do olhar na narrativa (ocorrência 3), encontramos a função da encenação de uma personagem específica da narração: o homem, constrangido, para evitar que o eu-passado da narradora causasse uma situação de embaraço. 5.4 Discussão geral dos dados O primeiro aspecto que analisamos nessa narrativa diz respeito às escolhas linguísticas de Marisa Orth nas ocorrências. Foi possível observar, por exemplo, a utilização de aspectos gramaticais. Esses aspectos gramaticais, conforme observamos nas ocorrências que analisamos nas seções anteriores, revelam como as escolhas de discurso direto e indireto ativam imagens e pontos de vista nas narrativas. No bloco “Exposição”, encontramos a presença da primeira pessoa do singular (“04 eu tava solteira”), que revela a representação da voz do narrador, e a forma imperativa (“24 não ri AL:to” e “34 faz um brinquINHO menor”), que revela a representação de uma das personagens na narração. No bloco “Clímax”, encontramos a presença de uma frase interrogativa seguida de uma interjeição comunicativa de negação (“87 MO:?hm?hm”), a forma imperativa (“92 RIA das piAdas DEle”), que revelam a representação da personagem x²; a presença da terceira do singular (“98 coMEçou a falar dos FIlhos”), o que revela a perspectiva da narradora; a presença da primeira pessoa do singular (“105 e eu feLIZ falei”), que representa um aspecto referente à personagem x¹; e uma frase vocativa (“13 é garÇOM”)que revela a representação da personagem x³. 137 Já no bloco “Desfecho”, encontramos um verbo impessoal (“137 MO: foi esse TEMpo”) cujo segmento de fala pode ser associado ao gesto metafórico que, no interior da narrativa, instancia o tempo como um objeto; a presença da primeira pessoa do singular (“152 comecei aí”), que revela a posição de narradora; e a presença do discurso direto em si (“183 não por favor”) que revela a posição da personagem x³. Nas ocorrências 2 e 3 do bloco “Exposição”, um fato que nos chamou a atenção é que, no momento em que ela diz “um homem muito recomendado” e, em seguida, utiliza o imperativo em sentenças, como, por exemplo, “não vai de saia curta”, podemos observar que a narradora encena as ações de uma personagem que não é necessariamente nomeada ao longo do curso narrativo. No entanto, as reencenações realizadas por ela evidenciam que se trata de uma personagem presente na história e, conforme mencionamos anteriormente, é corporificada como uma pessoa que deu instruções para que ela fosse ao encontro. 5.4 Discussão geral dos dados De acordo com Dancyinger (2011), todas escolhas linguísticas funcionam como construtores de espaços mentais, que, por sua vez, podem direcionar para o Ground, a exemplo do discurso indireto na ocorrência 1 do bloco “Exposição” (04 eu tava solteira), ou para o Espaço Narrativo, a exemplo do discurso direto na ocorrência 3 do bloco “Exposição” (34 faz um brinquINHO menor). Ainda, de acordo com a autora, a presença dessas marcas gramaticais é fundamental para que os interlocutores do narrador consigam distinguir entre a especulação e um relato, por exemplo, além de marcar o que ela denomina como “Espaços de Pontos de Vista”, que, nesta pesquisa, analisamos com base em McNeill (1995). Nesse sentido, a presença dessas marcas é substancial para a estrutura da narrativa, já que, nas palavras de Dancyinger (2011), elas se relacionam diretamente com as expectativas dos interlocutores. É nesse sentido, portanto, que julgamos importante analisar essas marcas linguísticas, uma vez que elas demonstram como a Teoria dos Espaços Mentais pode ser aplicada ao discurso narrativo, levando em consideração sua utilização na construção de sentido dentro de uma narrativa. Além disso, conforme explica Dancyinger (2012), esse tipo de análise oferece uma abordagem elegante e profícua para lidarmos com discursos narrativos complexos, a exemplo das narrativas multimodais, porque ela responde a diversas questões a respeito da conceptualização e marcação de Espaços Mentais. No caso desta pesquisa, foi possível articular os recursos narrativos da modalidade verbal com a estrutura da narrativa no nível multimodal, revelando como os mecanismos gestuais e visuais se relacionam com os mecanismos linguísticos. 138 Partindo para a inter-relação entre os articuladores multimodais analisados, foi possível verificar como a narradora, no contexto da narrativa que analisamos, não lida somente com a administração de escolhas linguísticas. É como se ela administrasse, também, o seu espaço gestual, os movimentos e ações corporais. Ainda, avaliando a configuração física da interação, foi possível observar também os recursos que ela utilizou para obter a interação de interlocutores específicos: no bloco “Exposição”, por exemplo, ela inicia a narração de sua história articulando uma conexão entre a história da convidada que havia acabado de contar uma história e, nesse momento específico da narração, ela direciona seu corpo, seu olhar e o gesto para a convidada que, nesse caso, é a atriz Samantha Schmütz. Essa interação, levando em consideração o Espaço-base, está situada no aqui-agora. 56 Verificar também Stec e Sweetser (2013). 56 Verificar também Stec e Sweetser (2013). 5.4 Discussão geral dos dados Além disso, a narradora representa o conteúdo do Espaço Narrativo, direcionando seu olhar para lugares específicos do espaço físico a depender da necessidade de reencenação (a exemplo da ocorrência 2 do bloco “Exposição”, em que ela direciona o olhar e as mãos para cima, quando menciona o conselho de não rir alto que recebeu). Conforme explicam Sweetser e Stec (2016), esses fenômenos que mencionamos são explicados a partir da representação de diferentes Espaços Mentais, utilizando o quadro teórico proposto por Fauconnier (2003 [1985]). Com base na Teoria dos Espaços Mentais, nos referimos aos momentos em que Marisa Orth interage com Fábio Porchat, Samantha Schümtz e Ivete Sangalo como o espaço físico compartilhado, situado no Espaço-base. Assim, nos referimos às informações relativas ao “mundo da história”, como Espaço Narrativo. Ainda, em concordância com os resultados de pesquisa de Sweetser e Stec (2016)56 identificamos que, em relação à divisão do espaço gestual, a linha Falante-Ouvinte, na narrativa que analisamos, corresponde ao locus dos gestos interacionais referentes ao Espaço-base, enquanto os gestos que ocorrem em um dos lados dessa linha dizem respeito ao Espaço Narrativo. Já no que diz respeito à direção do olhar, identificamos que ela é fundamental para diferenciar a marcação de Espaços Mentais, partindo do pressuposto de que o olhar direcionado a um dos lados da linha Falante-Ouvinte representa eventos que se configuram como reencenações que estão alocados no Espaço Narrativo. Sendo assim, ainda em conformidade com as autoras, quando o olhar está direcionado para a linha Falante-Ouvinte, em si, a informação está localizada no Ground. Foi possível observar, também, a exemplo da ocorrência 1 do bloco “Exposição”, que o olhar direcionado a uma pessoa específica, conjuntamente com um gesto manual, é uma 139 maneira muito pervasiva, intuitiva e evidente para iniciar uma interação face a face. De acordo com Goffman (1963), a direção do olhar, seja ela lançada para uma pessoa específica, ou, até mesmo para longe de uma pessoa específica, é um recurso fundamental para a iniciação ou inibição de um momento interacional, revelando, ainda, que o estabelecimento de entreolhares como uma condição necessária para que se haja a interação social ratificada (GOFFMAN, 1963). No caso das interações presentes no nosso corpus, conseguimos visualizar também o nível de engajamento dos convidados do programa a partir dos mecanismos gestuais e a relação estabelecida com os mecanismos linguísticos e, ainda, com os mecanismos de segmentação da direção do olhar. 5.4 Discussão geral dos dados Uma das principais contribuições do nosso trabalho é permitir que análises vindouras possam investigar a correlação entre os gestos e a direção do olhar a partir de uma perspectiva não apenas interacional, mas também cognitica. Partindo mais especificamente para a natureza das interações analisadas nessa pesquisa, consideramos que, entre os tipos de discursos, o discurso narrativo tem atraído especial atenção na abordagem cognitiva de várias áreas das ciências humanas, especialmente no que diz respeito à Linguística Cognitiva. Dessa maneira, os Espaços Narrativos, conforme estabelecemos, são compreendidos como uma variedade de Espaços Mentais, uma vez que domínios cognitivos são ativados pelo uso de formas linguísticas, pelos gestos e pela direção do olhar. No que diz respeito à fala, foi possível observar que determinadas expressões linguísticas, a exemplo do segmento 04 eu tava solteira da ocorrência 1 do bloco “Exposição” da narrativa que analisamos, funcionam como construtores de espaços mentais (space builders) que, de acordo com Ferrari (2016) são indicadores linguísticos responsáveis pela criação de espaços que “constituem domínios alternativos em relação ao Espaço-base” (FERRARI, 2016, p. 112). Em tempo, no que diz respeito à modalidade gestual, observamos como os gestos manuais criam espaços alternativos em relação ao Espaço-base, a exemplo da ocorrência 3 do bloco “Exposição” da narrativa que analisamos, observamos a marcação do Espaço Narrativo, a partir da representação de um elemento físico concreto da narrativa, acompanhada do turno 34 faz um brinquINHO menor. Em relação à marcação de espaços mentais por meio da direção do olhar, a fim de exemplificar, retomamos à ocorrência 1 do bloco “Clímax” na qual encontramos a marcação do Espaço Narrativo, a partir da representação da expressão facial de seu eu-passado, ao recusar a bebida, que coocorre com o segmento 87 MO:?hm?hm em resposta ao segmento 83 não bebe. Em linhas gerais, essas marcações de Espaços Narrativos se configuram como atividades elaboradas e esperadas ao decorrer da sequência narrativa, além disso, elas 140 contribuem para a estrutura multimodal da narrativa, fazendo com que haja a correta compreensão dos interactantes em relação aos focos narrativos e aos focos de interação pertencentes ao Espaço-base à medida em que a narradora recria a sequência temporal dos eventos recontados. Ou seja: concluímos que as expressões linguísticas, gestuais e relativas à direção do olhar possuem um papel privilegiado na narrativa pois elas são primordiais para a identificação de Espaços Narrativos específicos. 5.4 Discussão geral dos dados A narrativa multimodal é, por si só, muito complexa, já que pode ser elaborada desde um simples flashback até a menção de um momento muito específico do passado, fatos que podem ser interrompidos por interações que dizem respeito ao aqui-agora. Por isso, buscamos compreender como os articuladores multimodais (os gestos e a direção do olhar) funcionam no contexto do português brasileiro, tendo em vista um olhar mais particular para o Português Brasileiro. No que se refere, de forma mais específica, ao corpus que adotamos, os vídeos retirados do programa Que História É Essa Porchat, do canal GNT, foi possível observar que o movimento corporal e a postura dos interactantes, especialmente a narradora, passaram por um processo de afinação (fine-tuning), termo utilizado por Dancygier (2011) para definir o fenômeno no qual a funções, contextos e interações dos movimentos corporais entram em níveis específicos de sincronia interacional. Foi observar esse fenômeno na ocorrência 3 do bloco “Desfecho”, no qual, em coocorrência com o segmento 183 não por favor tanto a posição do torso quanto o olhar são direcionados, em sincronia, para baixo, a fim de representar a personagem do homem na reencenação. O espaço físico do programa também precisou ser considerado: embora o funcionamento e manutenção dos segmentos de falam tenham sido organizados a fim de emular uma roda de conversa, as interações possuem um objetivo específico de entretenimento, o que justifica a organização dos indivíduos em semicírculo para que as câmeras possam ser posicionadas. Sendo assim, as histórias contadas, embora possuam narrações muito espontâneas, como é o caso da que escolhemos para analisar, são previamente conhecidas pelo apresentador e produção técnica do programa. Julgamos que há um nível de espontaneidade que é passível de análise, uma vez que a plateia e os outros convidados do programa não conhecem previamente a história. Nesse sentido, conforme explica Kendon (1990) os participantes de interações desse tipo conseguem administrar e manter uma configuração espacial na qual o espaço entre eles proporciona um acesso exclusivo, igualitário e direto ao momento interacional. A partir da nossa pergunta, a hipótese que assumimos foi a de que no português brasileiro, assim como no inglês americano (SWEETSER; STEC, 2016), o olhar e os gestos manuais podem marcar Espaços Mentais independentes em uma mesma interação. 5.4 Discussão geral dos dados Essa 141 hipótese condiz com os nossos resultados, conforme concluímos a partir das seguintes ocorrências: i) ocorrência 3 do bloco “Exposição”; ii) ocorrência 2 do bloco “Clímax”; iii) ocorrência 4 do bloco “Clímax”; iv) ocorrência 5 do bloco “Clímax” e v) ocorrência 1 do bloco “Desfecho”. Para detalhar a discussão dos resultados em um panorama mais geral, decidimos apresentar, num primeiro momento, gráficos contendo as porcentagens relativas às marcações de Espaços Mentais pelos gestos e, em seguida, pela direção do olhar. Levando em consideração o nosso objetivo de analisar a marcação multimodal de Espaços Mentais na representação de múltiplos pontos de vista, a partir da análise de 11 sequências multimodais, chegamos aos seguintes resultados: Gráfico 1: Marcação gestual de Espaços Mentais Gráfico 1: Marcação gestual de Espaços Mentais Fonte: Elaborado pelo Pesquisador Fonte: Elaborado pelo Pesquisador Considerando o total das ocorrências (11 ocorrências), a marcação predominante de Espaços Mentais pelos gestos foi a marcação do Espaço Narrativo, totalizando em números absolutos, 8 ocorrências em que os gestos marcam o Espaço Narrativo (73%), contra 3 ocorrências em que os gestos marcam o Ground (27%). Nesse caso, é interessante observar como, na maior parte do tempo, os gestos produzidos pela atriz revelaram que ela se posiciona como narradora por meio dos gestos no interior da narrativa. Nesse sentido, como direção futura deste trabalho, buscaremos analisar narrativas proferidas por indivíduos que possuam outros estatutos sociais, ou seja, que não sejam artistas, como jornalistas, por exemplo, a fim de 142 estabelecer uma comparação para compreender se, por exemplo, o fato de Marisa Orth ser uma atriz influencia no resultado descrito pelo gráfico representado anteriormente. Partimos, agora, para o Gráfico 2, que representa a marcação de Espaços Mentais pela direção do olhar. Gráfico 2: Marcação visual de Espaços Mentais Gráfico 2: Marcação visual de Espaços Mentais Fonte: Elaborado pelo Pesquisador. Fonte: Elaborado pelo Pesquisador. Também considerando o total das ocorrências (11 ocorrências), a marcação predominante de Espaços Mentais pela direção do olhar foi a marcação do Ground, totalizando em números absolutos, 8 ocorrências em que a direção do olhar marca o Ground, (73%), contra 3 ocorrências em que a direção do olhar marca o Ground (27%). 5.4 Discussão geral dos dados Levando em consideração os percentuais, a marcação de Espaços Mentais por ambos os articuladores é inversamente proporcional, o que revela que a narradora, em termos do comportamento visual, preferiu interagir no Ground com os seus interlocutores, ao invés de produzir reencenações, por meio da direção do olhar. Conforme mencionamos anteriormente, estabelecemos como direção futura do nosso trabalho, analisar indivíduos que possuam estatutos sociais diferentes em situações narrativas diversas. Partimos agora, no Gráfico 3, para os dados que dizem respeito à simultaneidade na marcação de Espaços Mentais, quando consideramos as ocorrências, a partir da análise simultânea de ambos os articuladores. Essa simultaneidade foi analisada com base em 143 Dancygier (2011) que a denomina como fine-tuning – processo de afinação, em uma tradução livre – para definir o fenômeno no qual a funções, contextos e interações dos movimentos corporais entram em níveis específicos de sincronia interacional. Gráfico 3: Simultaneidade na marcação de Espaços Mentais Gráfico 3: Simultaneidade na marcação de Espaços Mentais Fonte: Elaborado pelo Pesquisador. Fonte: Elaborado pelo Pesquisador. Fonte: Elaborado pelo Pesquisador. Das 11 ocorrências, 3 possuem simultaneidade no que diz respeito à marcação do Ground, ou seja, nessas ocorrências, ambos os articuladores estão alocados, ao mesmo tempo no Ground. A simultaneidade na marcação do Espaço Narrativo também está presente em 3 ocorrências (27%), ou seja, nelas, ambos os articuladores estão alocados, ao mesmo tempo no Espaço Narrativo. Se consideramos a simultaneidade de marcação de Espaços Mentais por ambos os articuladores (54% no total, sendo 27% relativos a gestos e direção do olhar marcando o Espaço Narrativo e os outros 27% relativos à marcação do Ground, por ambos os articuladores), concluímos que, na narrativa, os gestos estão afinados à direção do olhar. Além disso, as 5 ocorrências restantes reafirmam a nossa hipótese de que, no Português Brasileiro, assim como já demonstrado anteriormente no inglês americano (SWEETSER; STEC, 2016), o olhar e os gestos manuais podem marcar Espaços Mentais independentes em um mesmo momento interacional. Em 46% dos dados (5 ocorrências), os gestos marcaram o Espaço Narrativo, enquanto a direção do olhar marcou o Ground. Por fim, vale salientar que não 144 encontramos, portanto, uma marcação do Ground pelos gestos em concomitância com a marcação do Espaço Narrativo pela direção do olhar. Partimos, agora, para o Gráfico 4 que diz respeito, especificamente, aos resultados que encontramos no Espaço Narrativo. 5.4 Discussão geral dos dados Esses dados se referem ao ponto de vista gestual (MCNEILL, 1995), conforme é possível observar a seguir: Gráfico 4: Pontos de Vista nos Gestos do Espaço Narrativo Gráfico 4: Pontos de Vista nos Gestos do Espaço Narrativo Gráfico 4: Pontos de Vista nos Gestos do Espaço Narr Fonte: Elaborado pelo Pesquisador. Fonte: Elaborado pelo Pesquisador. Das 8 ocorrências em que os gestos marcam o Espaço Narrativo, encontramos, em números absolutos, 6 ocorrências (75%) em que o ponto de vista da personagem é marcado, contra 2 ocorrências (25%) em que o ponto de vista do observador é marcado. Esse dado revela que, a atriz preferiu utilizar seu corpo como um representante das ações das personagens que fazem parte da sua narrativa, podendo ela ser as personagens x¹, x² e x³ que, respectivamente, se referem ao seu eu passado, às personagens responsáveis pelos conselhos que ela recebeu e o homem com o qual ela saiu para jantar. Mais uma vez, conforme mencionamos anteriormente, daremos seguimento a este trabalho, realizando uma análise mais completa que utilizará como corpus, narrativas proferidas por indivíduos que possuem outros estatutos sociais, como jornalistas e políticos, por exemplo. No gráfico 5, a seguir, é possível observar os resultados que encontramos, também relativos, especificamente, ao Espaço Narrativo. Nesse caso, trata-se das funções exercidas 145 pelos gestos no interior da narrativa, com base em Müller (2013), ou seja, apenas consideramos os gestos que marcam o Espaço Narrativo. A autora definiu “representação, expressão e apelo”, como as funções que os gestos no Espaço Narrativo podem exercer. Gráfico 5: Funções dos Gestos no Interior da Narrativa Gráfico 5: Funções dos Gestos no Interior da Narrativa Gráfico 5: Funções dos Gestos no Interior da Narrativa Fonte: Elaborado pelo pesquisador. Fonte: Elaborado pelo pesquisador. Das 8 ocorrências em que os gestos marcam o Espaço Narrativo, encontramos, em números absolutos, 5 ocorrências que o gesto possui a função representacional (62%), ou seja, nessas ocorrências, representa ações, objetos e eventos concretos ou abstratos. Em 2 ocorrências, encontramos gestos cuja função é expressiva (25%), ou seja, nessas ocorrências é possível verificar que o corpo se relaciona como um todo com a expressão marcada no nível verbal. E, apenas em uma ocorrência, encontramos a função apelativa (13%), ou seja, somente em uma ocorrência, verificamos a utilização do gesto como regulação do comportamento de outras pessoas. Por fim, no que diz respeito ao Gráfico 6, a seguir, verificamos, com base nas categorizações de Sweetser e Stec (2016), a função da direção do olhar no interior da narrativa. As autoras afirmam que o olhar de um falante, assim como a direção da cabeça e as expressões faciais podem representar ações relativas a uma personagem da narrativa, ou podem fazer parte da narração propriamante dita. Sendo assim, com base nos pressupostos das autoras, levamos em consideração duas funções principais do olhar na narrativa: i) encenação da personagem e ii) narração propriamente dita. 146 Gráfico 6: Função da Direção do Olhar no Interior da Narrativa 6 CONSIDERAÇÕES FINAIS A pesquisa que realizamos buscou responder à seguinte pergunta: Quais são as funções dos gestos manuais e do direcionamento do olhar na representação de Espaços Mentais em narrativas no português brasileiro?” e, a partir dela, definimos como objetivo específico de pesquisa investigar, a partir de dados multimodais, o comportamento dos gestos e da direção do olhar em narrativas multimodais do Português Brasileiro. Em seguida, ao analisar a marcação multimodal de Espaços Mentais; verificamos a relação entre os gestos e a direção do olhar na marcação de Espaços Mentais; e realizamos uma descrição a respeito da estrutura multimodal da narrativa, levando em consideração os gestos e a direção do olhar. Para tanto, utilizamos onze amostras de dados multimodais pertencentes a uma narrativa. Sendo assim, no que diz respeito ao referencial teórico, fundamentado no histórico dos Estudos de Gesto, que têm crescido, principalmente, no que diz respeito às funções que os gestos desempenham em coocorrência com a fala, realizamos o nosso estudo com base em Kendon (2004), McNeill (1979, 1995, 2005), Müller (2004, 2013, 2018) e Cienki (2008, 2017). Para nós, pesquisadores de gestos, eles são relevantes a partir do momento em que os consideramos, conforme explica Goldin-Meadow (2007) como uma “janela para a mente”, isto é, assumimos que os gestos revelam nuances e conteúdo da mente do falante, possibilitando um acesso imagético ao que está sendo falado. Esses fatores são determinantes para chegarmos às evidências a respeito do potencial linguístico dos gestos, uma vez que, como foi possível constatar com o estudo que realizamos, os falantes utilizam mecanismos gestuais como parte do seu empenho comunicativo e, também, para beneficiar a compreensão dos seus ouvintes. Desse modo, foi possível observar os modos com os quais os gestos e fala são inter-relacionados durante o ato de interação semiespontânea (considerando que, apesar de os nossos vídeos possuírem um conteúdo muito naturalístico, foi preciso considerar que, de certa maneira, existe um script e, além disso, existe um participante da interação que possui o papel de intermediar os segmentos de fala e as narrações sendo ele, nesse caso, o apresentador Fábio Porchat). Além disso, foi preciso compreender o contexto dessas interações para compreender como o gesto está presente nelas, levando em consideração o posicionamento temporal do gesto em relação ao segmento de fala, a relação semântica entre o gesto e a fala e a distribuição dos padrões gestuais em uma mesma interação. Gráfico 6: Função da Direção do Olhar no Interior da Narrativa Fonte: Elaborado pelo pesquisador. Fonte: Elaborado pelo pesquisador. Os resultados que encontramos revelaram que, das 3 ocorrências nas quais a direção do olhar marcou o Espaço Narrativo, em todas elas (100%) verificamos a função de encenação da personagem, o que revela que a atriz, em termos do comportamento visual, interagiu com maior frequência no Ground e, quando migrou para o Espaço Narrativo, preferiu reencenar o olhar de alguma personagem no interior da narrativa, ao invés de utilizar a direção do olhar como um olhar de narração propriamente dito. Esse dado foi muito revelador ao nosso ver, indicando que a ampliação do corpus, levando em consideração outros indivíduos e outros contextos de narração, será muito produtiva para que refinemos os resultados relacionados à função da direção do olhar encontrados aqui. 147 6 CONSIDERAÇÕES FINAIS A direção do olhar constitui outro articulador importante para a realização de nossa análise. No Brasil, existe uma escassez considerável no que diz respeito aos trabalhos que 148 investigam a relação dos mecanismos do eye-gaze e sua relação com os gestos manuais em contextos narrativos, o que justifica a importância e ineditismo de nossa pesquisa. Buscamos, principalmente, a partir das análises realizadas por Sweetser e Stec (2016) investigar os modos como a direção do olhar expressam a conexão entres indivíduos interactantes em dados multimodais. Nessa perspectiva, foi possível observar como o olhar revela aspectos importantes do narrador, como, por exemplo, o modo a partir do qual ele conceptualiza o mundo a partir de suas motivações, objetivos e pontos de vista. Considerando, ainda, o projeto temático do qual essa pesquisa faz parte – Linguística, Cognição e Multimodalidade – que trata da relação entre múltiplas modalidades da linguagem humana, nosso trabalho possui a relevância de agregar aos Estudos de Gesto no Brasil, tópicos relativos à emergência das pesquisas sobre o olhar (e, mais especificamente, à direção do olhar). É preciso destacar, ainda, que somente o advento de investigações sobre dados multimodais permitiu a realização do nosso trabalho, possibilitando uma metodologia capaz de capturar detalhes da interação, além de oferecer ferramentas para a observação e representação do olhar nas o o, principalmente no tocante à sua relação com a fala. No que se refere ao objetivo geral dessa pesquisa, que foi, investigar, a partir de dados multimodais, o comportamento dos gestos e da direção do olhar em narrativas multimodais do Português Brasileiro, concluímos que, de fato, no português brasileiro, assim como no inglês americano (Sweetser, Stec, 2016), o olhar e os gestos manuais podem marcar Espaços Mentais independentes em uma mesma interação, uma vez que, em 46 % do dados, os gestos marcaram o Espaço Narrativo, enquanto a direção do olhar marcou o Ground, conforme pode ser observado em: i) ocorrência 3 do bloco “Exposição”; ii) ocorrência 2 do bloco “Clímax”; iii) ocorrência 4 do bloco “Clímax”; iv) ocorrência 5 do bloco “Clímax” e v) ocorrência 1 do bloco “Desfecho”. 6 CONSIDERAÇÕES FINAIS Levando em consideração os nossos objetivos específicos, concluímos, ao analisar a marcação multimodal de Espaços Mentais na representação de Espaços Mentais que, considerando o total das ocorrências (11 ocorrências), a marcação predominante de Espaços Mentais pelos gestos foi a marcação do Espaço Narrativo, totalizando em números absolutos, 8 ocorrências em que os gestos marcam o Espaço Narrativo (73%), contra 3 ocorrências em que os gestos marcam o Ground (27%). Além disso, também considerando o total das ocorrências analisadas (11 ocorrências), concluímos que a marcação predominante de Espaços Mentais pela direção do olhar foi a marcação do Ground, totalizando em números absolutos, 8 ocorrências 149 em que a direção do olhar marca o Ground, (73%), contra 3 ocorrências em que a direção do olhar marca o Ground (27%). Ao verificar a relação entre os gestos e a direção do olhar na marcação de Espaços Mentais e, também, ao realizar uma descrição da estrutura multimodal da narrativa, levando em consideração os gestos e a direção do olhar, verificamos que das 11 ocorrências, foi possível observar a simultaneidade no que diz respeito à marcação do Ground em 3 ocorrências (27%). Do mesmo modo, foi possível observar a simultaneidade na marcação do Espaço Narrativo que está presente em 3 outras ocorrências (27%). Se consideramos a simultaneidade de marcação de Espaços Mentais por ambos os articuladores (54% no total, sendo 27% relativos a gestos e direção do olhar marcando o Espaço Narrativo e os outros 27% relativos à marcação do Ground, por ambos os articuladores), concluímos que, na narrativa, a maior parte dos gestos está afinada à direção do olhar, o que não invalida a nossa hipótese de partida, uma vez que, encontramos 46% das ocorrências nas quais os dois articuladores marcam, independentemente, Espaços Mentais diferentes, ao mesmo tempo. Na tentativa de refinar a explicação dos mecanismos responsáveis pela marcação de Espaços Mentais pelos gestos e pela direção do olhar, acrescentando, ainda, a construção de um repertório de gestos do ponto de vista do observador e do ponto de vista da personagem, produzidos em contextos narrativos presentes em interações formais e informais, possuímos a intenção de continuar as análises iniciadas nesta dissertação, no nível de Doutorado. 6 CONSIDERAÇÕES FINAIS Surgiram, então, novas perguntas que dizem respeito à estrutura multimodal das narrativas cujas respostas pretendemos obter ao fim de uma nova pesquisa: i) Levando em consideração os gestos do ponto de vista do observador e os gestos do ponto de vista da personagem, quais são os gestos produzidos com mais frequência em narrações presentes em contextos formais e informais de interação? ii) Em quais contextos o falante/gesticulador tende a trocar com mais frequência o ponto de vista gestual na mesma interação? iii) Acompanhados de qual dos dois pontos de vista gestual os gestos e a direção do olhar tendem a marcar com mais frequência o mesmo Espaço Mental? A fim de responder a essas questões, partimos das seguintes hipóteses: i) Em narrações mais formais, os gestos que ocorrem com mais frequência são os gestos do ponto de vista do observador (O-VPT), enquanto nas mais informais, os gestos que ocorrem com mais frequência são os gestos do ponto de vista da personagem (C-VPT). ii) Nos contextos mais formais, a troca de ponto de vista gestual acontece com menos frequência. Por outro lado, nos contextos mais informais, o falante/gesticulador utiliza a troca de ponto de vista gestual com mais frequência. 150 iii) A marcação de Espaços Mentais pelos gestos e pela direção do olhar é mais simétrica (isto é, marcam o mesmo Espaço Mental) em contextos narrativos informais em relação aos formais. Ainda no que diz respeito às direções futuras do trabalho, possuímos como objetivo geral, verificar e analisar a frequência de produção de gestos do ponto de vista do observador (O-VPT) e gestos do ponto de vista da personagem (C-VPT) em contextos narrativos televisionados. Apresentamos como objetivos específicos: i) Comparar a frequência de produção de gestos do ponto de vista do observador (O-VPT) e do ponto de vista do personagem (C-VPT), em ocorrências narrativas extraídas mais formais (como, por exemplo, programas jornalísticos como o “Jornal Nacional” e o “Roda Viva”) e mais informais (como, por exemplo, programas de entretenimento como o “De Frente com Gabi” e o “Que História É Essa, Porchat?”); ii) Compor um repertório de ocorrências gestuais em diferentes contextos narrativos; iii) Descrever como o espaço físico dos diferentes contextos de narração influencia na marcação de Espaços Mentais e no ponto de vista gestual. 6 CONSIDERAÇÕES FINAIS Conforme mencionamos anteriormente, a ampliação da pesquisa realizada até então permitirá que refinemos os parâmetros de análise e interpretação da relação entre os mecanismos gestuais com os mecanismos visuais na marcação de Espaços Mentais, levando em consideração os pontos de vista gestuais e as funções dos gestos e da direção do olhar no interior de diferentes narrativas. 151 REFERÊNCIAS BAVELAS, J; COATES, L; JOHNSON, T. 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Não tinha irmão, irmãos mais velhos... Tinha vivido um pouco... Né, com quinze anos... Fábio: E foi o suficiente, porque né... Dali em diante não ia viver mais nada... Só trabalho... Letícia: Exatamente! E eu tinha paixão por teatro, né... Eu queria ser atriz, eu entrei nesse trabalho porque eu queria me virar pra pagar meus cursos sozinha sem depender da minha mãe e do meu pai e aí, é... Eu fui trabalhar, eu tinha asma, né, bronquite alérgica desde criança. E nessa época ainda tinha algumas crises. E aí, eu fazia tudo pra ficar um tempo em casa, ou ficar fazendo teatro com a galera e eu nunca ia poder fazer a peça junto com eles, porque eu ia pro México junto com a Xuxa, e aí eu tava com bronquite... Letícia: ... Quando eu entrei, a sorte é que eu já tinha feito algumas coisas... tinha beijado na boca, né... Sempre fui uma pessoa que... Eu sempre prezei os prazeres da vida, assim. Não tinha irmão, irmãos mais velhos... Tinha vivido um pouco... Né, com quinze anos... E foi o suficiente, porque né... Dali em diante não ia viver mais nada... Só trabalho... Letícia: Exatamente! E eu tinha paixão por teatro, né... Eu queria ser atriz, eu entrei nesse trabalho porque eu queria me virar pra pagar meus cursos sozinha sem depender da minha mãe e do meu pai e aí, é... Eu fui trabalhar, eu tinha asma, né, bronquite alérgica desde criança. E nessa época ainda tinha algumas crises. E aí, eu fazia tudo pra ficar um tempo em casa, ou ficar fazendo teatro com a galera e eu nunca ia poder fazer a peça junto com eles, porque eu ia pro México junto com a Xuxa, e aí eu tava com bronquite... Fábio: Todo o Brasil queria ir pro México com a Xuxa e ela queria ficar aqui fazendo teatro... Letícia: Exatamente! Aí eu falei “não, gente, pro México, acabei de sair de uma crise de asma, eu não sei se eu sou capaz... Preciso ficar aqui, eu quero fazer teatro” E tal... Louca! Kiko Mascarenhas: Não faz o menor sentido! Letícia: Não faz... A pessoa podia ter conhecido o México. ANEXO A: TRANSCRIÇÃO DA NARRATIVA DE LETÍCIA SPILLER57 Fábio: Você de paquita tem uma miscelânea de coisas, né? p q , , g Fábio: Mas era... Era pressão... Era jogo duro ali, né? Letícia: Era pressão! Quando eu entrei, né, realmente no trabalho, eu não fazia ideia da dimensão de quanto eu ia ter que trabalhar, né, nessa época da minha vida, tinha quinze anos, né? Letícia: Era pressão! Quando eu entrei, né, realmente no trabalho, eu não fazia ideia da dimensão de quanto eu ia ter que trabalhar, né, nessa época da minha vida, tinha quinze anos, né? Fábio: Uhum... Fábio: Uhum... Não fui até hoje! E aí... Ainda vou, um dia! E aí... Eu vou prolongar um pouco minha crise, eu vou... Letícia: Não faz... A pessoa podia ter conhecido o México. Não fui até hoje! E aí... Ainda vou, um dia! E aí... Eu vou prolongar um pouco minha crise, eu vou... Fábio: Vou prolongar minha crise?! Ficou sem camisa na janela, tomando vento. Letícia: Aí, eu falei “Xuxa, eu não to me sentindo bem ainda, tô com falta de ar... Letícia: Aí, eu falei “Xuxa, eu não to me sentindo bem ainda, tô com falta de ar... Fábio: Mentindo no trabalho. Letícia: “... Eu vou pro México, não sei se vai ser uma boa ideia por causa da altitude e tal, aquilo tudo ”... “É... Não, tudo bem, fica tranquila...” Marlene né, principalmente... Fábio: Marlene! 57 Devido ao fato de não termos analisado essa narrativa, preferimos não transcrevê-la aos moldes do GAT 2 (SELTING et al., 2009). 156 Letícia: Tinha que falar, comunicar pra Marlene... Daí, tô eu lá com minha mãe, em casa, bate a campainha, quem era, Xuxa e Marlene, me fazer uma visita... Letícia: Tinha que falar, comunicar pra Marlene... Daí, tô eu lá com minha mãe, em casa, bate a campainha, quem era, Xuxa e Marlene, me fazer uma visita... Fábio: Mas e esse porteiro? Letícia: Daí ficou, ficamos lá conversando... Fábio: Mas eras foram lá sentir se você tava... Letícia: É... Foram... Não sei, foram me visitar... Kiko: Foram solidárias e dar uma checadinha... Kiko: Foram solidárias e dar uma checadinha... Letícia: Dar uma checadinha... Letícia: Dar uma checadinha... Fábio: Foram levar um termômetro... Mas você não podia fazer as coisas, porque você disse que já tinha beijado e tal, mas... Letícia: Não, é porque assim, a gente, a minha mãe contou nos dedos, o número de folgas que eu tive em um ano. Eu acho que eu tive duas folgas... É assim, era mui... Era show, show, show, show todo final de semana, às vezes tinha que viajar pra Argentina, fazer cinco programas na Argentina no fim de semana... Fábio: Programas. Explica só porque as pessoas podem... Letícia: Desculpa... obrigada... Programa de TV, gente, da Xuxa... E na Argentina, ainda era mais desgastante porque não tinha juizado de menor, então a gente tinha criança de um ano, dois, adolescentes... Fábio: Uhum... tudo misturado num palco e às vezes, a gente ficava assim, com neném no colo, falando, suando, daí trocava, trocava o público, né... Trocava o público, você tava suando... Fábio: E essas crianças eram infernais? Você já pisou no pé de uma? Fábio: E essas crianças eram infernais? Você já pisou no pé de uma? Letícia: Não... algumas eram mais quietinhas, não, eu nunca, graças a Deus... inclusive, inclusive, a Marlene um dia me chamou pra dedurar uma pessoa, uma das paquitas que tinha falado grosseiramente com uma criança, que a criança reclamou, não sei o que, ela falou “Pituxa, vem aqui” Aí, eu fui morrendo de medo e tal... Aí, ela falou, “quem foi que falou e fez isso e tal”... Aí, eu falei “eu não vou falar porque eu não sou dedo duro”. Aí, ela “então vá pra casa, quando você aprender a ser dedo duro, você volta”. o: Olha, delação premiada já naquela época. Fábio: Olha, delação premiada já naquela época. Letícia: Eu fui feliz da vida pra casa! Vou ficar uma tarde em casa, fui pra casa, voltei, no dia seguinte, como se nada tivesse acontecido, ela também não falou nada. Fábio: Olha que gostoso que era ser paquita, né, gente?! A pessoa preferia tomar um esporro... A pessoa falava “Graças a Deus”. E você foi pro México? 157 ANEXO B: TRANSCRIÇÃO DA NARRATIVA DE MARISA ORTH ANEXO B: TRANSCRIÇÃO DA NARRATIVA DE MARISA ORTH 01 MO: eu POSso conTAR minha hisTÓ[Ria]? 02 FP: [ahm] 01 MO: eu POSso conTAR minha hisTÓ[Ria]? ANEXO B: TRANSCRIÇÃO DA NARRATIVA DE MARISA ORTH 02 FP: [ahm] 03 MO: me lembro muito uma história sim 04 eu tava solteira 05 <<apontando com o dedo indicador da mão direita em 06 direção à direita>> 07 aí saí com um CAra 08 super recomendado pra mim 09 gente é ESse 10 mas olha: vai assim 11 é (.) primeira vez[né ] 12 SS: [aHAM] 13 MO: cê tá um tempo solteira 14 então é assim: 15 mas assim ã::: 16 não VAi de saia curta 17 não vai de calça muito justa 18 não vai de salto muito ALto que você já é [grande] 19 SS: [uhum ] 20 MO: né: vai vai vai mais [baixinha ] 21 FP: [hahahahaha] 22 MO: vai meio raste:ira 23 não bota cor muito for:te 24 não ri AL:to 25 <<levantando as duas para cima junto com a direção d 26 olhar>> 27 não abre a [boca] 28 SS: [gente] 29 MO: não deixa ele PERCEBER [que você é inteligente] 30 SS: [gente não VAle ] 31 né 32 IS: [esse é o cara pra você: ] ] 158 33 MO: [não deixa ele perceber que você é inteligente ] 34 faz um brinquINHO menor 35 <<levantando ambas as mãos em direção à sua própria 36 e seus dedos indicadores e polegares corporificam um 37 pequeno em cada lado do seu rosto>> 38 não BEbe 39 não [Fuma ] 40 FP: [gente] mas 41 mas foi no século deZOIto que: 42 MO: é mas hoje em dia é assim 43 pra você não assusTAR o homem 44 lá fui eu no Nude 45 FP: ((ri)) 46 SS: ((ri)) 47 IS: ((ri)) 48 MO: (--) 49 MO: tentando fazer a disCREta né 50 eu não sou boa nisso né 51 FP: marisa ORth [discreta ] 52 MO: [minha risada é ] GRANde 53 eu penso:: ai:: 54 mas fui e ele né tinha cara de execuTIvo 55 eu nun::ca pego os executivos 56 não É meu perfil: né 57 SS: aham 58 MO: e tinha um sapato BOM 59 DEdo bom 60 Uma cara BOA 61 AI AH VAmos ao teatro 62 depois vamos jantar:: 63 achei BOM que ele me levou ao teatro 64 ok:: o nome da peça era os cafaJEStes 65 eu deVIa ter me [ligado] 66 FP: [((ri))] [((ri))] 159 67 SS: [((ri))] 68 IS: [((ri))] 69 FP: ele que comprou o ingre::sso 70 MO: ele que escoLHEU 71 e comPROU 72 SS: não pois é:: 73 MO: e eu nem me toQUEI 74 falei AI tudo bem coisa de HÉ::tero né 75 SS: [ha] 76 IS: ((ri)) 77 FP: ((ri)) 78 MO: BURra 79 (.) 80 aí fomos no restaurante 81 papo bom e tal e eu NUde 82 ?hm?hm ?hm?hm 83 não bebe 84 FP: nã [hahahaha] 85 SS: [hahahaha] 86 IS: [hahahaha] 87 MO:?hm?hm 88 <<com o dedo indicador para cima em um movimento 89 repetitivo da esquerda para a direita>> 90 e né RI das PIadas de:le 91 vinha os aMIgos gays as aMIgas. ANEXO B: TRANSCRIÇÃO DA NARRATIVA DE MARISA ORTH 92 RIA das piAdas DEle 93 <<com as duas mãos com as palmas laterais e paralelas, 94 realiza um movimento descendente 95 ria [((incompreensível, aprox. 2 seg))] 96 SS: [leGENda ] 97 MO: e ah papo vem 98 coMEçou a falar dos FIlhos 99 <<com as duas palmas para cima realiza um gesto de 100 apresentar>> 67 SS: [((ri))] 68 IS: [((ri))] 69 FP: ele que comprou o ingre::sso 70 MO: ele que escoLHEU 71 e comPROU 72 SS: não pois é:: 73 MO: e eu nem me toQUEI 74 falei AI tudo bem coisa de HÉ::ter 75 SS: [ha] 76 IS: ((ri)) 77 FP: ((ri)) 78 MO: BURra 79 (.) 80 aí fomos no restaurante 81 papo bom e tal e eu NUde 82 ?hm?hm ?hm?hm 83 não bebe 84 FP: nã [hahahaha] 85 SS: [hahahaha] 86 IS: [hahahaha] 87 MO:?hm?hm 88 <<com o dedo indicador para cima em 89 repetitivo da esquerda para a direi 90 e né RI das PIadas de:le 91 vinha os aMIgos gays as aMIgas. 92 RIA das piAdas DEle 93 <<com as duas mãos com as palmas la 94 realiza um movimento descendente 95 ria [((incompreensível, aprox. 2 se 96 SS: [leGENda 97 MO: e ah papo vem 98 coMEçou a falar dos FIlhos 99 <<com as duas palmas para cima rea 100 apresentar>> [((ri))] [((ri))] 160 101 ele gostou MUIto 102 pior que ele adorou 103 ficou bem começou a falar dos Filhos 104 falou TEnho filhos 105 e eu feLIZ falei 106 <<ambas as palmas para cima em um movimento 107 ascendente>> 108 eu já pensando na minha mente doentia 109 que bom ele tem filhos eu também já tinha um 110 falei não vamos mais precisar ter 111 SS: [hahaha] 112 FP: [hahaha] 113 SS: [maravilhoso ] 114 MO: [mulher é bicho] doente 115 falei que baCAna 116 e eles moram com quem:: 117 moram com sua ex-mulher:: 118 não não 119 é moram coMIgo 120 e a mãe deles mora conosco também 121 garçom: 122 (--) 123 FP: co 124 MM: é eu olhei oi: 125 é a maneira MAis criativa 126 que eu já ouvi alguém me dizer que é Casado 127 SS: aham [a MÃE] dos meus filhos também é [ca 128 IS: [puts ] 129 MO: [não nã 130 os meus filhos moram coMIgo 131 e a MÃE DEles mora conosco também 132 é garÇOM 161 135 FP: um outro homem 136 um outro rapaz: 137 MO: foi esse TEMpo 138 <<mãos com as palmas diagonais em um movimento 139 ascendente>> 140 sentiram a biGORna que caiu na sala 141 SS: NOSsa 142 MM: foi mais ou menos essa biGORna que eu recebi 143 FP: e você planejando 144 mas daí você falou o que pra ele:: 145 MM: e aí começou a FARra 146 IS: aí você tirou o [nude ] 147 MM: aí começou [a farra] 148 aí o volume aumentou 149 estávamos num restaurante pequeno 150 eu comecei 151 já viu atraÇÃO fatal:: ((ri)) 152 comecei aí 153 <<ambas as mãos com as palmas para baixo em um movimento 154 para baixo>> 155 comecei na glenn CLOse 156 falei você não tem medo:: 157 eu sou atriz::: 158 você sabia que eu sou louca:: 159 SS: gente: 160 MO: aí ele começou se aproximando 161 qual é o problema você não é atriz:: 162 ele faLOU isso 163 qual é o problema você não é atriz: 164 eu falei então 165 e se eu descobrir onde seus filhos estudam:: 166 IS: ê::: 167: MO: comecei a fazer a louca 168 e ele por favor não sei o que lá 162 169 eu falei assim 170 aGOra comecei baixo 171 agora você vai enFIAR a MÃO no seu BOLso direito 172 e tiRAR a aliANça 173 daí eu fui aumentando o volume assim 174 e ele((incompreensível, aprox. [((ri))] [((ri))] 2 seg)) 175 não eu tô falando super baixo 176 que loucura agora 177 você vai enfiar a mão no seu BOLso direito 178 e tiRAR a alian::ça 179 ((incompreensível, aprox. 1 seg)) 180 eu falei 181 agora será que eu preciso repetir a terceira vez:: 182 ele falou 183 não por favor 184 <<com as palmas verticais, realiza um movimento 185 descendente>> 186 FP: ele [tinha a aliança no bol:so] 187 MO: [TInha a aliança no bolso ] 188 IS: [filho da pleuras ] 189 FP: O::lha só 190 MO: aí foi 191 tripudiei o quanto eu pude 192 a comida tava vin:do 193 eu falei não QUEro 194 brincando de aumentar a voz 195 não QUEro 196 IS ah eu ia pedir [tudo que é caro] 197 MO: [não não jamais] 198 não consegui 199 IS: coisa pra comer:: 200 MO: fui no caminho me leve pra casa imediatamente 201 fui no caminho e meio falando mal 202 GRAças a Deus não tinha acontecido NAda 163 203 foi me dando um ódio um ódio: 204 cheguei em casa falei o que eu pu:de falei mal DE:le 205 falei que eu tenho horror 206 cheguei chorei passou 207 foi isso 208 FP: ((ri)) 209 MO: o que eu vou fazer: 210 não é muito bom:: Que isso:: 211 achou que eu era a MAGda né 212 que eu não ia juntar o nome à pessoa 164 164 ANEXO C – PARÂMETROS DE ANÁLISE ANEXO C – PARÂMETROS DE ANÁLISE PARÂMETROS DE ANÁLISE OBJETIVO PARÂMETRO Realizar uma descrição da estrutura multimodal da narrativa, levando em consideração os gestos e a direção do olhar Trilha1: Trecho Narrativo Exposição, Clímax e Desfecho Responder à pergunta de pesquisa: Quais são as funções dos gestos manuais na marcação de de Espaços Mentais em narrativas no português brasileiro? [((ri))] [((ri))] Trilha 2: Stroke Simples, Frase Gestual Trilha 3: Transcrição Trilha 4: Formato da Mão Mão Aberta, Punho, Dedos Individuais, Combinação de Dedos Trilha 5: Orientação das Palmas Para Cima, Para Baixo, Diagonal, Lateral Trilha 6: Posição do Movimento 0 próprio corpo do falante, 1 distância próxima do corpo, 2 distância média ,3 distância longa Trilha 7: Tipo de Movimento Reto, Arqueado, Círculo, Espiral, Zigzag, Linha S Trilha 8: Direção do Movimento Ao longo do eixo horizontal (esquerda e direita, conforme a perspectiva do gesticulador); Ao longo do eixo vertical (para cima e para baixo); Movimentos ao longo do eixo sagital (em direção ao corpo e para fora do corpo) Trilha 9: Qualidade do Movimento Fluxo do movimento (fraco ou acentuado) PARÂMETROS DE ANÁLISE 165 OBJETIVO PARÂMETRO Trilha 10: Modo de Representação Gestual “encenar”, “corporificar”, “segurar/moldar” e “desenhar” Trilha 11: Marcação dos Espaços Mentais Ground, Espaço Narrativo Trilha 12: Ponto de Vista Gestual Observador, Personagem Trilha 13: Função na Narrativa Representação, Expressão e Apelo Responder à pergunta de pesquisa: Quais são as funções da direção do olhar na marcação de de Espaços Mentais em narrativas no português brasileiro? PARÂMETROS DE ANÁLISE Trilha 14: Direção do Olhar Linha Falante-Ouvinte, Esquerda, Direita, Cima, Baixo Trilha 15: Direção do Tronco Esquerda, Direita, Centro Trilha 16: Marcação dos Espaços Mentais Ground, Espaço Narrativo Trilha 17: Função da Direção do Olhar no Interior da Narrativa Narração, Encenação ANEXO D – TRILHAS DE ANÁLISE DO BLOCO “EXPOSIÇÃO” OBJETIVO ANEXO D – TRILHAS DE ANÁLISE DO BLOCO “EXPOSIÇÃO” 166 TRILHA OCORRÊNCIA 1 OCORRÊNCIA 2 OCORRÊNCIA 3 Trilha 1: Trecho Narrativo Exposição Exposição Exposição Trilha 2: Stroke Simples Simples Simples Trilha 3: Transcrição eu tava solteira não ri AL:to Faz um brinquinho menor Trilha 4: Formato da Mão Dedos Individuais Mão aberta Combinação de Dedos Trilha 5: Orientação das Palmas Para Baixo Lateral Lateral Trilha 6: Posição do Movimento 1 2 0 Trilha 7: Tipo de Movimento Reto Reto Reto Trilha 8: Direção do Movimento Direita Para Cima Para Cima Trilha 9: Qualidade do Movimento Fraco Acentuado Fraco Simples Simples Simples Combinação de Dedos 1 0 2 167 167 TRILHA OCORRÊNCIA 1 OCORRÊNCIA 2 OCORRÊNCIA 3 Trilha 10: Modo de Representação Gestual (Gesto Dêitico) Encenar Corporificar Trilha 11: Marcação dos Espaços Mentais Ground EN EN Trilha 12: Ponto de Vista Gestual - Personagem Observador Trilha 13: Função na Narrativa - Expressiva Representacional Trilha 14: Direção do Olhar Linha Falante-Ouvinte Para Cima Linha Falante-Ouvinte Trilha 15: Direção do Tronco Direita Centro Direita Trilha 16: Marcação dos Espaços Mentais Ground EN Ground Trilha 17: Função da Direção do Olhar no Interior da Narrativa - Encenação da Personagem - ANEXO E – TRILHAS DE ANÁLISE DO BLOCO “CLÍMAX” 168 TRILHA OCORRÊNCIA 1 OCORRÊNCIA 2 OCORRÊNCIA 3 Trilha 1: Trecho Narrativo Clímax Clímax Clímax Trilha 2: Stroke Repetição Gestual Simples Simples Trilha 3: Transcrição MO:?hm?hm RIA das piAdas DEle coMEçou a falar dos FIlhos Trilha 4: Formato da Mão Dedos Individuais Mão Aberta Mão Aberta Trilha 5: Orientação das Palmas Lateral Lateral Para Cima Trilha 6: Posição do Movimento 1 1 2 Trilha 7: Tipo de Movimento Arqueado Reto Arqueado Trilha 8: Direção do Movimento Esquerda e Direita Baixo Para Fora do Corpo Trilha 9: Qualidade do Movimento Fraco Acentuado Acentuado 168 Clímax Simples Simples Repetição Gestual Repetição Gestual Simples 1 2 169 169 TRILHA OCORRÊNCIA 1 OCORRÊNCIA 2 OCORRÊNCIA 3 Trilha 10: Modo de Representação Gestual Encenar (Gesto rítmico) (Gesto de apresentar) Trilha 11: Marcação dos Espaços Mentais EN EN Ground Trilha 12: Ponto de Vista Gestual Personagem Personagem - Trilha 13: Função na Narrativa Representacional Representacional - Trilha 14: Direção do Olhar Para Baixo Linha Falante-Ouvinte Linha Falante-Ouvinte Trilha 15: Direção do Tronco Centro Esquerda Esquerda Trilha 16: Marcação dos Espaços Mentais EN Ground Ground Trilha 17: Função da Direção do Olhar no Interior da Narrativa Encenação da Personagem - - OCORRÊNCIA 3 TRILHA OCORRÊNCIA 5 OCORRÊNCIA 4 OCORRÊNCIA 5 TRILHA OCORRÊNCIA 4 OCORRÊNCIA 5 OCORRÊNCIA 4 170 Trilha 1: Trecho Narrativo Clímax Clímax Trilha 2: Stroke Simples Simples Trilha 3: Transcrição e eu feLIZ falei é garÇOM Trilha 4: Formato da Mão Mão aberta Dedos Individuais Trilha 5: Orientação das Palmas Para Cima Para Baixo Trilha 6: Posição do Movimento 2 2 Trilha 7: Tipo de Movimento Reto Reto Trilha 8: Direção do Movimento Para Cima Para a Direita Trilha 9: Qualidade do Movimento Acentuado Fraco Trilha 10: Modo de Representação Gestual Encenar Encenar Trilha 11: Marcação dos Espaços Mentais EN EM Trilha 12: Ponto de Vista Gestual Personagem Personagem 170 Clímax Clímax Simples Simples Simples Simples 171 171 Trilha 13: Função na Narrativa Expressiva Apelativa Trilha 14: Direção do Olhar Linha Falante-Ouvinte Linha Falante-Ouvinte Trilha 15: Direção do Tronco Esquerda Esquerda Trilha 16: Marcação dos Espaços Mentais Ground Ground Trilha 17: Função da Direção do Olhar no Interior da Narrativa - - Trilha 13: Função na Narrativa 172 ANEXO F – TRILHAS DE ANÁLISE DO BLOCO “DESFECHO” TRILHA OCORRÊNCIA 1 OCORRÊNCIA 2 OCORRÊNCIA 3 Trilha 1: Trecho Narrativo Desfecho Desfecho Desfecho Trilha 2: Stroke Simples Simples Simples Trilha 3: Transcrição foi esse TEMpo comecei aí não por favor Trilha 4: Formato da Mão Mão Aberta Mão Aberta Mão Aberta Trilha 5: Orientação das Palmas Diagonal Para Baixo Vertical Trilha 6: Posição do Movimento 2 1 1 Trilha 7: Tipo de Movimento Reto Reto Reto Trilha 8: Direção do Movimento Para Cima Para Baixo Para Cima OCORRÊNCIA 3 Simples Simples Vertical 1 2 173 173 Trilha 9: Qualidade do Movimento Acentuado Acentuado Fraco Trilha 10: Modo de Representação Gestual Segurar/Moldar (Gesto Ritmico) Encenar Trilha 11: Marcação dos Espaços Mentais (Gestos Manuais) EM Ground EN Trilha 12: Ponto de Vista Gestual Observador - Personagem Trilha 13: Função na Narrativa Representacional - Representacional Trilha 14: Direção do Olhar Linha Falante-Ouvinte Linha Falante-Ouvinte Trilha 15: Direção do Tronco Esquerda Centro Centro Trilha 16: Marcação dos Espaços Mentais (Direção do Olhar) Ground Ground EN Trilha 17: Função da Direção do Olhar no Interior da Narrativa - - Encenação da Personagem
https://openalex.org/W4394854686	https://www.cambridge.org/core/services/aop-cambridge-core/content/view/D74E320EDD4C955D5E7E6EA2AD92F57A/S1479591422000523a.pdf/div-class-title-a-flexible-choice-of-comrades-the-dynamic-identity-of-the-muslim-huis-of-the-seventeenth-and-eighteenth-centuries-div.pdf	Chinese	null	A flexible choice of comrades: the dynamic identity of the Muslim Huis of the seventeenth and eighteenth centuries	International journal of Asian studies	2,024	cc-by	12,511	© The Author(s), 2024. Published by Cambridge University Press. 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, provided the original article is properly cited. Abstract Abstract Thispaperinvestigates howHuischolarsimaginedthemembershipoftheircommunityduringthe seventeenth and eighteenth centuries as sharing communal religious duties, such as Islamic education and funeral rites, which were correctively imposed by an Islamic doctrine on all Muslims. The analysis is mainly based on two texts. One is an inscriptive text drafted in Arabic and Persian by Ma Minglong in A.H. 1079 (1668– 1669) and inscribed by his son in 1673 in Wuchang, Hubei province. The other is Tianfang dianli (Commentary on Rites of Islam), written in Chinese by Liu Zhi (d. after 1724) based on Arabic and Persian Islamic books. The analysis shows that these Hui scholars have flexibly demarcated their communities from those whom they regarded as “other” Muslims, depending on various situations. Additionally, this paper illu- minates how Hui scholars’ various and flexible delineations of “us” facilitated their negotiations for advanta- geous positions toward Muslim rivals, as well as non-Muslims who suspected their orthodoxy. This relativizes the argument that Hui scholars understood themselves as being simultaneously Chinese and Muslim when they situated themselves vis-à-vis Chinese literati—an argument that has been often repeated in the study of Hui Muslims. Keywords: Chinese Islamic literature; Chinese-speaking Muslims; filial duty; Hui identity; Islamic law (funeral rites); Islamic law (religious education); Persian in China A flexible choice of comrades: the dynamic identity of the Muslim Huis of the seventeenth and eighteenth centuries Tatsuya Nakanishi Institute for Research in Humanities, Kyoto University, Kyoto-shi, Japan Email: nakanishi.tatsuya.7s@kyoto-u.ac.jp Institute for Research in Humanities, Kyoto University, Kyoto-shi, Japan Email: nakanishi.tatsuya.7s@kyoto-u.ac.jp (Received 31 August 2022; accepted 13 December 2022) International Journal of Asian Studies (2024), 1–16 doi:10.1017/S1479591422000523 RESEARCH ARTICLE Introduction Huis (Chinese-speaking Muslims) during the pre-modern period negotiated their Islamic beliefs and practices with the non-Muslim Han people, who often regarded Islam as heretical and its believers as dangerous. For example, Hui scholars have written Chinese works about Islamic teachings since the seventeenth century that reinterpret and express Islam as being harmonious with Confucianism, the dominant Chinese philosophy. Zvi Ben-Dor Benite’s pioneering study on Hui scholars’ identities examined their Chinese works, including those on Islamic teachings, and argued that they “understood themselves as simultaneously Chinese and Muslim” (Ben-Dor Benite 2005, pp. 7–8, 12–13, 110, 232, et passim). He explained that they recognized themselves as a group with a “Chinese Muslim body of knowledge” that is “one important branch of Chinese learning” or the “Dao of Muhammad” and identified themselves as among the Chinese literati in a broader sense (ibid., pp. 34–35, 78, 120, 141, et passim). In other words, they located themselves in “the overlapping space between an imagined and reified ‘Islam’ and ‘China’ (ibid., pp. 15, 213, 233) where they understood themselves as being “both as Chinese Tatsuya Nakanishi 2 men of letters and as members of a specifically Muslim branch of Chinese knowledge” (ibid., p. 3) simultaneously. Following this argument, other researchers discussed how Hui Muslim scholars’ ideas and discourses involved the simultaneity of Chineseness and Muslimness to legitimize their existence in China against objections from the non-Muslim Han people (Cieciura 2016, p. 135; Frankel 2011, pp. 15–17, 87, 121–22, 147–52, et passim; Lipman 2016; Tontini 2016, pp. 198, 205, 211). However, as Ben-Dor Benite (2005, pp. 8, 18-19, 104, 106, 111-12) also noted, such identity and group consciousness emerged among Hui scholars only when they situated themselves vis-à-vis Chinese intellectuals.1 We should go further than statically viewing the Chinese–Muslim simultaneity as the predominant Hui identity. It is unclear how they saw themselves in relation to “other” Muslims. This paper investigates how Hui scholars, during the seventeenth and eighteenth centuries, imagined the membership of their community as sharing communal religious duties, including Islamic educa- tion and funeral rites, which was correctively imposed by an Islamic doctrine on “all Muslims (al- jamī‘).”2 This paper will elucidate that Hui scholars have flexibly demarcated their communities from ones whom they regarded as “other” Muslims depending on various situations. 5For information on Mosque education in China and Hu Dengzhou, see Ben-Dor Benite (2005, pp. 35–43). Note that Hu Dengzhou’s foundation of mosque education in China is simply a discourse of his followers, and a similar system existed before him (Nakanishi 2013, p. 22, n.7). 1Lipman (1997, pp. 124, 133, 137, 152–54, 215, et passim) also demonstrated that the Hui flexibly redrew various bound- aries between “us” and “them,” depending on situations, thus concluding that “being a Muslim did not determine anyone’s behavior, though it probably influenced everyone’s.” 3I have discussed how Hui intellectuals, during the modern period, situated themselves (sometimes within and at other times outside) dār al-Islām and altered their relationship with the Umma in response to historical backgrounds (Nakanishi 2018, pp.121-44). 6For Liu Zhi and his Tianfang dianli, see Frankel (2011). 4For this inscription and its author, see Ma and Wang (2017). As for Ma Minglong, also see Ben-Dor Benite (2005, pp. 46, 100–6, et passim). 2As for the expression "all Muslims," see notes 13 and 17. 3 Introduction Thus, this paper illustrates the Huis’ multilayered and dynamic identities by scrutinizing how they contextually either articulated or obscured their fellow feeling for a group of Islamic believers vis-à-vis Muslim “outsiders.”3 This paper will illuminate how Hui scholars’ various and flexible delineations of “us” facilitated their negotiations for advantageous positions with Muslim rivals as well as non-Muslims who suspected their orthodoxy. First, an inscription written in Arabic and Persian by Ma Minglong 馬明龍(d. 1679) – a Hui scholar who lived in Wuchang 武昌, Hubei 湖北– is analyzed.4 In the inscription, he voices a sense of comradeship with “Muslim inhabitants in China,” celebrating a joint project involving many Hui Muslims from different backgrounds because of their common religio-intellectual ancestor, Hu Dengzhou 胡登洲(d. 1597), known as the founder of mosque education in China.5 Ma Minglong conceives the frame of “Muslim inhabitants in China” as an entity charged with maintaining Islamic knowledge via education, whereas Islamic jurisprudents usually identify "all Muslims" as the same entity. This related to his strategy to establish his superiority to other Hui Muslim scholars. Second, the way that Liu Zhi 劉智(d. after 1724) – a Hui Muslim scholar who lived in Nanjing – managed the notion of the collectivity of "all Muslims" in his Chinese work, Tianfang dianli 天方典禮, is examined.6 In this work, he persuades his co-religionist readers to band together with their own families, rather than the larger Muslim communities like the “Muslim inhabitants in China” and “all Muslims”, to collectively perform the funeral prayer. Liu Zhi reframes the unit for Islamic collect- ive obligations such as “Muslim inhabitants in China” and “all Muslims” as comprising each family, considering filial duty, which non-Muslim Chinese people have valued as one of the most important ethics in pre-modern China. 1Lipman (1997, pp. 124, 133, 137, 152–54, 215, et passim) also demonstrated that the Hui flexibly redrew various bound- aries between “us” and “them,” depending on situations, thus concluding that “being a Muslim did not determine anyone’s behavior, though it probably influenced everyone’s.” 2 3I have discussed how Hui intellectuals, during the modern period, situated themselves (sometimes within and at other times outside) dār al-Islām and altered their relationship with the Umma in response to historical backgrounds (Nakanishi 2018, pp.121-44). 5For information on Mosque education in China and Hu Dengzhou, see Ben-Dor Benite (2005, pp. 35–43). The inscription commemorating the relocation of Hu Dengzhou’s tomb The inscription commemorating the relocation of Hu Dengzhou’s tomb An English translation of the Arabic and Persian inscription written by Ma Minglong will now be examined ([1–46] represents the line numbers. The basmala is written before these 46 lines. The Persian translation is in bold. Regarding the Arabic and Persian transcription, see Appendix A).7 [Part one] [1] All praise be to God! He created the human being from the soil, then from a drop of sperm. He made them pairs and predestined hidden wisdoms and potential powers to appear among them [2] so as to raise them into a multitude of people organized into groups. He returns them to the soil with his firm hold on their destinations, brings them out [of the soil] while recreating [their bodies] after their death and questions them regarding inexcusable matters. [3] He is the Most Kind and the Best, who created death and life to test them [i.e., people] to know whether they commit evil. He is the Self- Sufficient and the Most Great. Oh, the Most Kind! May the forgiveness be from You, our Lord. We entrust You with our end. [4] Verily, You are the Merciful and the All-Forgiving. We give a benediction to Muhammad, His Prophet, who was created from his soil as a great cre- ation; to his family members and companions, all of whom are heirs to his view; [5] and to other Prophets and Saints before him and the Caliphs and Imams after him. May God’s blessings be upon all of them for as long as the tongue praising them continues to exist. As [6] the last station [of one’s journey through life] in this world is a level ground where the funeral is performed, the first station in the afterlife is also the flat soil of the tomb. The best one to visit [7] is this tomb among these tombs, because it is the tomb of the best of the forefathers and even the master of scholars in China; that is, His Holiness Hū Tāybābā Shams al-Dīn [i.e., Hu Dengzhou] <May God’s Mercy be upon him [8] until the Day of Judgement>. He is like the one who brought the teaching to China. 7The text has been transcribed and translated based on photos of the inscription, taken in 2009, by Takashi Aoki 青木隆 (Professor at Nihon University), and retaken in 2010 by a joint fieldwork team led by Takashi Kuroiwa 黒岩高(Professor at Musashi University), with the participation by Takashi Aoki, Minoru Satō 佐藤実(Professor at Otsuma Women’s University), Yōichi Yajima 矢島洋一(Professor at Nara Women’s University), and the author. Unfortunately, the photos, which are fragmented pictures of the inscription, cannot be published because many are so unclear that one cannot decipher characters on them without image enlargement. The author extends his appreciation to Yōichi Yajima, who modified certain parts of the transcription and translation. When I translated the inscriptional text into English, I partly consulted its Chinese translation by Ma and Wang (2017). However, the authors often give up translating word for word because many inscription characters are ambiguous. Thus, their Chinese translation fails to capture some important information from the original text. International Journal of Asian Studies International Journal of Asian Studies 3 Introduction Note that Hu Dengzhou’s foundation of mosque education in China is simply a discourse of his followers, and a similar system existed before him (Nakanishi 2013, p. 22, n.7). 6 International Journal of Asian Studies Self-awareness of “Muslim inhabitants in China” Self-awareness of “Muslim inhabitants in China” The inscription commemorating the relocation of Hu Dengzhou’s tomb Our Prophet, who makes [the truth] clear in his brilliant remark as follows: “Seek knowledge even in China.” However, all people of China were about [9] to profess themselves irreli- gionist due to deviation and misguidance. Most of them [understood Islam] ambiguously, and [the truth] unclearly. He (i.e., Hu) straightened the situations so that the column of this teaching rose. [10] All of their (i.e., people of China) problems, larger and smaller, were resolved by his instruction in these days. He is compared to the owner of the mineral veins of secrets of knowledge, who reveals them to the ones deserving of them, with lights that nullify [11] the value of pearls and make them cheap. All the powerful ones [i.e., those who have received the secrets of knowledge] are like treasuries with precious knowledge accumulated in terms of his education, rather like trees bearing fruits in the aspect of their student-hood. All of them [i.e., the powerful ones], in return [for his education], pay a part of their earnings [12] as alms. The diffusion of the fragrance, from pure flowers [of the trees], reaches the smell of humankind, and the fruits help him. May peace always be upon those who obtain [knowledge] from the excellent ones [13] and on all of those after him, among whom there will always be Islam. They are [Hu’s] successors, like Caliphs of the Prophet, each of whom teaches various kinds of knowledge to people annually. He is a man to whom their intention [of adherence] is turned, [14] day and night. He is like a sun that spreads benefits through its profitable brilliance, doing a favor for [all things] from the summit of heaven to beneath the seventh heaven. By his grace, the current Tatsuya Nakanishi 4 situation of learning in China is so great that the ramified parts [15] are never humiliated by [their unfavorable comparison with] the root from which all have originated. His heirs are scholars from his age to ours. All of them were heirs to his benefits. Now, when time has passed and their age has come, [16] it is obligatory for many teachers to thank the man who laid the foundation of the learning. Let those who seek knowledge in this age sincerely pray for [the person] who taught Muslims [17] knowledge. 10The original Persian expression “jagar lishte (licked the liver)” might be a mistake of the Persian expression “jagar khwurde (ate the liver; i.e., grieved)” under the influence of the Chinese expression “changdan 嘗胆(lick the gallbladder; i.e., endure pain to attain a purpose).” 9Kinjānfūy is a transliteration of Jingzhaofu 京兆府, the old name of Xi’an 西安, which is the capital of the Shanxi 陝西 province. In Persian sources, the Shanxi province was often called Kinjānfū. See Honda (1991, p. 464). 8For the Old Practice 古行and New Practice 新行, see Nakanishi, Morimoto, and Kuroiwa (2012) and Nakanishi (2013, pp. 143–80). 9 府安 The inscription commemorating the relocation of Hu Dengzhou’s tomb Verily, God characterized scholars with the highest rank in his following remark: “[He elevates] those who were taught knowledge to degrees of rank.” [Qur’ān, 58: 11] The gratitude [to Hu Dengzhou] obliged us to perform a litany, reciting [18] a petition [for a divine mercy] on him, after a prayer during the day and night, and to serve to him [i.e., Hu] by tears for him in addition to the witr prayer at breakfast before going to the predawn prayer. Oh God, we request You - Oh Lord, [19] You are the All-Forgiving and the Merciful – to show mercy to Your slave by Your great forgiveness; and to make the rotation of moons, the shining and moistening of day and night, the appointed time of the appearance of stars, [20] their constellation after their appearance, the glorification of angels from the heaven to the earth, the thunderclap pealing all over the earth, the laudation of beasts in the earth’s hidden places, [21] the withering of plants growing from the ground, the blowing of wind with rains, and their sounds with [those of] leaves of trees, all of them, as longings for Your eternal mercy toward him [22] and as desires for your everlasting kindness toward him. I [i.e., Ma Minglong] wrote this to establish it for pilgrims. You are the most Merciful among owners of mercy. Kinjānfūy is a transliteration of Jingzhaofu 京兆府, the old name of Xi an 西安, which is the capital of the Shanxi 陝西 province. In Persian sources, the Shanxi province was often called Kinjānfū. See Honda (1991, p. 464). 10The original Persian expression “jagar lishte (licked the liver)” might be a mistake of the Persian expression “jagar khwurde (ate the liver; i.e., grieved)” under the influence of the Chinese expression “changdan 嘗胆(lick the gallbladder; i.e., endure pain to attain a purpose).” 8For the Old Practice 古行and New Practice 新行, see Nakanishi, Morimoto, and Kuroiwa (2012) and Nakanishi (2013, 143 80) [Part two] That man [i.e., Hu Dengzhou], who is perfect as an educator of excellent persons and complete as [23] a teacher of scholars <May God put him down in a site of his Garden and make him resident and peaceful in the Garden>, left this place of ephemerality in accordance with the mandate of “Everything on the earth will perish” [Qur’ān, 55: 26]. [24] Since he turned his face toward the world of perpetuity, one hundred years have already passed. However, the people of the religion do not forget the traces of him, using their mouths to narrate his legacy. As for his great achievement, the inhabitants [25] of China do not neglect it, reflecting on it in their minds. Suddenly, a group of merchants reached the city [i.e., Wuchang, Hubei], where an inconsiderable and worthless abject one [i.e., Ma Minglong], who is more trivial than the pellicle of a date, lived. They were distin- guished members of [the school of] the Old Practice and sincere members [26] of [the school of] the New Practice.8 After [my] meeting with them, they explained the following situation [to me]: Our cities in Shanxi 陝西province (Kinjānfūy)9 are usually covered with pebbles. However, this year, the following unusual situation occurred. Due to the power of God the Most High, it incessantly rained [27] for more than forty days and nights, and [God] made the ground suffer the trouble of a flood. Every lowland changed into a river. Every highland [28] absorbed water and broke at every spot. Wherever divine judgment and predestination reached remained in such a state for a long time. Because of this heavenly trial, most graves [29] were deva- stated. All the buried bones were exposed. The aforementioned man [i.e., Hu Dengzhou] also grieved10 for this, as the same fate was about to happen [to him]. Thus, scholars and other pious people [30] in the city burned their hearts with compassion for him. Distinguished persons, whom they call “the best one of all the present-day leaders, [or] the beautiful essence of the present day [people],” and our scholars, more in number [than the distinguished persons], gathered. [31] They bought a burial site that they had agreed to buy. [Part three] [Part three] [40] As for the content of this elegy, it was written [by] the poor one. He was a miserable sum- moner, Muhammad al-Mahdī b. Imām al-ʿAtā’ Allāh [i.e., Ma Minglong] <May God’s Mercy be on both>, who is a trivial one [41] among humankind and a base one, trodden under legs. [It was written] in 1079 [1668-1669] of the hijra calendar of the selected person, Muhammad <May God bless him and show mercy to him>. [42] The copy of this written satisfied elegy and ode was finished by the hand of a feeble slave. He was a son and scribe of the author. [43] He was Ahmad al-Nūrī. He was taught studies and manners by the author. As for his relationship with the praised one, the author is a disciple of his [i.e., Hu’s] disciple, whom he complementally called Hūnlaw Santay Bābā [44] from al-Kinjānfūyī, a blessed region, and Tālib b. ʿAbd al-Rahmān, which is one of the fortunate auspicious names. g May God forgive him [i.e., Hūnlaw Santay Bābā] [45] with all predecessors and successors by per- fect pardon and all-embracing forgiveness, as He forgave the praised one in reward for his merits that nobody but scholars among the predecessors knows. A couplet: [46] Oh God, please forgive all of us [i.e., Ma Minglong and his son] and the two teachers [i.e., Hu Dengzhou and Hūnlaw Santay Bābā] among our predecessors, if they transmit useful knowl- edge to us and our posterity, as they [i.e., their predecessors] did. [Part two] They then dug up the ephemeral body of the International Journal of Asian Studies 5 5 scholar, who pertained to the Lord, together with [bodies of] his highborn family members and sons, from the nearly devastated tomb, and buried all of them in the newly bought graveyard. [32] Then, their minds were humbled by these bodies, and their eyes became cautious [regarding them]. Their hearts led them to be preoccupied with rendering what was in their hearts into a text. Therefore, toward these visitors, [33] they stressed [the plan]. However, no text had yet been sent. Thus, this feeble one composed an elegy. I dedicate it to the leader of the prayer. At that time, they were building a place to pray for His Holiness. [34] They were promulgating meritorious deeds of that estimable nonpareil and furthermore narrating his episodes. Their good deeds will also be known at every place. When I heard of this story, [35] I regarded it as an example to follow. If I had not passed on the tale about them in writing, nobody would have heard of it. Thus, I wrote that [i.e., the aforementioned elegy] in Arabic, combined it with this Persian text [about their good deeds], dedicated the former to his mausoleum [36] as an elegy, and showed the latter for pil- grims to the tomb as a pathetic song. Thus, I publicized his prominent scholarship for the predecessors and expounded the prominence of his meritorious deeds to the successors. All praise be to God! He put knowledge as a means by which people approach [37] the rank of the Prophets and favored our praised one [i.e., Hu Dengzhou] with the name of “introducer [of Islam]” before other scholars. Oh God, please forgive our praised one with noble ancestors [38] and descen- dants by perfect grace and all-embracing forgiveness. I sincerely narrate his meritorious deeds for him and recite something good as a closing supplication for his comfort in the afterworld so that the people of the religion continue to tell his story [39] beyond centuries, until the Settlement Day, by the grace of God, who is the Unique, the Subduer the Transcendent, and the Creator, as with Muhammad, the elite monotheist, his family, and other honorable ones. “Muslim inhabitants in China” as a unit for the communal mission of education This Arabic and Persian text is inscribed on a stone, located on a wall of Ma Minglong’s tomb at Wuchang. The same stone has a Chinese text written by Ma Minglong and inscribed by the author’s son, Ma Fanglong 馬房龍, in 1673, below the Arabic and Persian text. The Chinese inscription records that the “virtuous gentlemen ( junzi 君子)” of the Shanxi 陝西province donated the relocation of Hu Dengzhou’s tomb to avoid a flood from the Wei river; this sincere generosity allowed them to finish Tatsuya Nakanishi 6 the tomb relocation operation before the original graveyard became part of the Wei river 渭水.11 The Arabic and Persian text was written by Ma Minglong in 1668–1669, and inscribed by Ma Fanglong, likely along with the Chinese inscription in 1673. The Arabic and Persian text comprises three sections. The first praises Hū Tāybābā Shams al-Dīn while urging Muslim scholars in China to thank him for his transmission of Islamic knowledge to them. We can identify Hū Tāybābā (i.e., Hu taibaba 胡太爸爸) with Hu Dengzhou, by consulting the second section and the Chinese text, both of which mention the removal of his tomb. This section is written predominantly in Arabic and partly in Persian. The second section narrates why and how the tomb of Hu Dengzhou was relocated and the inscrip- tion text produced. This section is written predominantly in Persian and partly in Arabic. According to it, the author composed the first section in Arabic when the people who were concerned with the tomb’s reconstruction came from Shanxi 陝西province and revealed their plan to put their inmost admiration and gratitude to Hu Dengzhou into words. Afterward, the author wrote the inscription’s second section in Persian and annexed it to the first section. He intended to offer the two sections as an epitaph of the new tomb to let visiting pilgrims know the achievements of the bur- ied as well as his followers’ activities extoling him. The third section is a kind of postscript informing the reader that Muhammad al-Mahdī b. al-Imām ʿAtā’ullāh drafted the first and second sections in A.H. 1079 (1668–1669) and that his son, Ahmad al-Nūrī, inscribed the text onto the stone. In the third section as well, both Muhammad al-Mahdī b. al-Imām ʿAtā’ullāh and Ahmad al-Nūrī likely played the same role. “Muslim inhabitants in China” as a unit for the communal mission of education When consulting the Chinese text of the inscription, Muhammad al-Mahdī is identified with Ma Minglong and Ahmad al-Nūrī with Ma Fanglong. According to the Chinese text, Ma Fanglong wrote the entire Arabic and Persian text onto the stone in 1673. At the end of the third section, Ma Minglong prayed for God to forgive him, his son, and his teachers, such as Tālib b. ʿAbdurrahmān, alias Hūnlaw Santai Bābā from al-Kinjānfūyī (Feng Laosantai baba 馮老三太爸爸from Jingzhaofu 京兆府or Shanxi 陝西province; i.e., Feng Bo’an 馮伯菴),12 and Hu Dengzhou as well as the future Muslims who received intellectual benefits from these scholars. The inscription’s Arabic and Persian text conveys that Ma Minglong was conscious of his affiliation with “Muslim scholars of China” at times and with the “Muslim inhabitants in China” in general at other times. For example, in the first section (ll.17–18), Ma Minglong says, “The gratitude [to Hu Dengzhou] obliged us to perform a litany reciting a petition [for a divine mercy] on him after a prayer day and night….” In this sentence, “us” signifies the “Muslim scholars of China (ʿulamā’ al-Sīn)” (l.7) who inherited knowledge through their master-disciple lineage from Hu Dengzhou to become his “successors (khulafā’)” (l.13). The inscription compares them to “ramified parts (ajzā’) who are never humiliated by [their unfavorable comparison with] the root from which all originated (laisa mukhzā min nujār al-kull min asl-hi)” (ll.14-15). In this sentence, the author remembers Hu Dengzhou as “our” religio-intellectual ancestor and the “Muslim scholars of China” as “we” who equally belong to the same genealogical tree of education stemming from him. q y g g g g In the second section (l.37), Ma Minglong called Hu Dengzhou “our praised one (mamdūh-nā).” This “our” refers to the “people of the religion (ahl al-dīn)” who are “inhabitants in China (abnā’ al-Sīn)” (ll.24–25) including scholars and other pious people (ʿālimān wa dīndārān)” (l.29) . In other words, the “us” indicates Muslim inhabitants in China in general who more or less received reli- gious knowledge to “approach the rank of the Prophets” from Hu Dengzhou (ll.36-37) through his “successors,” “each of whom teaches various kinds of knowledge to people” (l.13). The self-awareness of belonging to “Muslim inhabitants in China” was likely prompted in Ma Minglong’s mind when he heard of people who worked hard to preserve Hu Dengzhou’s tomb (ll.26–34). 11The Chinese text is recorded in Da (2007, p. 68). 12For more on Feng Bo’an, see Ben-Dor Benite (2005, pp. 45–46). “Muslim inhabitants in China” as a unit for the communal mission of education The cognizance of such people led Ma Minglong reconfirm the existence of Islamic believ- ers who shared the same origin of knowledge that the entombed master embodied. 7 International Journal of Asian Studies International Journal of Asian Studies Additionally, a more concrete image of the religio-intellectual community might have occurred to Ma Minglong when he met Shanxi merchants from schools of the Old and New Practices who visited Wuchang to consult on the contribution of an elegy for Hu Dengzhou (ll.25–26). The fact that the two rivaling factions for the Islamic orthopraxis in China cooperated to publicly honor their common founding father may have impressed Ma Minglong regarding the unity of “Muslim inhabitants in China.” Furthermore, Ma Minglong seems to have intensively envisaged the Muslim community in China while boasting his fulfillment of the liability toward it for transmitting the Islamic knowledge inherited from Hu Dengzhou to future generations. Islamic jurisprudents have often counted learning and teaching Islamic sciences among the communal duties called “furūdkifāya (collective obligations),”13 the singular form of which is “fardkifāya (collective obligation).”14 God imposed this type of obligation upon all Muslims collect- ively rather than on individual believers. If a Muslim fulfills this type of obligation, the others are exempt from it; however, if nobody fulfills it, all Muslims are deemed sinful. In certain cases, even if Muslims in a region fulfill a communal obligation, those in other regions are not exempt from it.15 Ma Minglong might have had the latter viewpoint in mind for the collective obligation of educa- tion. He apparently thought that Muslims in China are never exempted from that obligation, even though co-religionists in other countries maintain Islamic knowledge. In other words, he seemingly categorized the “Muslim inhabitants in China” as a unit that was collectively obliged to transmit Islamic knowledge and shared the same soteriological destiny, independently from other similar units outside China. This is why the inscription extols Hu Dengzhou and his scholarly successors’ efforts to improve the learning situation for “all people of China (ahl al-Sīn kullu-hum)” (ll.8-15) and additionally implores God to forgive not only Hu Dengzhou and Feng Bo’an but also the latter’s “predecessors” (al-aslāf) and “successors” (al-akhlāf) (ll. 44–46). “Predecessors” refers to scholars in the generations preceding that of Feng Bo’an, those who transmitted knowledge to him and enabled him to do similar work. 13It is highly possible that Ma Minglong recognized this legal interpretation. For example, Baydāwī (1998, vol. 3, p. 102) comments on a part of the Qur’ānic verse 9: 122 “It is not possible for the believers to go forth all together. Why, then, does not a party from every section of them go forth that they [i.e., the rest] may become well versed in religion, and that they may warn their people when they return to them? as follows: “In this verse, there is proof that learning the Islamic jurisprudence and reminding people of it (al-tafaqquh wa al-tadhkīr) are counted among communal obligations ( furūdal-kifāya).” According to Jingxue xichuanpu 經學繋傳譜, Ma Minglong gave special importance to this exegesis (Gesui 咯遂i.e., Tafsīr al-Qādī) (Zhao 2005, p. 43). Moreover, Kanz al-ʿibād (Fasl fī al-masā’il al-mutafarriqa), which Ma Minglong likely consulted, cites the following opin- ion from al-Kubrā: “Teaching the whole Qur’ān is a communal obligation ( fardkifāya), and learning indispensable knowl- edge of the Islamic jurisprudence is an individual obligation ( fardʿayn)” (Ghūrī 1908, p. 455). Kanz al-ʿibād is enumerated in Arabic inscriptions at Zhuxianzhen and Kaifeng (Henan province), erected by followers of Ma Minglogn (Nakanishi, Morimoto, and Kuroiwa 2012, pp. 96, 101–2). Ma Minglogn likely recognized what the collective obligation meant. Baydāwī (1998, vol. 2, pp. 31–32) interpreted the Qur’ānic verse 3: 104 “Let there be among you (min-kum) a group of people who call [others] to goodness, command right, and forbid wrong. They are successful” as follows: “‘Among (min)’ signifies dividing [‘you,’ i.e., Muslims] into parts (tab‘īd), because commanding right and forbidding wrong is counted among the collective obligations (furūdal-kifāya), and not everyone is qualified for it… It is imposed on everyone in the sense that all of them become sinful (athimū jamīʿan) as soon as they abdicate it, but they become exempted from it if someone from among them performs it.” 14Th ll ti bli ti i j t d ith th i di id l bli ti (f d ʿ ) th t i i d h M li I l i “Muslim inhabitants in China” as a unit for the communal mission of education In addition, the couplet at the end of the inscription (l.46) humbly assumes Ma Minglong’s success in handing down knowledge from his predecessors to successors. Thus, it in fact boasts that this success has already come true and exon- erated not only himself but also his two teachers, Feng Bo’ab and Hu Dengzhou, from the sin against the divine order of the intellectual transmission among each Muslim community. Minglong, deserve God’s tolerance because they transmitted knowledge to posterity, thereby saving the “Muslim inhabitants in China” from being sinful regarding the collective duty of education, and enabled Ma Minglong’s disciples to accomplish the same task. In addition, the couplet at the end of the inscription (l.46) humbly assumes Ma Minglong’s success in handing down knowledge from his predecessors to successors. Thus, it in fact boasts that this success has already come true and exon- erated not only himself but also his two teachers, Feng Bo’ab and Hu Dengzhou, from the sin against the divine order of the intellectual transmission among each Muslim community. Through the imploration and couplet, the author authorizes himself as a genuine heir to Hu Dengzhou, the first savior of “all people of China” in the Islamic educational mission, and as a retro- spective corroborator of the precursor’s accomplishing of the mission in question. In the context of this self-legitimization, Ma Minglong envisioned the community of “Muslim inhabitants in China” as people whom he saved as a successor to Hu Dengzhou. Additionally, Ma Minglong needed the category of the “Muslim scholars of China,” including him- self, to demonstrate Hu Dengzhou’s achievement in the same mission and thereby enhance the authority of succeeding him. Indeed, the inscription stresses that Hu Dengzhou’s educational activity yielded so great a result that his “successors” could never be unfavorably compared to their founder (ll.14-15). Here, Ma Minglong appears to have qualified the “Muslim scholars of China” equally as Hu Dengzhou’s successors despite their division into Old and New Practice branches. However, Ma Minglong’s actual purpose in the categorization of the “Muslim scholars of China” was not to make his position inconspicuous among them. Ma Minglong likely needed this categorization to highly valorize the successorship to Hu Dengzhou itself before he eventually implied that he is more authentic than other scholars in terms of that successorship. “Muslim inhabitants in China” as a unit for the communal mission of education “Successors” refers to those who received knowledge from him and will continue his pedagogical effort. The imploration implies that Hu Dengzhou, Feng Bo’an, and their successors, including Ma Moreover, Kanz al-ʿibād (Fasl fī al-masā’il al-mutafarriqa), which Ma Minglong likely consulted, cites the following opin- ion from al-Kubrā: “Teaching the whole Qur’ān is a communal obligation ( fardkifāya), and learning indispensable knowl- edge of the Islamic jurisprudence is an individual obligation ( fardʿayn)” (Ghūrī 1908, p. 455). Kanz al-ʿibād is enumerated in Arabic inscriptions at Zhuxianzhen and Kaifeng (Henan province), erected by followers of Ma Minglogn (Nakanishi, Morimoto, and Kuroiwa 2012, pp. 96, 101–2). Ma Minglogn likely recognized what the collective obligation meant. Baydāwī (1998, vol. 2, pp. 31–32) interpreted the Qur’ānic verse 3: 104 “Let there be among you (min-kum) a group of people who call [others] to goodness, command right, and forbid wrong. They are successful” as follows: “‘Among (min)’ signifies dividing [‘you,’ i.e., Muslims] into parts (tab‘īd), because commanding right and forbidding wrong is counted among the collective obligations (furūdal-kifāya), and not everyone is qualified for it… It is imposed on everyone in the sense that all of them become sinful (athimū jamīʿan) as soon as they abdicate it, but they become exempted from it if someone from among them performs it.” 14The collective obligation is juxtaposed with the individual obligation (fardʿayn) that is imposed on each Muslim. Islamic legal duties are classified as either of these two types. 15For example, Badā’iʿ al-sanā’iʿ (Kitāb al-sayr) states, “When [the jihād] is a collective obligation ( fardʿalā kifāya), the caliph (imām) must not empty any border from a group of conquerors (ghuzāt) who have a wealth and ability to fight enemies; when they engage in it, others are exempted from it” (Kāsānī al-Hanafī 2003, vol. 9, p. 381). In other words, as al-Nahr al-fā’iq (Kitāb al-jihād) interpreted, “It is not proper to imagine that people of India are exempted from [the jihād] when people of Anatolia engage in it” (Ibn Najīm al-Hanafī 2002, vol. 4, p. 199). Tatsuya Nakanishi 8 Minglong, deserve God’s tolerance because they transmitted knowledge to posterity, thereby saving the “Muslim inhabitants in China” from being sinful regarding the collective duty of education, and enabled Ma Minglong’s disciples to accomplish the same task. 16This chapter is a developed version of part of my previous study (Nakanishi 2017). “Muslim inhabitants in China” as a unit for the communal mission of education Certainly, the author had some rivals among Hu Dengzhou’s “succes- sors.” Ma Minglong may have been concerned with the Old Practice, and his authority appears to have been challenged by other contemporary religio-intellectual descendants of Hu Dengzhou, such as Chang Zhimei 常志美and She Qiling 舎起靈, who were founders of the New Practice (Nakanishi et al. 2012, pp. 101-2; Nakanishi 2013, pp. 121-24). Ma Minglon also competed with Chang Zhimei and She Qiling in interpreting Sufism (Nakanishi 2007, pp.63-64). Ma Minglong’s imagination of the “Muslim inhabitants in China” as ones whom he saved as a proper successor to Hu Dengzhou likely aimed to claim his superiority to such rivals among the “Muslim scholars of China.” In short, in the context of this self-authorization, Ma Minglong in his inscription delineated the “Muslim inhabitants in China” as in contrast with co-religionists outside the country and associated him- self with the “Muslim scholars of China” among the former. As mentioned in the Introduction, according to Ben-Dor Benite, the Hui Muslim scholars, including Ma Minglong (Ben-Dor Benite 2005: 100-6), iden- tified themselves as a special group among non-Muslim Chinese literati to prevent the latter from despis- ing the former. However, this identity changed depending on the context. Ma Minglong at least imagined his affiliation not with a part of non-Muslim Chinese literati but rather with that of the “Muslims inha- bitants in China,” or a unit among “all Muslims,” to compete with other Hui scholars. The collective funeral obligation The collective funeral obligation As noted above, Ma Minglong likely applied all Muslims’ communal liability for knowledge transmission to “Muslim inhabitants in China.” However, how did the other Huis express the collect- ivity of all Muslims or joint liability for Islamic legal obligations? Pre-modern Hui scholars doubtlessly recognized the care of co-religionist dead bodies as a collective obligation imposed on all Muslims from the Hanafī school’s Islamic jurisprudent International Journal of Asian Studies 9 books, which Hui Muslims followed exclusively.17 Ma Boliang 馬伯良, in Jiaokuan jieyao 教款捷要 (finished in 1678), gave a relatively precise explanation of the collective funeral obligation, as follows: A classic work says as follows: Washing and shrouding a dead body, the funeral prayer (dian janāzaﺟﻨﺎﺯﻩ )and inhumation are the second heavenly mandate (futian[ming] 副天[命]).18 If anyone performs it, everybody will be relieved from the binding [of the mandate]. If nobody performs it, all believers in the region (or district) [where the mandate has not been carried out] (benfang jiaozhongren 本方敎中人) cannot be free from it (Ma Boliang 2005, p. 243/f. 93b). Notably, Ma Boliang did not regard all Muslims as those in charge of the funeral rites but rather Muslims in a specific region or district. This suggests that he was strongly conscious of the unity of either the “Muslim inhabitants in China,” like Ma Minglong, or that of a Hui community around a mosque. Liu Zhi seems to have described the communal funeral duties differently. He neither closely repro- duced the cooperative aspect of all Muslims that his Hanafite references’ interpretation of this collect- ive obligation implies nor conceived of the unity of the “Muslim inhabitants in China” who were collectively liable for their funerals; this is unlike Ma Minglong, who suggested the same unity con- stituted sharing liability for knowledge transmission among that community. This can be seen in an explication of this Islamic funeral rule in Liu Zhi’s Tianfang dianli. p f g Liu Zhi wrote this Chinese work based on Arabic and Persian Islamic books, which he enumerates in the reference list. Books of jurisprudence, among the references, consist of exclusively those from the Hanafī school, including al-Hidāya, Sharhal-Wiqāya, Targhīb al-salāt, Majmūʿ-i Khānī, Salāt-i Masʿūdī (Leslie and Wassel 1982). In Tianfang dianli, the author’s explanation of the inhumation faithfully follows Targhīb al-salāt and Majmūʿ-i Khānī. 17As for the funeral rite, which is categorized not as an individual obligation (fardʿayn) but a collective obligation (fard kifāya), for example, Sharhal-Wiqāya (Bāb al-janā’iz) states the following: “The [funeral] prayer is a collective obligation. If someone performs it, it is omitted from everybody else. However, if nobody performs it, everybody (al-jamīʿ) becomes sinful (ya’thamu)” (Sharīʿa 2009, vol. 1, pp. 252–53). Sharhal-Wiqāya was circulated among the Hui during the late Chinese imperial era (Nakanishi, Morimoto, and Kuroiwa 2012, pp. 86, 95). 18 18The original text of Ma Boliang 2005 is missing the character ming. I added this character based on another version of Jiaokuan jieyao (Ma Bokiang 1919, pp.41-42). The collective funeral obligation Targhīb al-salāt (Fasl 49 ahkām-i janāza-hā) states, “According to Fatwā-yi sirājī, it is permissible that a body of a deceased person be kept in a house [of his family] for [their] mourning for three days, but it is better that it is not kept even for three days [but buried quickly]” (Zāhidī (1556)., f. 119a). Majmūʿ-i Khānī (Bāb 32 dar bayān-i namāz-i janāza) states, “It is prohibited to open a grave [of a Muslim] after his death,” unless water overwhelms his grave (Nāgawrī 1896–1897, vol. 1, p. 119). Based on these descriptions, Tianfang dianli (Liu 2005, p. 180/vol. 20, Sangzangpian, f. 8a) wrote, “Bury [the body of a person] within three days [after his death],” commenting as follows: https://doi.org/10.1017/S1479591422000523 Published online by Cambridge University P The body becomes peaceful just when it is buried. The utmost term for keeping it in a house is three days. If he died on his trip, and [his body] is carried to his home town [to be buried there], the time limit is not applied. However, it is proper to choose a place in a region where he died on his trip and bury his body there. Moreover, Liu Zhi, in his gloss of this passage, admonished that the dead traveler’s relatives should not bring the deceased’s body to his own hometown, either to bury or rebury it there (ibid.). Moreover, Liu Zhi, in his gloss of this passage, admonished that the dead traveler’s relatives should not bring the deceased’s body to his own hometown, either to bury or rebury it there (ibid.). These comments mean that, if a Muslim traveler dies in a place that none of his family members can reach within three days after his death, he must be buried there by someone who is co-religionist but unrelated to him. In short, the author of Tianfang dianli suggests that every Muslim is liable for the inhumation of a deceased co-religionist, even if the latter is a stranger to the former. This discourse Tatsuya Nakanishi 10 was compatible with the Chinese custom of mutual aid for the inhumation of a person who died in a land distant from his homeland. 21According to al-Hidāya, guardians (awliyā’) have precedence for the funeral leadership “in the order mentioned for mar- riage (nikāh)” (Marghīnānī 1996–1997, vol. 2, p. 144; 2006, p. 232). “The order in the case of ʿasabāt with respect to nikāhis the same as that of residuaries in inheritance, with the more distant being excluded by the nearer” (Marghīnānī 1996–1997, vol. 3, p. 41; 2006, p. 496). As for the guardianship of marriage of an insane woman, Muhammad al-Shaybānī gave prece- dence to her father, while Abū Hanīfa and Abū Yūsuf gave it to her son because of his precedence among residuaries (Marghīnānī 1996–1997, vol. 3, p. 50; 2006, p. 500). Salāt-i Masʿūdī (Bāb 45 dar bayan-i namāz-i janāza) states, “If a person died and his father and son are alive, it is desirable that the father perform the funeral prayer” (Samarqandī 1897–1898, vol. 2, p. 189). Majmūʿ-i Khānī (Bāb 32 dar bayān-i namāz-i janāza) states, “If the religious head of the community (imām-i hayy) is not attendant, a guardian of the deceased (walī-yi mayyit) such as the father, son, brother, and uncle leads the funeral prayer” (Nāgawrī 1896–1897, vol. 1, p. 110). The collective funeral obligation He was often buried where he died or brought back to his homeland and re-buried by benevolent individuals.19 Regarding the collective obligation for the inhumation of co-religionists, Liu Zhi agreed that it is imposed on all Muslims, as Targhīb al-salāt and Majmūʿ-i Khānī do. However, this is not the case for the funeral prayer. 19However, this type of mutual aid was expected of people from the same homeland or kinship group, but it was not imposed on the deceased person’s “co-religionists” in Confucian, Buddhist, or Taoist communities (Fuma 1997, pp. 651, 654-56, 709-39; Chen 2009). 20As for the Islamic legal discourses and historical tensions around who should lead the funeral prayer, see pp. 168-79). However, this type of mutual aid was expected of people from the same homeland or kinship group, but it was not imposed on the deceased person’s “co-religionists” in Confucian, Buddhist, or Taoist communities (Fuma 1997, pp. 651, 654-56, 709-39; Chen 2009). 20As for the Islamic legal discourses and historical tensions around who should lead the funeral prayer, see Halevi (2007, pp. 168-79). 21According to al-Hidāya, guardians (awliyā’) have precedence for the funeral leadership “in the order mentioned for mar- riage (nikāh)” (Marghīnānī 1996–1997, vol. 2, p. 144; 2006, p. 232). “The order in the case of ʿasabāt with respect to nikāhis the same as that of residuaries in inheritance, with the more distant being excluded by the nearer” (Marghīnānī 1996–1997, vol. 3, p. 41; 2006, p. 496). As for the guardianship of marriage of an insane woman, Muhammad al-Shaybānī gave prece- dence to her father, while Abū Hanīfa and Abū Yūsuf gave it to her son because of his precedence among residuaries (Marghīnānī 1996–1997, vol. 3, p. 50; 2006, p. 500). Salāt-i Masʿūdī (Bāb 45 dar bayan-i namāz-i janāza) states, “If a person died and his father and son are alive, it is desirable that the father perform the funeral prayer” (Samarqandī 1897–1898, vol. 2, p. 189). Majmūʿ-i Khānī (Bāb 32 dar bayān-i namāz-i janāza) states, “If the religious head of the community (imām-i hayy) is not attendant, a guardian of the deceased (walī-yi mayyit) such as the father, son, brother, and uncle leads the funeral prayer” (Nāgawrī 1896–1897, vol. 1, p. 110). the Islamic legal discourses and historical tensions around who should lead the funeral prayer, see Halevi (20 ) Between the communal obligation and the filial duty Tianfang dianli’s explanation regarding the funeral prayer leader20 was undoubtedly based on – but subtly different from – an account from an authoritative Arabic book of Hanafī jurisprudence, Sharhal-Wiqāya (Bāb al-janā’iz), which explained as follows: The following are qualified for leadership of the funeral prayer (imāma): the ruler (sultān), then the judge (qādī), then the religious head of the community (imām al-hayy), and then a guardian of the deceased (walī), who is chosen from among the paternal male relatives of the deceased based on the legal order (ʿalā tartīb al-ʿasabāt). There is no problem with his [the guardian’s] entrusting the leadership of the funeral prayer to others. When any person other than them per- forms the funeral prayer, the guardian can reconduct it if he wants to do so. Subsequently, any person other than him cannot perform the prayer [again] (Sharīʿa 2009, vol. 1, p. 254). According to the Hanafī school, the term walī (guardian) primarily refers to the deceased person’s father or sons in the context of the funeral prayers.21 Sharhal-Wiqāya privileges the walī in that he can conduct the funeral prayer for his ward once again after others have already conducted it. Still, the Arabic book permits any Muslim throughout the world to lead and perform a funeral prayer for a deceased co-religionist, regardless of prior approval from the walī. This is in line with the Islamic legal principle collectively obliging all Muslims to conduct the funeral of a deceased co-religionist. However, Liu Zhi seems to object to this Islamic doctrine regarding the collective obligation in fun- erals and thus, by extension, the concept of the collectivity of all Muslims. In Tianfang dianli, he trans- lates the Arabic explanation cited above into Chinese as follows: The funeral prayer leader is sure to be the governor of the city [where the funeral prayer is per- formed]. If the governor does not attend the funeral prayer, it is sure to be [passed on to] the religious head of the community [where the funeral prayer is performed]. If the religious head of the community does not attend the funeral prayer, it is sure to be [passed on to] the chief of the family in mourning (zhuren 主人). 22Zhu Xi in Jiali 家禮(vol. 4, Zangli) states the following (the main texts are in bold): Between the communal obligation and the filial duty The chief has a liability to take charge of the funeral 19However, this type of mutual aid was expected of people from the same homeland or kinship group, but it was not imposed on the deceased person’s “co-religionists” in Confucian, Buddhist, or Taoist communities (Fuma 1997, pp. 651, 654-56, 709-39; Chen 2009). International Journal of Asian Studies 11 prayer by himself (you duren binli zhi ze 有独任殯礼之責). If any other person leads the funeral prayer without the chief’s agreement, either the latter can perform it again, or the sons of the deceased [other than the eldest son] can choose to reperform the funeral prayer. (Liu 2005, p. 181/vol. 20, Sangzangpian, f. 10b) Zhuren means “the chief of the family in mourning.” According to Zhu Xi’s 朱熹understanding, which was influential in Chinese society during the Qing period, the eldest son or the son’s eldest son (if the eldest son is not present) takes charge of the chiefship when the parent is being mourned.22 When anyone in a family dies, if the father is alive, he takes charge of the chiefship. When the mother dies, the chief for the mourning is her husband if he is alive and the eldest son if her husband has already died.23 Zhuren almost corresponds to walī. Sharhal-Wiqāya, the Arabic sourcebook that Liu Zhi con- sulted, requires any Muslim to perform a funeral prayer for his co-religionists in the first stage and privileges the eldest son to redo his parents’ funeral prayer as the walī, if the deceased’s father is not alive.24 However, Liu Zhi remarked that the eldest son alone is liable for organizing his father’s (or mother’s) funeral as the zhuren, if the deceased’s father is (or the deceased’s father-in- law and husband) not alive, thus requiring the eldest son to direct the funeral prayer. This requires him, rather than grants him a privilege, to redo the rites after others have done it without his permis- sion. Additionally, Liu Zhi suggested that the other sons can also start their parent’s funeral afresh in certain cases. Liu Zhi accepted other Muslims’ leading of the funeral prayer, but this is acceptable only under the supervision or approval of the eldest son. It is obvious here that Liu Zhi – circumventing the To establish the chief in mourning (sangzhu 喪主). [Note:] The chief (zhuren 主人) denotes the eldest son of the deceased person. 24al-Hidāya explains that the guardians (awliyā’) have a right (haqq) to redo their ward’s funeral (Marghīnānī 1996–1997, vol. 2, p. 144; 2006, p. 233). To establish the chief in mourning (sangzhu 喪主). [Note:] The chief (zhuren 主人) denotes the eldest son of the deceased person. If the eldest son has died, his eldest son will take charge of the important duty and present offer- ings [to the deceased]… [To establish] the female chief in mourning (zhufu 主婦). [Note:] That denotes the wife of the deceased or the wife of the chief if the former has already died. (Zhu 2010a, p. 902) Between the communal obligation and the filial duty If the eldest son has died, his eldest son will take charge of the important duty and present offer- ings [to the deceased]… [To establish] the female chief in mourning (zhufu 主婦). [Note:] That denotes the wife of the deceased or the wife of the chief if the former has already died. (Zhu 2010a, p. 902) 23Hui’an xiansheng Zhu Wengong wenji 晦庵先生朱文公文集(vol. 43) records Zhu Xi’s following remark: 23Hui’an xiansheng Zhu Wengong wenji 晦庵先生朱文公文集(vol. 43) records Zhu Xi’s following remar According to the mourning rite (sangli 喪禮) [i.e., “Rules on Hurrying to the Mourning Rites (bensang 奔喪),” a chapter in the Book of Rites (Liji 禮記)], “In all mourning [in a family], if the father is alive, he becomes the chief, and if the father is alive, his son is never in charge of it.” [That chapter] also says as follows: “If the father has already died, and his sons live together (xiongdi tongju 兄弟同居), each of them takes charge of the chiefship in mourning.” [Zheng Xuan’s 鄭玄] commentary on it says as follows: Each of them takes charge of the chiefship in the mourning for his wife and children. According to this, in all mourning of the wife, her husband becomes the chief. Currently, making the son the chief in the mourning [of the mother when her husband is alive] seems insuf- ficient. (Zhu 2010b, vol. 22, p. 1947) Zhu Xi in Jiali (vol. 4, Zangli) writes about the rite of the second day of mourning (xiaolian 小斂), as follo The chief and the femal chief in mourning incline forward and embrace the body [with the breast of the former attached to the heart of the latter], while crying and beating their breasts. [Note:] …In all cases, [the behavior] of the son for the father or mother is embracing the body; either [that of] the father and mother for the son, or the husband for the wife, is shaking the shroud [around the heart]; [that of] the daughter-in-law for the father-in-law and the mother-in-law is holding up the shroud [around the heart]; [that of] the father-in-law for the daughter-in- law is pushing [the heart]; [that of anyone] for the brothers is shaking the shroud [around the heart]…. (Zhu 2010a, p. 907; cf. Hu et al. 1988, p.580/ vol. 21, f. 27b) This passage suggests that when the mother died after her husband, the son embraces the body as the chief, and his wife holds up the shroud as the female chief. 24al-Hidāya explains that the guardians (awliyā’) have a right (haqq) to redo their ward’s funeral (Marghīnānī 1996–1997, vol. 2, p. 144; 2006, p. 233). Tatsuya Nakanishi 12 Islamic law – introduced the Confucian supreme principle of filial duty and attempted to present Islam in an understandable way for non-Muslim Chinese readers. Chinese people have traditionally believed that when a person dies, his/her spirit must be provided with ritual offerings in funerals,25 and then eternally through regular rites, by his/her descendants. In these rites, ancestral spirits cannot enjoy offerings from any person other than their descendants (Shiga 1967, pp. 34-37). Additionally, only the descendants can call their ancestors’ spiritual energies back for the rites, as these spiritual energies usually disperse.26 Thus, the family unit is collectively liable for ensuring an adequate funeral for its ancestral member. The eldest son, or his descendants, is liable for his father’s (or mother’s) funeral if the deceased’s father is (or the deceased’s father-in-law and husband) not alive. Other sons must perform it on behalf of the eldest son if he and his descendants have died.27 This “collective obligation” imposed on the family unit constitutes an important part of the Confucian filial duty. If the funeral is entrusted to someone other than family members, offerings and calls to the decea- sed’s spirit are ineffective. 25The chief in mourning (eldest son or grandson) must provide offerings for his parent’s spirit during the mourning period (three years), through various methods. For example, Liji 禮記(Tanggong shang) states, “The offering of the morning is at sunrise, and that of the evening is before sunset 朝奠日出，夕奠逮日.” Qingding Liji yishu 欽定禮記義疏introduces the following comment of Fang Que 方慤: “The offering of the morning symbolizes the breakfast, and that of the evening the dinner. The dutiful son serves his dead parent as he serves a live one” (Qinding Liji Yishu 1988, p. 345/vol. 11, f. 42b). 26Zhuzi yulei 朱子語類(vol. 3, Guishen) records the following statements of Zhu Xi: 27Wan Sida 萬斯大, a Confucian scholar of the Qing period, states the following in his Xueli zhiyi 學禮質疑: The second son can only become the chief in mourning for his parent when there is no other choice because the [deceased] eldest son has no son and the [deceased] eldest grandson or the [deceased] eldest great-grandson has no son. (Wan 1988, p. 468/vol. 2, Disun chengzhong yi, f. 43a) Descendants are composed of their ancestor’s vital energy. Even after his vital energy disperses, his root is there. If [his descendants] fully show their sincerity and veneration toward him, they can invite and condense his vital energy there. (Li 1994, vol. 1, p. 47) 28See Kuwabara (1977, pp. 19-30, 56-65). Daqing lüli 大清律例, the code of the Qing dynasty, stipulates various punish- ments for unfilial behaviors of the sons mourning their parent. Zhu Xi in Jiali (vol. 4, Zangli) writes about the rite of the second day of mourning (xiaolian 小斂), as follo This indicates an abandonment of the collective filial duty; such abandon- ment is meant to starve the parent’s spirit, thus constituting his/her sons’ sin of being unfilial toward him/her (Shiga 1967, pp. 112–13). Moreover, in pre-modern China, the consequences of neglecting filial duties were perceived not only as ethically lacking, and hence antisocial, but also as politically rebellious to the Chinese Emperor.28 Therefore, every family unit was a community that shared a com- mon destiny, in that the whole becomes sinful if nobody performs the ancestral funeral. y y p Thus, in the preceding passage, Liu Zhi suggests that if the funeral prayer of a dead Muslim is per- formed not by his eldest son but someone else, then the former must redo his father’s funeral prayer. Thus, the author states that the eldest son “can perform it again,” a remark that is consistent with his Arabic and Persian reference books, which state that the redo is his privilege. However, Liu Zhi impli- citly invalidates certain unqualified Muslims’ first performances and regards the eldest son’s redoing as the definitive fulfillment of the funeral duty. In other words, unlike his Arabic and Persian reference books, he virtually eliminates the value and necessity of the mutual aid of unrelated Muslims as regards the funeral prayer when sons of the deceased are not involved. Liu Zhi thus released his Muslim readers from the obligation of the mutual aid offered at funeral prayers by all Muslims. Instead, the Hui author transferred the “collective obligation” for the funeral prayer, which is accorded to all Muslims in the original source text, to the deceased individual’s entire family. Regarding the companions for whom his Muslim readers should perform the collective funeral obligation, Liu Zhi guided them to opt for their families rather than any other community of Muslims. Descendants are composed of their ancestor’s vital energy. Even after his vital energy disperses, his root is there. If [his descendants] fully show their sincerity and veneration toward him, they can invite and condense his vital energy there. (Li 1994, vol. 1, p. The second son can only become the chief in mourning for his parent when there is no other choice because the [deceased] eldest son has no son and the [deceased] eldest grandson or the [deceased] eldest great-grandson has no son. (Wan 1988, p. 468/vol. 2, Disun chengzhong yi, f. 43a) Conclusion According to Ben-Dor-Benite, the Hui scholars, including Ma Minglong, presented themselves as a group differentiated from the Chinese literati in a narrow sense while identifying with them in a broad sense through the “Dao of Muhammad.” Thus, they tried to reconcile themselves with non-Muslim Han people while being Muslim. Meanwhile, in the abovementioned Arabic and Persian inscription, Ma Minglong expressed a sense of belonging to the “Muslim inhabitants in China” including non-scholars, who were described as a unit for the collective obligation of Islamic education, which he contrasted with the other co-religionists outside China. Additionally, he situated himself as a prominent member of the “Muslim scholars of China,” who were leading members, or saviors, of the former unit that shared the same soteriological destiny. Thus, he tried to demonstrate his competence to Muslim rivals to survive. g y p In contrast, Liu Zhi weakened the sense of unity of his Hui readers with Muslim groups beyond that of the family, such as “all Muslims” or “Muslim inhabitants in China,” by suspending the former’s involvement with mutual aid within the latter regarding funeral prayers. In Tianfang dianli, he sug- gested the communal duty of the inhumation of co-religionists in harmony with the Chinese custom of mutual aid for the tentative burial of travelers, thus urging his readers to stand together with “all Muslims.” However, he encouraged his co-religionist readers to neglect the collectivity of the funeral prayer liability that his Hanafite references prescribed as another communal duty of all Muslims. He intended to loosen, if not sever, his Muslim readers’ bond with unrelated co-religionists to follow the Confucian prerequisite of filial duty. He categorized each Muslim family as a unit that shares funeral obligations, from which he excluded “other” Muslims without kinship to avoid the Chinese literati’s contempt and hostility toward Islam and its adherents. Liu Zhi, in his discussion of filial duty, prior- itized his Muslim readers’ affiliation with families over larger groups, such as “all Muslims,” the “Muslim inhabitants in China,” and the “Muslim scholars of China.” These cases show that the Hui scholars assumed dynamic identities to distinguish themselves from Muslim “others” and shifted these identities depending on with whom they negotiated their legitim- acy. The following two points are particularly remarkable in comparison with the paradigmatic argu- ment of Ben-Dor Benite (2005) on Hui identity. 29We need to reconsider Frankel’s (2005, p. 152) argument based on Liu Zhi’s statement of marriage and funerary rites that “Chinese Muslims who adopted certain Chinese customs and shared many Chinese mores, while maintaining the core praxis of Islam, have effected their special Chinese-Islamic simultaneity.” The concept of “maintaining the core praxis of Islam” is problematic. Liu Zhi at least thought that abandoning the normative Muslim unity for the collective obligation of funerals is not contradictory with keeping the Chinese–Islamic simultaneity. Zhu Xi in Jiali (vol. 4, Zangli) writes about the rite of the second day of mourning (xiaolian 小斂), as follo 47) an Sida 萬斯大, a Confucian scholar of the Qing period, states the following in his Xueli zhiyi 學禮質疑: The second son can only become the chief in mourning for his parent when there is no other choice because the [deceased] eldest son has no son and the [deceased] eldest grandson or the [deceased] eldest great-grandson has no son. (Wan 1988, p. 468/vol. 2, Disun chengzhong yi, f. 43a) 28See Kuwabara (1977, pp. 19-30, 56-65). Daqing lüli 大清律例, the code of the Qing dynasty, stipulates various punish- ments for unfilial behaviors of the sons mourning their parent. International Journal of Asian Studies 13 In this regard, he invalidated their membership in any community beyond the family. He denied the joint liability of “Muslim inhabitants in China” for the funeral prayer, unlike Ma Minglong, who conceived of the same community’s collective liability for education of its members. In other words, Liu Zhi dissolved the imaginal framework of “Muslim inhabitants in China.” Conclusion First, the Hui scholars’ self-identification as being Muslim was not always coexistent (or compat- ible) with their understanding of themselves as being simultaneously among Chinese literati and Muslims. The Arabic and Persian inscription that Ma Minglong wrote, while they express his sense of belonging to the “Muslim inhabitants in China,” hardly manifest an interest in presenting the author as one with cultural Chinese-ness or being part of the Chinese literati (cf. ibid., pp. 100–6). Liu Zhi’s Chinese work also sacrificed the solidarity of the "Muslim inhabitants in China" for securing “the overlapping space of Islam and China."29 Second, Hui scholars flexibly changed the contours of “us” when situating themselves vis-à-vis “other” Muslims through their negotiations with Muslim rivals and non-Muslims in China. Ma Minglong pre- tended to be part of the Muslim faction of the Chinese literati in front of non-Muslim Han people and behaved as a leading member of the Chinese unit of Muslims against rival co-religionists. Liu Zhi expanded the idea of “we” who share collective obligations to contain “all Muslims” in one case and restricted it to “Muslim family” in other cases through negotiation with the non-Muslim Han people. 14 Tatsuya Nakanishi Further studies on the flexibility of the Hui’s identities will provide deeper insights into their strat- egies for competition and reconciliation with “others.” Acknowledgement. This work was supported by the Kyoto University Foundation and JSPS KAKENHI (Grant numbers 21401026, 17K03130, 20H05825, and 21K00906). Acknowledgement. This work was supported by the Kyoto University Foundation and JSPS KAKENHI (Grant numbers 21401026, 17K03130, 20H05825, and 21K00906). References Baydāwī Nāsir al-Dīn Abū al-Khayr ʿAbdullāh b. ʿUmar b. 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Tatsuya Nakanishi 16 Cite this article: Nakanishi T (2024). A flexible choice of comrades: the dynamic identity of the Muslim Huis of the seven- teenth and eighteenth centuries. International Journal of Asian Studies 1–16. https://doi.org/10.1017/S1479591422000523 Appendix A (1)–(46) represents the line numbers. [] means a conjectural restoration of a worn-out part of the inscription. Proper nouns d li d (1)–(46) represents the line numbers. [] means a conjectural restoration of a worn-out part of the inscription. Proper nouns d l d (1)–(46) represents the line numbers. [] means a conjectural restoration of a worn-out part of the inscription. Proper nouns are underlined. are underlined. (1)ﺍﻟﺤﻤﺪ ﺍﻟﻠﺬﻯﺧﻠﻖﺍﻻﻧﺴﺎﻥﻣﻦﺗﺮﺍﺏﺛﻢﻣﻦﻧﻄﻔﺔﺛﻢﺟﻌﻠﻬﻢﺍﺯﻭﺍﺟﺎﻭﻗﺪﺭﺑﻴﻨﻬﻢﺧﻔﺎﻳﺎﺍﻟﺤﻜﻤﺔﻭﺧﺒﺎﻳﺎﺍﻟﻘﺪﺭﺓﻳﻨﺒﺮﻫﻢ (2)ﺟﻤﻬﺮﺓﺍﻓﻮﺍﺟﺎﺛﻢﺍﻋﺎﺩﻫﻢﻓﻰﺍﻟﺘﺮﺍﺏﺑﻘﺒﻀﺔ]ﻗﻀﺎﺋﻬﻢ[ ﺍﺣﺮﺍﺟﺎﻭﺍﺧﺮﺟﻬﻢ]ﻣﻦ[ ﺍﻟﺘﺮﺍﺏﺑﻌﺪﺍﻟﻤﻮﺕﺍﺣﺪﺍﺛﺎﻓﺴﺄﻝﻣﻦﺍﺷﻴﺎْ ﺍﺍﻟﺨﻠﻖﻣﺎﻻﻋﻠﺔ (3)ﻟﻬﺎﻭﻫﻮﺍﻟﻠﻄﻴﻒﺍﻟﺨﻴﺮﺍﻟﻠﺬﻯﺧﻠﻖﺍﻟﻤﻮﺕﻭﺍﻟﺤﻴﻮﺓﻟﻴﺒﻠﻮﻫﻢﺍﻧﻬﻢﻻﺣﺴﻦﻋﻤﻼﻭﻫﻮﺍﻟﻐﻨﻰﺍﻟﻜﺒﻴﺮﻳﺎﻟﻄﻴﻔﺎﻛﻦﻏﻔﺮﺍﻧﻚﺭﺑﻨﺎﻭﺍﻟﻴﻚﺍﻟﻤﺼﻴﺮ (4)ﺍﻧﻚﺍﻧﺖﺍﻟﺮﺣﻴﻢﺍﻟﻐﻔﻮﺭﻭﻧﺼﻠﻰﻋﻠﻰﻧﺒﻴﻪﻣﺤﻤﺪﺧُﻠﻖﻓﻰﺧﻠﻖﻋﻈﻴﻢﻣﻦﺗﺮﺍﺑﻪﻭﻋﻠﻰﺁﻟﻪﻭﺻﺤﺒﻪﻛﻠﻬﻢﻣﺘﻮﺍﺭﺛﻮﻥﻓﻰﺭﺍﻳﻪﻭﻋﻠﻰ (5)ﺍﻻﻧﺒﻴﺎﺀﻭﺍﻻﻭﻟﻴﺎﺀﻗﺒﻠﻪﻭﺍﻟﺨﻠﻔﺎﺀﻭﺍﻳﻤﺔﺑﻌﺪﻩﺻﻠﻮﺍﺕ ﻋﻠﻴﻪﻭﻋﻠﻴﻬﻢﺟﻤﻌﺎﻣﺎﺩﺍﻣﺖﻟﻬﻢﻟﺴﺎﻥﺍﻟﺜﻨﺎﻫﻤﭽﻨﺎﻧﻜﻪ (6)ﺁﺧﺮﻣﻨﺰﻝﺍﺯﻣﻨﺎﺯﻝﺩﻧﻴﺎﻫﻢ]ﻏﺎﻟﻰﺳﻬﺐ[ ﻣﺠﻨﺰﺍﺳﺖﻛﻪﻧﻴﺰﺍﻭﻝﻣﻨﺰﻝﺍﺯﻣﻨﺎﺯﻝﺁﺧﺮﺓﻫﻤﻮﺍﺭﺧﺎﻛﻰﮔﻮﺭﺍﺳﺖﻭﺑﻌﺪﻓﺨﻴﺮﺍﻟﻘﺒﻮﺭﺯﻳﺎﺭﺓً (7)ﻫﻰﻫﺬﻩﺍﻟﺘﺮﺑﺔﻓﻰﻫﺬﻩ]ﺍﻟـﺘﺮﺍﺑﺎﺕ[ ﻻﻧﻬﺎﺗﺮﺑﺔﻣﻦﻫﻮﺧﻴﺮﺍﻵﺑﺎﺀﺑﻞﺍﺳﺘﺎﺫﻋﻠﻤﺎﺀﺍﻟﺼﻴﻦﺍﻋﻨﻰﺣﻀﺮﺓﺣﺆﺗﻰﺑﺎﺑﺎﺷﻤﺲﺍﻟﺪﻳﻦﺭﺣﻤﺔ ﻋﻠﻴﻪ (8)ﺍﻟﻰﻳﻮﻡﺍﻟﺪﻳﻦﻛﺎﻧﻪﻣﻮﺻﻞﺍﻟﺪﻳﻦﻓﻰﺍﻟﺼﻴﻦﻣﺎﻗﺎﻝﻧﺒﻴﻨﺎﺍﻟﻤﺒﻴﻦﻓﻰﻗﻮﻟﻪﺍﻟﻤﻨﻴﺮﺍﻃﻠﺐﺍﻟﻌﻠﻢﻭﻟﻮﺑﺎﻟﺼﻴﻦﻭﻟﻘﺪﻛﺎﺩﺍﻫﻞﺍﻟﺼﻴﻦﻛﻠﻬﻢﺍﻥﻳﻜﻮﻧﻮﺍ (9)ﺿﺎﻟﻴﻦﻭﻣﻀﻠﻴﻦﻭﺑﺎﻟﻼﺩﻳﻦﻣﺘﺪﻳﻨﻴﻦﻭﻛﺎﻥﺍﻛﺜﺮﻫﻢ]ﻣﻐﻤﻮﺿﻴﻦ[ ﻭﻓﻰﺍﻟﺤﻖﻏﻴﺮﻣﺤﺼﺤﺼﻴﻦﻭﺑﺎﻗﺎﻣﺘﻪﺑﺘﻠﻚﺍﻻﺣﻮﺍﻝﻗﺎﻡﻋﻤﺎﺩﻫﺬﺍﺍﻟﺪﻳﻦﻭ (10)ﺑﺎﺭﺷﺎﺩﻩﻓﻰﺫﻟﻚﺍﻟﺰﻣﺎﻥﺻﻠﺢﻣﻨﻬﻢﺍﻣﻮﺭﺍﻻﻭﻟﻴﻦﻭﺍﻵﺧﺮﻳﻦﻓﻤﺜﻠﻪﻛﻤﺜﻞﺫﻯﺍﻟﻤﻌﺎﺩﻥﻓﻰﺍﺳﺮﺍﺭﺍﻟﻌﻠﻮﻡﻳﻈﻬﺮﻋﻠﻰﺍﻟﻤﺴﺘﺤﻘﻴﻦﺑﺎﻧﻮﺍﺭﻋﻔﺎ (11)ﺍﻟﻨﻔﻴﺴﺔﻣﻦﺍﻟﺠﻮﺍﻫﺮﺭﺧﺼﺎﻭﺍﻟﺸﺪﻳﺪﻭﻥﻛﻠﻬﻢﻛﻤﻌﺪﻥﺫﺧﺮﺍﻓﻰﺍﻓﺎﺩﺗﻪﺍﻟﻨﻮﺍﺩﺭﺑﻞﻛﺎﺷﺠﺎﺭﺍﻟﻔﺎﻛﻬﺔﻓﻰﺍﻟﻤﺴﺘﻔﺎﺩﺓ]ﻳﻨﻔﻖﺿﺪﺍ[ ﻣﻦﺍﻟﻤﻨﻔﻌﺔ (12)ﻟﺰﻛﻮﺓﻓﻮﺻﻞﺍﻟﻰﻣﺸﺎﻡﺍﻻﻧﺎﻡﻧﺸﺮﺍﻟﻔﻮﺣﺎﺕ]ﻣﻦ[ ﻭﺭﺩﻫﺎﺍﻟﺼﻔﻴﺔﺍﻟﻰﺍﻥﺛﻤﺮﻫﺎﻳﻨﺼﺮﻩﺍﻟﺴﻼﻡﻛﻞﺯﻣﺎﻥﻋﻠﻰﺍﻟﻤﺤﺼﻠﻴﻦﻣﻦﺍﻟﻔﻀﻼﺀ (13)ﻭﻛﻞﺑﻌﺪﻩﻓﻰﺑﻴﻨﻬﻢﺍﻻﺳﻼﻡﻛﻞﺍﻭﺍﻥﻛﺎﺧﻼﻑﺍﻟﻨﺒﻰﻣﻦﺍﻟﺨﻠﻔﺎﺀﻭﻛﺎﻥﻳﻌﻠّﻢﺍﻟﻨﺎﺱﺑﺎﻧﻮﺍﻉﺍﻟﻌﻠﻢﻓﻰﺟﻤﻴﻊﺍﻻﻋﻮﺍﻡﻭﻫﻮﻣﺮﺍﺩﻫﻢ (14) ]ﻣﻦ[ ﺍﻟﻤﺴﺎﺀﻭﺍﻟﺼﺒﺎﺡﻛﺎﻧﻪﺷﻤﺲﻓﻀﻞﺗﺸﺮﻕﺑﺎﺷﺮﺍﻗﻬﺎﺍﻟﻤﻔﻴﺪﺓﻣﻦﺍﻭﺝﺍﻟﺴﻤﺎﺀﺗﺤﺖﺍﻟﺴﻤﺎﺀﺍﻟﺴﺎﺑﻊﻓﻌﻠﻢﺍﻵﻥﻓﻰﺍﻟﺼﻴﻦﻟﻪﻛﺎﺟﺰﺍﺀ (15)ﻟﻴﺲﻣﺨﺰﻯﻣﻦﻧﺠﺎﺭﺍﻟﻜﻞﻣﻦﺍﺻﻠﻪﻣﻦﺍﻭﺭﺛﻪﺍﻟﻌﻠﻤﺎﺀﻣﻦﺯﻣﺎﻧﻪﺍﻟﻰﺍﻟﺰﻣﺎﻧﻨﺎﻫﺬﺍﻛﻞﻣﻦﺍﻟﻮﺍﺭﺛﻴﻦﻣﻦﻓﻀﻠﻪﻓﻠﻤﺎﺗﺎﺧﺮﺍﻟﻰﻣﺮﻭﺭﻫﻢﻓﻰﻫﺬﺍ (16)ﺍﻟﺰﻣﺎﻥﻋﻠﻰ]ﺩﻫﻢﺍﻟﻤﻌﻠﻢ[ ﺍﻥﻳﺸﻜﺮﻭﺍﻣﻦﻛﺎﻥﻳﺄﺳﺲﺍﻟﻤﺒﺎﻧﻰﻓﻰﺍﻟﻌﻠﻢﻭﻟﻴﺨﻠﺺﺍﻟﻤﺘﻌﻬﺪﻭﻥﻋﻨﺪﺍﻻﻭﺍﻥﻋﻠﻰﺍﻟﻌﻠﻢ]ﻷﻥ[ ﻳﺪﻋﻮﺍﻟﻤﻦﻛﺎﻥﻳﻌﻠﻢ]ﺍﻟﻤﺴﻠﻤﻮﻥ[ ﻟﻪ (17)ﺑﺎﻟﻌﻠﻢﻓﺎﻥ ﺗﻌﺎﻟﻰﻭﺻﻒﺍﻟﻌﻠﻤﺎﺀﻓﻰﻗﻮﻟﻪﺑﺎﻋﻠﻰﺍﻟﺪﺭﺟﺎﺕﻭﺍﻟﺬﻳﻦﺍﻭﺗﻮﺍﺍﻟﻌﻠﻢﺩﺭﺟﺎﺕﻓﺎﻭﺟﺐﺍﻟﺸﻜﺮﻋﻠﻴﻨﺎﺍﻥﻧﺴﻴﺮﻭﺭﺩﻗﺮﺍﺀﺓ (18)ﺍﻟﺘﻀﺮﻉﺍﻟﻴﻪﺁﻧﺎﺀﺍﻟﻠﻴﻞﻭﺍﻃﺮﺍﻑﺍﻟﻨﻬﺎﺭﻧﺴﺘﺨﺪﻣﻪﺑﺎﻟﺪﻣﻮﻉﻋﻠﻴﻪﺧﺪﻣﺔﺍﻻﻧﻮﺗﺮﻋﻨﺪﺗﺴﺤﺮﻻﺑﻜﺎﺭﻧﺴﺄﻟﻚﺍﻟﻠﻬﻢ]ﻳﺎﺭﺏ[ (19)ﺍﻧﺖﺍﻟﻐﻔﻮﺭﺍﻟﺮﺣﻴﻢﺍﻥﺗﺮﺣﻢﻋﺒﺪﻙﺑﻐﻔﺮﺍﻧﻚﺍﻟﻌﻈﻴﻢ]ﻭﺍﻥ[ ﺗﺠﻌﻞﺩﻭﺭﺍﻥﺍﻟﻘﻤﺮﻳﻦﻭﺍﻻﺷﺮﺍﻕﻭﺍﻟﺘﺮﻃﺐﻓﻰﺍﻟﻤﻠﻮﻳﻦﻭﻣﻮﻗﺖﺍﻟﻜﻮﺍﻛﺐﻓﻰﺍﻟﻈﻬﻮﺭ (20)ﻭﺍﻓﻮﻟﻬﺎﺑﻌﺪﺍﻟﻈﻬﻮﺭﻭﺗﺴﺒﻴﺢﺍﻟﻤﻼﺋﻜﺔﻣﻦﺍﻟﺴﻤﺎﺀﺍﻟﻰﺍﻻﺭﺽﻭﺍﺻﻮﺍﺕﺍﻟﺮﻋﺪ]ﻋﻠﻰ[ ﻣﻼﺀﺍﻻﺭﺽﻭﺗﺤﻤﻴﺪﺍﻟﺪﺍﺑﺔﻓﻰﺍﻟﻤﻮﺍﺿﻊﺍﻟﺨﻔﻴﺎﺕﻣﻦﺍﻻﺭﺽ (21)ﻭﻣﻮﺕﺍﻟﻨﺒﺎﺗﺎﺕﺍﻟﻠﺘﻰﻧﺸﺄﺕﻣﻦﺍﻻﺭﺽﻭﺍﺭﺳﺎﻝﺍﻟﺮﻳﺎﺡﻣﻊﺍﻻﻣﻄﺎﺭﻭﺩﻭﻳﻬﺎﻣﻊﺍﻭﺭﺍﻕﺍﻻﺷﺠﺎﺭﻛﻠﻬﺎﺷﻴﺌـﴼﻟﻪﺍﻟﻰﺭﺣﻤﺘﻚﺍﻟﺒﺎﻗﻴﺔ (22)ﻭﻣﻄﻤﻌﴼﻟﻪﺍﻟﻰﻟﻄﻔﻚﺍﻟﺪﺍﺋﻤﺔﺛﻢﺍﺧﻄﻬﺎﺛﺎﺑﺘﺔﻟﻠﺰﺍﻳﺮﻳﻦﻭﺍﻧﺖﺍﺭﺣﻢﺍﻟﺮﺍﺣﻤﻴﻦ. ﺍﻣﺎﺁﻥﻛﺎﻣﻠﻰﺍﺯﻣﺮﺑﻰﺍﻟﻔﻀﻼﺀﻭﺁﻥﻣﻜﻤﻠﻰﺍﺯ (23)ﻣﻌﻠﻢﺍﻟﻌﻠﻤﺎﺀﺍﻧﺰﻟﻪ ﻣﻨﺰﻝﺭﺿﻮﺍﻧﻪﻭﺍﻭﻃﻨﻪﻳﻄﻤﺄﻥﺟﻨﺎﻧﻪﺑﺮﻗﻀﻴﻪﻛﻞﻣﻦﻋﻠﻴﻬﺎﻓﺎﻥﺍﺯﻳﻦﺟﺎﻯﻓﺎﻧﻰﺍﺭﺗﺤﺎﻝﻛﺮﺩﻩﺍﺳﺖﻭ (24) ]ﺍﺯ[ ﺭﻭﻯﺁﻭﺭﺩﻥﺑﺪﺍﻥﺩﺍﺭ ﺻﺪﻭﺍﻓﺰﻭﻧﻰﺳﺎﻝﮔﺬﺷﺘﻪﺍﺳﺖﻭﻟﻜﻦﺁﺛﺎﺭﻩﻓﻰﺍﻓﻮﺍﻩﺍﻫﻞﺍﻟﺪﻳﻦﻻﻳﻨﺴﻮﻧﻬﺎﺫﻛﺮﻫﺎﻭﻣﺂﺛﺮﻩﻓﻰﻗﻠﻮﺏﺍﺑﻨﺎﺀ (25)ﺍﻟﺼﻴﻦﻻﻳﻐﻔﻠﻮﻧﻬﺎﻓﻜﺮﻫﺎﻧﺎﮔﺎﻩﮔﺮﻭﻫﻰﺍﺯﺗﺠﺎﺭﺑﺸﻬﺮﺍﻳﻦﻛﻤﻴﻨﻪﺣﻘﻴﺮﻧﻘﻴﺮﻛﻤﺘﺮﺍﺯﻗﻄﻤﻴﺮﺭﺳﻴﺪﻧﺪﻛﻪﺍﻳﺸﺎﻥﺍﺯﺧﺼﺎﻥﻗﺪﻳﻤﻰﻭﺧﻠﺼﺎﻥ (26)ﺣﺪﻳﺜﻰﻣﻰ]ﺑﻮﺩﻩ[ ﺍﻧﺪﺑﻌﺪﺍﺯﻣﻼﻗﺎﺕﺁﻥﻗﺼﻪﺷﺄﻥﺭﺍﺑﺎﺯﮔﻔﺘﻨﺪﻛﻪﺷﻬﺮﻫﺎﺀﻣﺎﺍﺯﻛﻨﺠﺎﻧﻔﻮﻯﻫﺮﺳﺎﻝﺍﺯ]ﻣﺤﺼﺒﺔ[ ﺑﻮﺩﻧﺪﺧﺎﺻﻪﺍﻣﺴﺎﻝﺑﻘﺪﺭﺓﺧﺪﺍﻯﺗﻊ (27)ﭼﻬﻞﻭﺍﻓﺰﻭﻧﻰﺷﺒﺎﺭﻭﺯﺑﺎﺭﺍﻥﺩﻳﻤﻪﺑﺒﺎﺭﻳﺪﻭﺁﺯﺍﺭﺳﻴﻠﺶﺭﺍﺩﺭﺯﻣﻴﻨﻬﺎﺀﺑﺨﻮﺍﺭﺍﻧﻴﺪﻫﺮﺁﻧﺠﺎﻛﻪﭘﺴﺖﺑﻮﺩﺭﻭﺩﺷﺪﻩﺍﺳﺖﻭﻫﺮﺁﻥﺟﺎﻯﻛﻪﺑﻠﻨﺪﺑﻮﺩﺍﺯﺍﻥ (28)ﺁﺏﺧﻮﺍﺭﺍﻧﻴﺪﻥﺍﺯﻫﺮﺟﺎﺑﺸﻜﺎﻓﺘﻪﺍﺳﺖﻫﺮﺟﺎﻯﻛﻪﻗﻀﺎﻭﻗﺪﺭﺣﻖﺗﻌﺎﻟﻰﺑﺪﻭﺑﺮﺳﺪﺑﻬﻤﺎﻥﺣﺎﻟﺶﺩﻳﺮﻳﻨﻪﺑﻤﺎﻧﺪﻭﺑﻴﺸﺘﺮﻣﺰﺍﺭﺍﺯﺁﻥﺁﺯﻣﺎﻳﺶﺳﻤﺎﻭﻯ (29)ﻭﻳﺮﺍﻥﮔﺸﺘﻪﺍﻧﺪﻭﺍﺳﺨﻮﺍﻧﻬﺎﺀﻣﺪﻓﻮﻥﻫﻤﻪﭘﻴﺪﺍﺷﺪﻩﺍﻧﺪﺗﺎﺁﻥﻛﺎﻣﻞﻣﺬﻛﻮﺭﻫﻢﺑﺮﻳﻦﺟﮕﺮﻟﺸﺘﻪﻭﻧﺰﺩﻳﻜﺴﺖﻛﻪﻫﻤﻮﻭﺍﻗﻊﺷﺪﻩﭘﺲﻋﺎﻟﻤﺎﻥﻭﺩﻳﻨﺪﺍﺭﺍﻥ (30)ﻛﻪﺩﺭﺍﻥﺷﻬﺮﺑﺎﺷﺪﺩﻝﺳﻮﺯﺍﻥﺷﻮﻧﺪﻭﺁﻥﻋﺰﻳﺰﺍﻥﻛﻪﺍﻳﺸﺎﻥﻏﺮﺓﻭﺟﻮﻩﺍﻟﻴﻮﻡﻭ]ﺯﺑﺪﺓ[ ﻭﺟﻴﻪﺍﻟﻴﻮﻡﺧﻮﺍﻧﻨﺪﺟﻤﻊﺷﻮﻧﺪﻭﻋﺎﻟﻤﻨﺎﺀﺑﻴﺸﺘﺮﮔﺮﺩﻛﺮﺩﻧﺪﻭﮔﻮﺭﺳﺘﺎﻥﺍﺯﺁﻥ (31)ﺟﺎﻯﻛﻪﻣﻮﺍﻓﻘﺖﺑﻮﺩﺧﺮﻳﺪﻧﺪﺗﺎﺟﺴﻢﻓﺎﻧﻰﺁﻥﻋﺎﻟﻢﺭﺑﺎﻧﻰﺑﺎﺍﻫﻞﻭﺍﻭﻻﺩﺍﻭﻛﻪﺍﺯﺍﺷﺮﺍﻓﻨﺪﺍﺯﺁﻥﮔﻮﺭﻫﺎﻧﺰﺩﻭﻳﺮﺍﻧﻰﺑﻜﺎﻭﻧﺪﻫﻤﻪﺭﺍﺑﺪﺍﻥﮔﻮﺭﺳﺘﺎﻥﻧﻮﺧﺮﻳﺪﻯ (32)ﺩﻓﻦﻛﺮﺩﻧﺪﻭ]ﺑﻌﺪ[ ﺫﻟﻚﻓﺎﻟﻘﻠﻮﺏﺑﻬﺎﺧﺎﺷﻌﺔﻭﺍﻻﺑﺼﺎﺭﻟﻬﺎ]ﺣﺎﺿﺮﺓ[ ﺛﻢﺍﻫﻤﺘﻬﻢﻗﻠﻮﺑﻬﻢﺑﺎﻥﻳﻨﺼﻮﺍﻣﺎﻓﻰﺻﺪﻭﺭﻫﻢﻻﺟﺮﻡﻣﺮﻳﻦﺁﻳﻨﺪﻫﮕﺎﻥﺭﺍ (33)ﺗﺄﻛﻴﺪﮔﻔﺘﻨﺪﻭﻟﻜﻦﺭﻗﻌﻪء ﻧﻤﻰﻓﺮﺳﺘﻨﺪﺗﺎﺍﻳﻦﺿﻌﻴﻒﻣﺮﺛﻴﺔﺭﺍﺗﺼﻨﻴﻒﻛﺮﺩﻡﻭﺑﻪ]ﭘﻴﺶﻧﻤﺎﺯ[ ﻋﺮﺿﻪﻛﻨﻢﺁﻧﮕﺎﻩﺑﻪﻣﺸﺮﻗﻴﻪﺁﻥﺣﻀﺮﺕﺑﺮﭘﺎﻯﻣﻰﻛﺮﺩﻧﺪ (34)ﻣﻨﺎﻗﺒﻬﺎﺀﺁﻥﻋﺰﻳﺰﻳﮕﺎﻧﻪﺍﻋﻼﻡﻣﻴﻜﺮﺩﻧﺪﻭﻗﺼﻬﺎﺀﺍﻭﺭﺍﻋﻠﻰﻟﺤﺪﻩﺣﻜﺎﻳﺖﻣﻰﻛﻨﻨﺪﻭﺍﻓﻌﺎﻝﺧﻴﺮﺍﻳﺸﺎﻥﻧﻴﺰﺑﻬﺮﺟﺎﻯﺑﺪﺍﻧﻨﺪﭼﻮﻥﺍﻳﻦﻗﺼﻪﺭﺍ (35)ﺷﻨﻴﺪﻡﺍﻣﺘﺜﺎﻝﻻﺯﻡﺷﻤﺮﺩﻡﻭﻟﻮﻟﻢ]ﺍﺭﻭﻳﻬﻢ[ ﻛﺘﺎﺑﺎﻻﺳﻤﻊﻟﻬﻢﺧﻄﺎﺑﺎﻓﻜﺘﺒﺘﻬﺎﻋﺮﺑﻴﺔﻭﻭﺻﻠﺘﻬﺎﺍﻟﻴﻬﺎﻋﺠﻤﻴﺔﻭﻋﺮﺿﺘﻬﺎﻋﻠﻰﻗﺒﻞﻗﺒﺘﻪ (36)ﻣﺮﺛﻴﺔﻭﺑﻴّﻨﺘﻬﺎﻟﺰﻳﺎﺭﺓﻗﺒﺮﻩﺩﻣﻌﻴﺔﻧﺸﺮﴽﻟﻔﻀﻞﻋﻠﻤﻪﻻﻟﺴﻼﻑﻭﻓﺴﺮﴽﻟﻔﻀﻞﻣﻨﺎﻗﺒﻪﻋﻨﺪﺍﻻﺧﻼﻑﻭﺍﻟﺤﻤﺪ ﺍﻟﺬﻯﺟﻌﻞﺍﻟﻌﻠﻢﻗﺮﺑﺔً (37)ﻻﻫﻠﻪﺍﻟﻰﺩﺭﺟﺔﺍﻻﻧﺒﻴﺎﺀﻭﺧﺼﺺﺍﻟﻤﺜﻨﻰﻋﻠﻴﻪ]ﺑﺎﺳﻢ[ ﺍﻟﻤﻮﺻﻞﻣﻦﻛﻞﺍﻟﻌﻠﻤﺎﺀﻓﺎﻏﻔﺮﺍﻟﻠﻬﻢﻟﻤﻤﺪﻭﺣﻨﺎﺑﺎﻟﻔﻀﻞﺍﻟﺘﺎﻡﻭﺍﻟﻤﻐﻔﺮﺓﺍﻟﻌﺎﻡﻣﻊﺍﺳﻼﻓﻪ (38)ﺍﻟﻜﺮﺍﻡﻭﺍﺧﻼﻓﻪﺍﻟﻜﺮﺍﻡﻭﺍﺟﻌﻞﻟﻪﻟﺴﺎﻥﺻﺪﻕﻓﻰ]ﺍﻟﻨﺎﻗﺒﺔ[ ﻭﺍﺧﺘﻢﻟﻪﺑﺨﻴﺮﻓﻰﺣﺴﻦﺍﻟﻌﺎﻗﺒﺔﺣﺘﻰﻭﺍﻇﺐﺫﻛﺮﻩﺑﺎﻟﺴﺎﻥﺍﻫﻞﺍﻟﺪﻳﻦﻣﻦﻗﺮﻥ (39)ﺍﻟﻰﻗﺮﻥﺍﻟﻰﻳﻮﻡ]ﻟﻠﻌﺪ[ ﺑﻔﻀﻞ ﺍﻟﻮﺍﺣﺪﺍﻟﻘﻬﺎﺭﺍﻟﻤﺘﻨﺰﻩﻭﺍﻟﺨﺎﻟﻖﻧﺤﻮﻣﺤﻤﺪﺍﻟﻤﻮﺣﺪﺍﻟﻤﺨﺘﺎﺭﻭﺁﻟﻪﻭﻏﻴﺮﻩﺍﻻﻣﺠﺎﺩ (40)ﺍﻳﻦﻋﺒﺎﺭﺍﺕﻣﺮﺛﻴﺔﻛﻪﻓﻘﻴﺮﺍﻟﺤﻘﻴﺮﻧﻮﻳﺸﺘﻪﺷﺪﻩﺍﺳﺖﻭﻫﻮﺍﻟﺪﺍﻋﻰ]ﺍﻟﻨﻜﺪﻯ[ ﻣﺤﻤﺪﺍﻟﻤﻬﺪﻯﺍﻻﻣﺎﻡﻋﻄﺎﺀ ﻋﻠﻴﻬﻤﺎﺭﺣﻤﺔ ﺻﻐﻴﺮ (41)ﺑﻴﻦﺍﻻﻧﺎﻡﺣﻘﻴﺮﺗﺤﺖﺍﻻﻗﺪﺍﻡﺩﺭﺗﺎﺭﻳﺦﻫﺠﺮﻯﺍﺯﻣﺤﻤﺪﻣﺼﻄﻔﻮﻯﺻﻠﻰ ﻋﻠﻴﻪﻭﺳﻠﻢﻫﺰﺍﺭﻭﻫﻔﺘﺎﺩﻭﻧﻪﺳﺎﻝﺑﻮﺩﺳﻨﻪ ٠٠٠١ ٠٧ ٩ (42)ﻭﻗﻊﺍﻟﻔﺮﺍﻍﻣﻦﻛﺘﺎﺑﺔﻫﺬﻩﺍﻟﻤﺮﺛﻴﺎﺕﻭﺍﻟﻤﺪﺣﺎﺕﺍﻟﻤﺴﻄﻮﺭﺍﺕﺍﻟﻤﺮﺿﻴﺎﺕﻋﻠﻰﻳﺪﺍﻟﻌﺒﺪﺍﻟﻀﻌﻴﻒﺍﻟﺬﻯﻫﻮﺍﺑﻦﻣﺼﻨﻔﻬﺎﻭ]ﻧﺎﺳﺦﺭﻗﻴﻢ[ ﻣﺼﻨﻔﻬﺎ (43)ﺍﺣﻤﺪﺍﻟﻨﻮﺭﻯﺍﻟﻤﺘﻌﻠﻢﺑﺘﻌﻠﻴﻤﺎﺗﻪﻭﺍﻟﻤﺘﺄﺩﺏﺑﺂﺩﺍﺏﺗﺄﺩﻳﺒﺎﺗﻪﺍﻣﺎﺍﻟﻤﺼﻨﻒﻓﻬﻮﻓﻰﺣﻖﺍﻟﻤﻤﺪﻭﺡ (44)ﻣﻦﺍﻟﻜﻨﺠﺎﻧﻔﻮﻯﺍﻟﻤﻴﻤﻮﻧﺔﻭﺳﻤﻰﺑﻄﺎﻟﺐﺑﻦﻋﺒﺪﺍﻟﺮﺣﻴﻢ-ﻣﻦﺑﻴﻦﺍﺳﺎﻣﻰﺍﻟﻤﺮﻏﻮﺳﺔﻭﺍﻟﻬﻤﺎﻳﻮﻧﺔﻏﻔﺮ– ﻟﻪ]ﻏﻔﺮﺍﻧﺎ[ ﺗﺎﻣّﺔﻭﻣﻐﻔﺮﺓﻋﺎﻣﺔﻣﻊﺟﻤﻴﻊ (45)ﺍﻻﺳﻼﻑﻭﺍﻻﺧﻼﻑ]ﻛﻤﺎ[ ﻏﻔﺮﻟﻠﻤﻤﺪﻭﺡﻋﻠﻰﻣﺜﻮﺑﺎﺗﻪﻓﻰﺍﻻﺛﺎﺑﺔﺣﻴﺚﻻﻳﻌﻠﻢﺍﻻﺍﻟﻤﻌﻠﻤﻴﻦﻣﻦﺍﻻﺳﻼﻑﻓﻰﺍﻟﺒﻴﺖ. 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W2060603688.txt	null	fr		Toxicité au méthotrexate au cours d’une leucémie aiguë lymphoblastique	The Pan African medical journal	2,014	cc-by	425	Open Access Images in medicine Toxicité au méthotrexate au cours d’une leucémie aiguë lymphoblastique Mouna Lamchahab1,&, Said Benchekroun 1 1 Service d’Hématologie et d’Oncologie Pédiatrique, Hôpital 20 Août 1953, CHU Ibn Rochd, Casablanca, Maroc & Corresponding author: Mouna Lamchahab, Service d’Hématologie et d’Oncologie Pédiatrique, Hôpital 20 Août 1953, CHU Ibn Rochd, Casablanca, Maroc Key words: Toxicité, méthotrexate, leucémie aiguë lymphoblastique Received: 01/04/2013 - Accepted: 09/05/2013 - Published: 15/01/2014 Pan African Medical Journal. 2014; 17:16 doi:10.11604/pamj.2014.17.16.2681 This article is available online at: http://www.panafrican-med-journal.com/content/article/17/16/full © Mouna Lamchahab et al. The Pan African Medical Journal - ISSN 1937-8688. 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, distributi on, and reproduction in any medium, provided the original work is properly cited. Image en medicine Mlle K.M, âgée de 19 ans, est suivie au service d'hématologie pour une LAL diagnostiqué sur les données cliniques (syndrome d'insuffisance médullaire, un syndrome tumoral important) et paracliniques (hyperleucocytose majeure à 145 720/mm3 et 95% de blastes). Le myélogramme et l'immunophénotypage confirmait le diagnostic de LAL biphénotypique T et B. Le caryotype était complexe. La patiente a été traitée selon le protocole national des LAL (protocole MARALL) avec une rémission complète après l'induction. Suite à son traitement de consolidation par MTX à haute dose (5g), la patiente présentait une fièvre à 40°c, une mucite grade 4 avec des ulcérations buccales douloureuses à surface fibrineuse, touchant le palais et la muqueuse jugale et débordant sur les lèvres empêchant l'alimentation. Ont noté également, une mélanodermie diffuse, hépato-splénomégalie et colite toxique (diarrhées profuses). Le bilan biologique objectivait une aplasie profonde, insuffisance rénale et cytolyse hépatique. Le diagnostic de toxicité grave au MTX cutanée, hématologique et viscérale a été retenu. Le traitement a consisté en une hydratation, alimentation parentérale, repos digestive, antalgiques, antibiotiques et acide folinique avec une très bonne évolution. Actuellement la patiente est en rémission complète maintenue. Le MTX est un cytostatique de la famille des antifolates, utilisé à haute dose en onco-hématologie. Ses effets secondaires sont dose-dépendants. Différents organes peuvent être touchés. La thrombopénie et la leucopénie sont les signes les plus précoces suivies de la stomatite ulcéreuse. Les signes cutanés sont à type de réaction anaphylactique, érythème et desquamation palmo-plantaires, associés à une mucite importante, un décollement bulleux voire une érythrodermie. Figure 1: En haut: ulcérations buccales et chéilite érosive; En bas: amélioration après traitement Pan African Medical Journal – ISSN: 1937- 8688 (www.panafrican-med-journal.com) Published in partnership with the African Field Epidemiology Network (AFENET). (www.afenet.net) Page number not for citation purposes 1
https://openalex.org/W2783546114	https://content.sciendo.com/downloadpdf/journals/cejpp/11/2/article-p2.pdf	English	null	Policy, learning and regime change: Western concepts and CEE experience	Central European Journal of Public Policy	2,017	cc-by	7,700	Cent. Eur. J. Public Policy 2017; 11(2): 2–10 DOI: 10.1515/cejpp-2016-0037 H.K. Colebatch1 Policy, learning and regime change: Western concepts and CEE experience Abstract: This article offers an outsider’s perspective on the place of policy in the analysis of governing in Central and Eastern Europe, both before the change from a communist to a post-communist order, and since. It explores the way in which ‘policy’ is used as a construct in both the practice of governing and the analysis of that practice. It argues that we have to recognise multiple strands – authority, structured interaction, and collective problematisation - in the construction of ‘policy’. It points to a distinction between ‘formal’ and ‘practical’ perspectives, and argues that this distinction reflects structural tensions in the process of ’putting together’ the shared understandings and relationships which make g for ‘governing’, It argues for the importance of continuing research, empirically based and theoretically informed, into the way that governing is ‘put together’ in Central and Eastern Europe, and how both participants and the governed ‘make sense’ of this process. Keywords: practice of governing; policy; policy analysis; concepts in practice; sense-making Received 24 May 2017; accepted 29 September 2017. Received 24 May 2017; accepted 29 September 2017. Received 24 May 2017; accepted 29 September 2017. The much-anticipated passage from capitalism to socialism had been theorised ad nauseam in academies, universities and coffee bars from Belgrade to Berkeley, but no-one had thought to offer a blueprint for the transition from socialism to capitalism (Judt, 2006, p. 685). 1 H.K. Colebatch: a Visiting Professorial Fellow in the School of Social Science, University of New South Wales. Email: h.colebatch@unsw.edu.au 2017 licensee De Gruyter Open. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 License. Introduction The emergence of the Central European Journal of Public Policy reflected a perceived need for a venue for discussion of the distinctive nature of governing in the post-Communist states of central Europe. Ten years on, the editors chose to invite this contribution from an outsider – a ‘Westerner’ (though Australia is clearly south-east of CEE), and not a ‘regional’ specialist, but one whose focus is on the analysis of governing and the place of ‘policy’ within it. This article offers an outsider’s perspective on the problematics of governing, which arose in the Central and Eastern Europe over these years, and to which this journal has addressed itself. It does not attempt to be a survey of what has happened in this region, either in the current regimes or previously, but flags some of the questions that (analytically) need to be addressed, and suggests some points that emerge from an outsider’s scan of the Central and Eastern European experience. It was felt, both by the editors and the author, that this could be a valuable spur to the continuing discussion in the journal. The collapse of the Communist regimes across Central and Eastern Europe was a dramatic challenge to both the practitioners in the governmental process and to the observers of it. How could they make sense of the process of governing, how were people to relate to it, and how could it be talked about? Paraphrasing Judt’s observation (above), there was no blueprint for the transition from a Communist to a post-Communist political order. Governing is performed by not only the governors, but also by the governed, and reflects shared understandings about ‘the order of things’. How did the collapse of the Communist order change the way in which participants in the governing of CEE ‘put together’ the new mode of governing? And how has the concept of ‘policy’ been mobilised in the practice of governing, and in the analysis of that practice? 2 Cent. Eur. J. Public Policy 2017; 11(2): 2–10 DOI: 10.1515/cejpp-2016-0037 The approach that is being followed focuses not primarily on policy, but also on governing, and on governing, which is a process; and not government, which may be seen as an outcome of this process, but is more commonly seen as an institutional formation (‘the government’), regarded as an actor that produces the outcome. The starting point: the post-war map It is best to start with the stories that everyone knows: the ‘official account’ of government in the CEE countries, and in their counterparts in the West. In the CEE countries, the official accounts pointed to constitutions that provided for elections and legislatures, presidents and prime ministers, courts and police, and a state bureaucracy. In some cases, these also made specific provision for the protection of the rights of individual citizens. But they also usually recognised the ‘leading role’ of the Communist Party (or its local name) in the governing of the country. Governing, then, was the work of the party, the constitutionally-recognised leaders, and the bureaucracy, carrying out well-publicised ‘plans’. It does not appear that ‘policy’ was a significant concept in the description of analysis of governing; there was no word to identify ‘policy’ as a distinct aspect of governing in most of the CEE languages, and in the official account, there was less interest in recognising alternatives or innovation. It was a narrative of progress along a known road, led by the party, expressed in the plans and programs of the government, and demonstrated in well-publicised statistics. While this was an ‘official story’ that everyone knew, few saw it as a guide to behaviour – either their own, or that of the actors it described. It was clear that legislatures and ministers were of less significance than the party hierarchy, and that the police and courts were more concerned with maintaining political control than enforcing the law (Goetz, 2001; Goetz & Wollman 2001; Nunberg, 1999, 2000). But there were also silences – known but not talked about. One (with the partial exception of Yugoslavia) was the Soviet system: the political, economic and military dominance of the USSR, whose Red Army had been the army of occupation through which nearly all these regimes were founded, and which had demonstrated that it could, and would, intervene with military force to compel CEE governments to change direction. This also gave rise to a ‘horizontal’ dimension in CEE, in that the governing in one country could be influenced by events and experiences in others, whether as positive or negative cues.fi At the same time, there was what might be thought of as a parallel ordering of practice, at variance with the official story – well described in Janos Kenedi’s Do It Yourself (1982). Introduction In this, we follow the lead of the organisational scholar Karl Weick (Weick, 1979), who points to the utility of focusing on the activity (‘organising’), which produces the phenomenon (‘organisation’), rather than attributing it to the activity of an intentional actor (‘the organisation’), which is seen as controlling the actions of the participants so as to attain the desired outcomes for the collectivity. In this perspective, ‘policy’ is seen as a particular discourse mobilised in both the accomplishment and the explanation of governing. In the liberal democratic states of the west, it is used to present action as being validated by manifesting the aspects of authority (the action was taken by an authoritative person of his/her subordinates), order (the action is not random or idiosyncratic, but is the normal response), and problematisation (the action is in response to a known problem, which has been addressed in an appropriate way). These elements are variable, and the policy is likely to draw on these elements in different combinations at different points of time and contexts – which is why, the policy is frequently described as ‘complex’ and ‘difficult to define’.h The analytic approach being taken might be described as ‘interpretive’ or ‘social construction’, but it is not adopted as a fashion choice; rather, experience shows that it is better at making sense of patterns of governing – particularly where these are changing – than more systematic approaches, anxious to fit each empirical example into a categoric box. It is more alert to the ways in which local actors frame their own responses to problematic situations.h This is clearly an outsider’s perspective, and an analytical questioning, not an empirical report of practice; it aims to stimulate questioning from researchers working in the region. It does not pretend that Central Europe (let alone Central and Eastern Europe) has a single and unique political experience, but uses the term as a convenient way of focusing attention. Similarly, it does not assume that there was a single, cataclysmic change of governing practice in 1989, any more than (for instance) that the Reformation ‘happened’ in Germany in 1517 or in England in 1534; there was change before that date and continuity after it, but the date helps to focus attention on the nature and extent of the change. 2017 licensee De Gruyter Open. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 License. The starting point: the post-war map As Kenedi points out, the state-sanctioned collective enterprises struggled to provide the food and services needed, and a significant private sector could be seen, sometimes referred to as the ‘second economy’, tolerated in various ways, but not officially recognised. In this mode of practice, people found their own ways to discover and obtain what they needed, using contacts, networks, exchange, side payments, and a web of shared understanding. The gap between the official account 3 Cent. Eur. J. Public Policy 2017; 11(2): 2–10 DOI: 10.1515/cejpp-2016-0037 and the realities of practice was also felt in the state system. The rigidities of the system of central planning and quotas meant that in order to ensure that they would be able to meet imposed targets, factory managers would conceal reserves of raw material and labour from higher authority (Judt, 2006, p. 579). and the realities of practice was also felt in the state system. The rigidities of the system of central planning and quotas meant that in order to ensure that they would be able to meet imposed targets, factory managers would conceal reserves of raw material and labour from higher authority (Judt, 2006, p. 579). But operating in this shadow world of ‘do it yourself’ always ran the risk of official discovery and sanctions, which could be very substantial. So people had to learn how to pay deference to the official story, but find ways to ‘get by’ outside the official system without provoking an official reaction. So, the question ‘how does governing happen?’ always had to be answered in a specific context: how, in this context, did the participants take note of these three accounts: of official collective activity, of individually negotiated practice, and of the significance of coercion – to guide their practice, and the account they gave of this practice. Being attentive to, and knowledgeable about, all three accounts, became an essential life-skill. And the official, systematic account of governing was recognised as significant but inadequate; you would need more than that to get what you wanted, probably by making side-payments to the officials who found themselves in a position to be gatekeepers. fi The official account of governing in the West differentiated a realm of governing (‘the state’) from a realm of self-interested exchange (‘the market’), and a realm of freely-chosen norms and relationships (the ‘private sphere’/’civil society’/’the community’). The starting point: the post-war map The realm of governing was presented as a necessary complement to the other two, dealing with those matters that could not be managed either by the self-stabilizing working of market exchange, or the moral force of community norms. The official account sometimes refers to the ways in which this separation into market, governing and the private is institutionalised and documented. Since there might be divergent views on what should be governed, and how this should be done, this account posited a chain of authority and responsibility, ultimately resting on some expression of the views of the citizens: the citizens elect a legislature, which in turn appoints the executive, or the electorate may also choose the top executive. These authoritative leaders – the legislature and the executive – make choices about the modes of governing, responding to (or anticipating) the preferences of the citizens; these choices are ‘policy’, which is implemented by the subordinate officials. Governing is therefore presented in this account as the execution of choices made by the authoritative (usually elected) leaders, in response to (or anticipation of) the preferences of the public, with policy as the aggregation of these choices. It is not clear to what extent this account was believed, but there were few sanctions for publicly rejecting it. Electoral outcomes were held to demonstrate popular support for the proclaimed programs of the victors (‘the electoral mandate’), and the absence of overt censorship was seen as demonstrating a free contest of ideas. Scholarly research challenged some of the key assumptions, for example, that the voters’ choices were determined by policy preferences, or drew attention to the participation of non-government actors through ‘policy communities’ - but these were seen as marginal subtleties rather than major challenges. Those involved in the governing process seemed to be able to manage the discrepancies between the official account of an open competition of ideas and the much more structured and constrained observable practice by accepting that different accounts might ‘make sense’ in different contexts. p p gf gf The most substantive overt challenge to the official account came from the market-oriented politicians, arguing (in ‘principal- agent’ terms) that the bureaucracy was likely to be more attentive to its own institutional agendas than to the preferences of political superiors. 2017 licensee De Gruyter Open. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 License. The challenge of regime change The collapse of the Communist regime in the USSR and CEE took many observers by surprise, and there was much puzzling about its implications for governing. The most dramatic interpretation was that of Francis Fukuyama, who saw it as ‘the end of history’. What we may be witnessing is not just the end of the Cold War, or the passing of a particular period of post-war history, but the end of history as such; that is, the end point of mankind’s ideological evolution and the universalization of Western liberal democracy as the final form of human government (Fukuyama, 1989). There has been much disputation since then about what this prediction meant and how analytically helpful it has been. As an outsider, I have had a partial and hence distorted access to the European experience, but let me offer some perceptions and invite critique. Perhaps the first observation is that what this uprising was against was more evident than what it was for. To some extent, this reflected a strategic choice by dissidents, anxious to avoid provoking further repression by open advocacy of a different political order, but it also reflected variations in motivation. Antipathy to repressive rule and party dominance, and discontent with the shortages and quality of food, consumer goods and housing were felt everywhere, though perhaps in different proportions. In CEE, there was also an anticolonial spur, a reaction to Russian domination. In some cases, ethnic consciousness was part of the story – positive in the case of East Germany, negative in Czechoslovakia and Yugoslavia. The Yugoslavian case also had overtones of regional resentment, with the more-developed Slovenia and Croatia coming to feel that they would be better-off not having to contribute to the needs of the four less-developed states in the federation (Judt, 2006, 670). But as Fukuyama demonstrated, there was a tendency for Western observers to see it as a resolution of the long Cold War rivalry with the USSR; this was a rejection of the Soviet model, and an embrace of the Western alternative: liberal democratic capitalism. What it meant in Central and Eastern Europe is another question. There had been little empirical research in these countries on the processes of governing before 1989, and there was not a great deal after 1989 on the working of the old order. The challenge of regime change For both practitioners and researchers, there were questions about the understandings, practices and skills that had served them well in the old order, and how they could adjust to the changing circumstances. Anyone giving an account of change would have to account for their own part in it. But while there might have been some reticence in CEE about the nature and extent of the change, there were a number of Western institutions ready to instruct the former socialist states on this transition. The World Bank had been actively promoting an agenda of ‘good governance’ among its client states in the third world, which was focused on creating the institutional basis for a free market, advising governments to remove any constraints on market forces, privatise state functions, reduce public employment, and enforce the rule of law, and it seemed easily applicable to the countries of the ‘second world’ (to use a designation that had never caught on). This was reinforced by the complementary agenda of the World Trade Organisation, which defined the rules for market competition, had procedures for enforcement, and demanded acceptance of these as a condition of entry to the club. p , p , p y This reform agenda embodied a zeal for codifying the institutional dynamics of liberal democratic governme With the collapse [of communism,] the focus of international agencies in the former Soviet Union was on establishing the mechanisms of a capitalist economy through privatization and the creation of free markets. By the mid-1990s, it was clear that markets embedded in corrupt or dysfunctional states would themselves become corrupt and dysfunctional. … [There was] a renewed appreciation of the importance of governance … But you cannot fix what you cannot measure, and so underpinning these more visible developments was a quieter but equally important movement to develop indicators of good governance (Buduru & Pal, 2010, p. 514). This has involved not only aid agencies and ‘official transnational’ bodies like the OECD in the codification of good practice, but has also drawn in non-government bodies like the Soros-sponsored Open Society Foundations and Transparency International. So, there have been a number of overlapping and broadly similar templates being applied to the CEE experience by participants from outside (Pottůček et al., 2016). Cent. Eur. J. Public Policy 2017; 11(2): 2–10 DOI: 10.1515/cejpp-2016-0037 2017 licensee De Gruyter Open. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 License. The starting point: the post-war map This gave rise to a rhetoric of organisational change, termed ‘the New Public Management’, which incorporated competitive contracting agencies (see Pollitt, 2007; Colebatch, 2005), quantitative indicators and payment by results. The term has had a high profile in academic discussion (e.g., Christensen & Laegreid, 2002, 2007), though its utility was questioned (e.g., Hood & Peters, 2004), particularly for CEE (e.g., Drechsler, 2009; cf. Pollitt & Dan, 2011; Dan & Pollitt, 2015). But the reform impetus seems to have subsided, and the term ‘post-NPM’ is starting to appear. So, both in the West and in CEE, official account was broadly accepted in the post-war years. In CEE, as we have seen, people had to incorporate the official account into their ‘working knowledge’. In the West, while the accuracy of the official account as a representation of practice or a guide to behaviour might be questioned by both practitioners and academics (perhaps for different reasons), it was not challenged as an account. This was seen as an appropriate way to account for governing in public and official contexts. It identified a rationale for governing, and a sphere in which it was practised. It validated the outcome of governing, and the actions of (and the deference to) the authoritative leaders who presided over it. And it provided a channel for critique and innovation: to achieve change in the way governing was carried out, it would be necessary to persuade the leaders to adopt a different policy, or install new leaders who would do this. There is less evidence of how this Western account was used within CEE, but I suspect that in official circles, it was a target for challenge, and in private, it served more as a comparator, an ideal to contrast with their own experience, and so its empirical accuracy was both less evident, and of less significance. 4 Making sense of governing on changing terrain For both participants and observers (and citizens), while the ending of the communist regimes may not have been the end of history, it has added to the uncertainty about how we are governed. There are questions about what are the significant institutions, practices and actors, how they contribute to governing, and in what way ‘policy’ becomes part of the story. There was not an established culture for the critical examination of governing practices, nor recognisable methodologies and discourses. There had been a recognised way of making sense of governing; that had now been dethroned, but the substitute on offer was not well-known or well-established, and in any case, did not seem to offer much guidance to the mode of governing. Here, the real problem was the relationship between the model of practice, and the experience of practice, and the questioning of the model by central Europeans was paralleled by a similar questioning in the West of a model that presented governing in terms of authoritative choices by a goal-oriented government. A better-informed public had become more aware that while it was convenient to speak of ‘the government’, public authority is exercised through an array of organisational forms, linked in various, and not always direct, ways to central political authority. These distinct bodies tend to have specialised and often competing agendas, and are often more closely linked to their functional counterparts outside the government than to the other organisational components of the government. Empirically, the government is more of an arena, where organised voices contend for recognition and resources, than an actor. Observers also became aware that while the hierarchical authority of government is widely asserted, it is not always clear that this claim is sustained, or that this is the way in which governing is accomplished. For instance, contemporary governments tend to prohibit the use of a range of drugs, but this prohibition seems to have little impact on the use of these drugs, other than to raise the price, and drug use in prisons (where prisoners are under the absolute control of the government) is often as prevalent as it is outside. Even within the official bureaucracy, appeal to hierarchical authority (e.g., to resolve conflicts between agencies) is of limited utility; political leaders are reluctant to expend their time and political capital in struggles with their counterparts, and expect their own bureaucrats to find a mutually-acceptable course of action. The challenge of regime change But the most explicit source of modelling has been the European Union, whose acquis communautaire sets out (in 35 chapters) the laws and regulatory principles of the EU, and which provides the basis for the negotiations between the 5 2017 licensee De Gruyter Open. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 License. Cent. Eur. J. Public Policy 2017; 11(2): 2–10 DOI: 10.1515/cejpp-2016-0037 CEE candidate states and the EU representatives (and the negotiations may begin before the candidacy) (see Meyer-Sahling, 2009; Madarevic, 2015). But alongside this formal handbook of legal requirements, there is a looser concept of the ‘European mode of governance’, which refers to the ways of governing ‘that are not based on legislation and include private actors in policy formulation’ and ‘include the open method of coordination, voluntary accords, and regulatory fora’ (Heritier, 2004) – a way of governing by negotiation, as compared with the acquis communautaire, which stresses its obligatory and coercive nature. Thus, there has been a very specific reform agenda held out to the countries of Central and Eastern Europe by a range of outside reformers. 2017 licensee De Gruyter Open. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 License. Passing laws, appointing high officials, or signing international agreements are part of the performance of ‘the state’, demonstrating its coherence and authority: the state as actor. But participants know that behind these public performances is a great deal of structured interaction, often painful, aimed at minimising overt resistance to these outcomes (see, e.g., Degeling, Baume & Jones, 1993). Consequently, accounts of governing have to be appropriate to the context in which they are used. There is a public presentation that sees governing as governments, acting on accurate diagnoses of problems, prescribing clear remedies (‘policy’), which will lead to a beneficial outcome. But there is a counter-account, to which the insiders are more attentive, which focuses on the process – the appropriateness of the problematisations, participants, linkage, commitments, and resources – as much as the outcome, and is concerned with the maintenance of the collective, interactive and continuing managing of the problematic aspects of social life. This means that participants are likely to mobilise different discourses in different contexts, and to be aw of acting in this way. As one policy worker told a researcher: articipants are likely to mobilise different discourses in different contexts, and to be aware of the significanc As one policy worker told a researcher: This means that participants are likely to mobilise different discourses in different contexts, and to be aware of the significance of acting in this way. As one policy worker told a researcher: this model would not be of great use. …These words are so neutral. It’s not about consultation. It’s really about stakeholder this model would not be of great use. …These words are so neutral. It’s not about consultation. It’s really about stakeholder engagement (Howard, 2005, p. 10). This distinction between ‘insider’ and ‘outsider’ discourses about governing is well-recognised in the empirical literature, for example, ‘front-stage’ and ‘back-stage’ (Goffman, 1959), ‘normative rules’ and ‘pragmatic rules’ (Bailey, 1969), and ‘sacred’ and ‘profane’ discourses (Degeling & Colebatch, 1984), though its significance for systematic modelling of governing is less well understood. But while there appear to be two quite different perspectives, in many ways, they are two sides of the same coin, reflecting structural tensions in the governing process: between process and outcome, between central authority and context, between informal ways of working and formal ways of enacting outcomes, and between formal (‘sacred’) discourses and informal (‘profane’) activity. Cent. Eur. J. Public Policy 2017; 11(2): 2–10 DOI: 10.1515/cejpp-2016-0037 accomplished (Feldman & March, 1981). Goal statements seem to interact with practice: they may be derived from practice (Spicker, 2006), or generated in response to a procedural requirement (Chapman, 1993). accomplished (Feldman & March, 1981). Goal statements seem to interact with practice: they may be derived from practice (Spicker, 2006), or generated in response to a procedural requirement (Chapman, 1993). So, the explanatory power of the official model is also doubted in the West. There have been different sorts of responses to this. One is a reform agenda: to the extent that practice deviates from the model, it should be made to conform. All activities should state clear objectives, and should be evaluated to confirm that these have been achieved. The government must be seen to govern. Another response has been to see the disjunction between model and experience as evidence of a historical shift: once, the model had been a good description of practice, but circumstances have changed, governing is now more complex, and practice has had to meet the new circumstances. The fullest exposition of this perspective is the argument by Rhodes (1997) that government by authoritative direction has been replaced (or is giving way to) governance by negotiation among self-organising networks, though Rhodes later moved away from this assertion (Bevir & Rhodes, 2010), and there have been few attempts to produce evidence of the time when practice did resemble that authoritative, instrumental model. A third possibility recognises the place of interpretation in the process of governing: that it calls for people to ‘make sense’ of the world and their place in it. This recognises that participants may ‘make sense’ in more than one way, and will need to reach an accommodation with other participants with whom they will be continuing to interact in the managing of this concern. The instrumental assumption that behaviour is governed by perceptions of outcome is challenged by the suggestion that people choose behaviour that is ‘appropriate’ in the context in which they find themselves (March and Olsen 1989). All three of these responses have been in play in both practice and scholarship. What we see here is that while these models of governing can be seen as analytic categories, to be finely honed in the seminar room, they are better viewed as constructs in use, mobilised in the accomplishment of governing. Participants learn that accounts need to be appropriate to context, and to match the discourse to the situation. For political leaders, ‘authority’ will always be a central concern, so the government being seen to have acted appropriately will be regarded as policy success. Central officials are likely to be more concerned with securing acceptance of the policy among the relevant participants (coherence); researchers in that policy field may be more interested in the impact the policy has had on the identified problem (problematisation). All the participants (and all the observers) have a particular and partial view of the action. 2017 licensee De Gruyter Open. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 License. Making sense of governing on changing terrain It is evident, too, that there are other forms of authority than the legal-rational authority of the government – most obviously, kinship groups (the family), ethnic and religious formations, occupations and economic groupings — and that actors in the government seek to recruit these into the process of governing (see, e.g., Colebatch, 2002). Governments may endow these groups with official authority, for example, vesting regulatory authority in a professional association or industry body. In some cases, these bodies are called into existence by the government, and may even receive government funding, which makes it difficult to distinguish between ‘government’ and ‘non-government’. It is clear, too, that government actors (particularly political leaders) are very sensitive to the preferences of organised interests, recognise them as ‘stakeholders’, and seek their prior consent before exercising authority over them – and that failure to do this – described as ‘failure to consult’ - is sufficient grounds for resisting the exercise of authority. In the liberal democracies, those claiming hierarchical authority learn that it is wise not to place too much reliance upon it (which may point to a very significant difference in the cultural inheritance of the CEE states, not only because of the immediate Communist background, but also because of their long exposure to the European Rechtsstaat tradition). A second assumption about liberal government, its instrumentality – that governments are there to achieve objectives – had also become less credible. The enunciation of goals has become more important for both political leaders and organisations, both inside and outside the official sphere and the social sciences have developed more specific ways of measuring their achievement. But the public tend to be sceptical of these claims, and empirical research often casts doubt on the extent to which governing can be seen as the pursuit of specific goals. Goals are often difficult to discern, either being unstated, or stated in terms so broad or ambiguous that it is difficult to attach any specific meaning to them. Leaders may enunciate goals, but show little interest in whether they have been 6 The implications for the analysis of policy in CEE The first point to note here is that systematic presentations of modes of governing (‘systems’) are logical constructions, and not empirical descriptions. So, while it is important to note (and as far as possible, to explain) the differences between observable 7 Cent. Eur. J. Public Policy 2017; 11(2): 2–10 DOI: 10.1515/cejpp-2016-0037 practice and the model in particular cases, this is not an evidence of a ‘failure’ or ‘deficiency’ of practice, any more than it shows the ‘inaccuracy’ or ‘failure’ of the model; it simply shows that they are different sorts of representation. So, the question, when new institutions and practices are introduced into CEE, is not ‘are they being correctly used?’, but ‘how are they being used, and in what way does their introduction change practice?’ practice and the model in particular cases, this is not an evidence of a ‘failure’ or ‘deficiency’ of practice, any more than it shows the ‘inaccuracy’ or ‘failure’ of the model; it simply shows that they are different sorts of representation. So, the question, when new institutions and practices are introduced into CEE, is not ‘are they being correctly used?’, but ‘how are they being used, and in what way does their introduction change practice?’ Linked to this is Rose and Miller’s point that Government is a congenitally failing operation … We do not live in a governed world so much as in a world traversed by the ‘will to govern’, fuelled by the constant registration of ‘failure’, the discrepancy between ambition and outcome, and the constant injunction to do better next time (Rose & Miller, 1992, pp. 190-191) - and they give an example of the welfare reform in UK: - and they give an example of the welfare reform in UK: - and they give an example of the welfare reform in UK: … welfare was not a coherent mechanism that would enable the unfolding of a central plan. The networks were assembled from diverse and often antagonistic components … This was no ‘state apparatus’, but a composition of fragile and mobile relationships and dependencies making diverse attempts to link the aspirations of authorities with the lives of individuals (ibid. p. 193). This does not suggest that the world has becomes too complex for the state, but that attempts to impose systematic state governing encounter the diversity of human life. Items for the agenda The editors have invited me to conclude with some personal viewpoints and reflections, and I thought I might review some of the more significant questions that might be appropriately covered in the journal. This is, of course, a very personal list and should not be taken as an implicit rejection of topics not mentioned. The editors have invited me to conclude with some personal viewpoints and reflections, and I thought I might review some of the more significant questions that might be appropriately covered in the journal. This is, of course, a very personal list and should not be taken as an implicit rejection of topics not mentioned. Since the term ‘policy’ originated in the Anglosphere, there is little agreement on the precise meaning of the term; though it is distinguished from both ‘politics’ and ‘administration’, it is worth asking what is it being used to denote in the CEE, and what is it being distinguished from. A number of translations of policy texts into CEE languages have not translated ‘policy’, but simply used the English word. It would be interesting to track how the meaning is explained in the language of the translation. (See Hale 1988 on the emergence of ‘public policy’ in degree programs in the US.) Similarly, ‘policy analysis’ is a term that has no inherent meaning, even in its American birthplace, where it emerged from RAND as ‘systems analysis’. It is tempting to think it became ‘policy analysis’ in response to Lasswell’s call (1951) for a science of policy, but in any case, Radin (2000) found that those employed as ‘policy analysts’ were engaged in a wide variety of forms of work, not necessarily related to the systematic comparison of options taught in North American graduate courses in ‘policy analysis’, and the term now seems on the verge of being supplanted by ‘policy design’ (Howlett, 2014). It would be valuable to track how the term was introduced, and what sort of activity it has been used to describe. Particular attention should be given to the way these terms are part of a transnational discourse of governing, emerging from the OECD, the EU, the WTO, the UN family, or the wide array of donor organisations. It would be important to flag the cultural assumptions that are implicit in these introduced terms. The implications for the analysis of policy in CEE This fuels the interest in interpretive approaches, which asks how participants in the process of governing perceive the task – what is to be governed and why, what is known, who should be involved, what is an appropriate response, and how can the governing be judged? In this perspective, governing is less the pursuit of specific goals, and more a matter of appropriate process in the collective managing of the problematic – ‘making sense together’ (Hoppe, 1999); goals are significant, but as part of the process. 2017 licensee De Gruyter Open. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 License. Items for the agenda For instance, does the idea of ‘self-organising networks’, or community organisation more generally, assume a relatively high level of interpersonal trust, which may not be found in former socialist societies recovering from systemic disruption (see, e.g., Grdesic & Koska, 2008, 2009)? How do existing attitudes, skills and practices relate to the assumptions in the introduced model? The question about policy (and policy analysis) would be not ‘has this concept been successfully introduced into CEE?’, but ‘in what way has this concept been introduced? What is it being used to denote? What organisational positions, practices and outcomes does it validate? And how does it relate to the previous practice? Does it, for instance, impel the codification of ‘normal practice’ – and in so doing, constrain the autonomy of field staff? ‘In other words, ‘what part does it play in the practices of governing?’ 2017 licensee De Gruyter Open. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 License. 8 Cent. Eur. J. Public Policy 2017; 11(2): 2–10 DOI: 10.1515/cejpp-2016-0037 In this connection, it may now be possible to attempt some systematic mapping of governing practice under the ancien regime (i.e., before 1989). What factors made situations ‘attention-worthy’? How were commitments of resources secured, or extended, or evaded? It may be possible to secure the cooperation of the participants to produce some micro-level historical studies of governing, which can then be compared to how such matters are governed under the new order. Finally, it would be appropriate to be attentive to the link between theorising and practice, and to theorising as part of practice – that is, the way that participants draw on models as part of practice. Boxelaar et al. (2006) describe a case where agricultural extension staff had worked with farmers to develop an agreed harvesting practice that would reduce the risk of fire. They reported this as an example of collective norm-setting by farmers, but their managers insisted that this be re-written to describe it as a case of the department delivering ‘services’ to its ‘customers’ – which would better validate its activities in the eyes of the minister and Treasury than presenting it as empowering a group of farmers. In this way, managers draw on a more favourable conceptualisation to represent (re-present) their activities in a more favourable light. As Kurt Lewin is reputed to have said ‘There is nothing more practical than a good theory’. References Degeling, P., Baume, P., & Jones, K. (1993). Staging an official inquiry for policy change: The case of the Drug Evaluation Review in Australia. Policy and Politics, 21(4), 259-274. Bailey, F.G. (1969). Stratagems and Spoils. Oxford: Basil Blackwell. 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https://openalex.org/W2743485416	https://europepmc.org/articles/pmc5612106?pdf=render	English	null	The unintended mitochondrial uncoupling effects of the FDA-approved anti-helminth drug nitazoxanide mitigates experimental parkinsonism in mice	Journal of biological chemistry/The Journal of biological chemistry	2,017	cc-by	11,341	The unintended mitochondrial uncoupling effects of the FDA-approved anti-helminth drug nitazoxanide mitigates experimental parkinsonism in mice From the Centre for ‡Chemical Biology and §Pharmacology and Toxicology, Council of Scientific a Indian Institute of Chemical Technology, Uppal Road, Tarnaka, Hyderabad – 500 007, Telangana From the Centre for ‡Chemical Biology and §Pharmacology and Toxicology, Council of Scientific and Industrial Research (CSIR)– Indian Institute of Chemical Technology, Uppal Road, Tarnaka, Hyderabad – 500 007, Telangana State, India Edited by Paul E. Fraser Mitochondria play a primary role in the pathophysiology of Par- kinson’s disease (PD), and small molecules that counteract the ini- tial stages of disease may offer therapeutic benefit. In this regard, we have examined whether the off-target effects of the Food and Drug Administration (FDA)–approved anti-helminth drug nita- zoxanide (NTZ) on mitochondrial respiration could possess any therapeutic potential for PD. Results indicate that MPP-induced loss in oxygen consumption rate (OCR) and ATP production by mitochondria were ameliorated by NTZ in real time by virtue of its mild uncoupling effect. Pretreatment of cells with NTZ mitigated MPP-induced loss in mitochondrial OCR and reactive oxygen species (ROS). Similarly, addition of NTZ to cells pretreated with MPPcouldreverseblockinmitochondrialOCRandreactiveoxy- gen species induced by MPP in real time. The observed effects of NTZ were found to be transient and reversible as removal of NTZ from incubation medium restored the mitochondrial respiration tothatofcontrols.ApoptosisinducedbyMPPwasamelioratedby NTZ in a dose-dependent manner. In vivo results demonstrated that oral administration of NTZ (50 mg/kg) in an acute MPTP mouse model of PD conferred significant protection against the loss of tyrosine hydroxylase (TH)-positive neurons of substantia nigra.Basedontheaboveobservationswebelievethatrepurposing of NTZ for PD may offer therapeutic benefit. chondria were found to play an indispensable role in the onset and progression of PD (1–3). In fact, PD mimetics, such as MPP and rotenone, happen to be the direct inhibitors at the complex-I of mitochondria (1–3). Also, some of the PD-associ- ated genes, such as parkin and pink-1 have been shown to directly affect the mitochondrial integrity by regulating its qual- ity control, and mutations in these genes were found to be asso- ciated with familial PD (4–6). Indeed, complex-I activities were reported to be significantly reduced in postmortem samples of SNPc and platelets of PD patients (7, 8). Further, the catalytic subunits of complex-I derived from PD patients were found to be oxidatively damaged (8). Results 3 Theabbreviationsusedare:PD,Parkinson’sdisease;NTZ,nitazoxanide;ROS, reactive oxygen species; H2DCF-DA, dichlorofluorescein diacetate; OCR, oxygen consumption rate; DNP, 2,4-dinitrophenol; MPP, 1-methyl-4-phe- nylpyridinium ion; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; ANT, 2-amino-5-nitrothiazole; OAS, O-acetyl salicylamide; CAT, carboxyatractylo- side; SNPc, substantia nigra pars compacta; TH, tyrosine hydroxylase; UCP, uncouplingprotein;DPPH,2,2-diphenyl-1-picrylhydrazyl;TMPD,N,N,N,N-te- tramethyl-p-phenylenediamine;DPBS,Dulbecco’sphosphate-bufferedsaline. 3 Theabbreviationsusedare:PD,Parkinson’sdisease;NTZ,nitazoxanide;ROS, reactive oxygen species; H2DCF-DA, dichlorofluorescein diacetate; OCR, oxygen consumption rate; DNP, 2,4-dinitrophenol; MPP, 1-methyl-4-phe- nylpyridinium ion; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; ANT, 2-amino-5-nitrothiazole; OAS, O-acetyl salicylamide; CAT, carboxyatractylo- side; SNPc, substantia nigra pars compacta; TH, tyrosine hydroxylase; UCP, uncouplingprotein;DPPH,2,2-diphenyl-1-picrylhydrazyl;TMPD,N,N,N,N-te- tramethyl-p-phenylenediamine;DPBS,Dulbecco’sphosphate-bufferedsaline. NTZ prevents MPP-induced block in mitochondrial respiration and restores ATP production by its mild uncoupling effect Based on some of the recent reports on the ability of NTZ in enhancing mitochondrial oxygen consumption rate (OCR), we J. Biol. Chem. (2017) 292(38) 15731–15743 15731 cro cro ARTICLE The unintended mitochondrial uncoupling effects of the FDA-approved anti-helminth drug nitazoxanide mitigates experimental parkinsonism in mice Moreover, the pathogenesis of PD appears to be converging on common pathways, such as mito- chondrial dysfunction followed by oxidative stress and protein aggregation, which ultimately leads to apoptosis (9, 10). Ther- apeutic strategies employing antioxidants and iron chelators have been utilized in PD animal models with limited success (11–13). Our earlier results demonstrated the neuroprotective effectsofthemitochondria-targetedantioxidantMitoQincellular and animal models of PD (14). In this regard, targeting the very initial steps of disease pathology employing small molecules capa- bleofenhancingmitochondrialrespirationandmitigatingreactive oxygen species (ROS) appears to be the promising strategy in pre- venting the downstream biochemical abnormalities. A recent study identified the mitochondrial respiration–enhancing effects of some of the FDA-approved drugs, including the anti-helminth drug nitazoxanide (NTZ) (15). Moreover, NTZ happens to be a highly bioavailable drug through oral route and has a better safety profile with a half-life of nearly 3.5 h and can reach the CNS (16, 17). In the present study, we have investigated the efficacy of NTZ in dopaminergic cell lines against MPP-induced alterations in the mitochondrial bioenergetics as well as in an acute MPTP mouse model of PD in both preventive and therapeutic models. OurfindingsdemonstratethatthemilduncouplingeffectsofNTZ could mitigate MPP-induced deleterious effects in both cellular and animal models of PD. Parkinson’s disease (PD)3 is one of the most common neuro- degenerative disorders that is characterized primarily by the loss of dopaminergic neurons in the substantia nigra pars com- pacta (SNPc) leading to dopamine deficit in the striatum. Mito- 2 To whom correspondence should be addressed: Tel.: 91-040-2719 1865; Fax: 91-040-2716 0387; E-mail: kalivendi@iict.res.in. This work was supported by CSIR, India XII FYP projects MiND (BSC-0115) and SMiLE(CSC-0111)andbytheDepartmentofBiotechnology,IndiaResearch project. This work was also supported by research fellowships from the University Grant Commission, Government of India (to S. T.) and from CSIR, India(toS. N. P.andR. L. V.).Theauthorsdeclarethattheyhavenoconflicts of interest with the contents of this article. 1 These authors contributed equally to this work. This work was supported by CSIR, India XII FYP projects MiND (BSC-0115) and SMiLE(CSC-0111)andbytheDepartmentofBiotechnology,IndiaResearch project. This work was also supported by research fellowships from the University Grant Commission, Government of India (to S. T.) and from CSIR, India(toS. N. P.andR. L. V.).Theauthorsdeclarethattheyhavenoconflicts of interest with the contents of this article. J. Biol. Chem. (2017) 292(38) 15731–15743 15731 2 To whom correspondence should be addressed: Tel.: 91-040-2719 1865; Fax: 91-040-2716 0387; E-mail: kalivendi@iict.res.in. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. Nitazoxanide mitigates experimental parkinsonism TZ and its chemical moieties on mitochondrial OCR. A, chemical structures of nitazoxanide, O-acetyl sal -dependent effect of NTZ on mitochondrial OCR: SK-N-SH cells in 24-well plates were treated with differe ular OCR was measured using Seahorse extracellular flux analyzer. C, effect of NTZ (1 and 5 M) and its chemica mino-5-nitrothiazole (ANT) (1, 5, and 10 M), on cellular OCR was measured using Seahorse extracellular flux a umes of buffer in place of compounds. Data are representative of the mean S.D. of samples analyzed in t ents. Figure 1. Effect of NTZ and its chemical moieties on mitochondrial OCR. A, chemical structures of nitazoxanide, O-acetyl salicylamide, and 2-amino-5- nitrothiazole. B, dose-dependent effect of NTZ on mitochondrial OCR: SK-N-SH cells in 24-well plates were treated with different concentrations of NTZ (0.25–10 M) and cellular OCR was measured using Seahorse extracellular flux analyzer. C, effect of NTZ (1 and 5 M) and its chemical moieties, salicylamide (1, 5, and 10 M) and 2-amino-5-nitrothiazole (ANT) (1, 5, and 10 M), on cellular OCR was measured using Seahorse extracellular flux analyzer. Control cells were treated with equal volumes of buffer in place of compounds. Data are representative of the mean S.D. of samples analyzed in triplicates (n 3) from two independent experiments. Figure 1. Effect of NTZ and its chemical moieties on mitochondrial OCR. A, chemical structures of nitazoxanide, O-acetyl salicylamide, and 2-amino-5- nitrothiazole. B, dose-dependent effect of NTZ on mitochondrial OCR: SK-N-SH cells in 24-well plates were treated with different concentrations of NTZ (0.25–10 M) and cellular OCR was measured using Seahorse extracellular flux analyzer. C, effect of NTZ (1 and 5 M) and its chemical moieties, salicylamide (1, 5, and 10 M) and 2-amino-5-nitrothiazole (ANT) (1, 5, and 10 M), on cellular OCR was measured using Seahorse extracellular flux analyzer. Control cells were treated with equal volumes of buffer in place of compounds. Data are representative of the mean S.D. of samples analyzed in triplicates (n 3) from two independent experiments. examined whether this property of NTZ could ameliorate MPP-induced dysfunction in mitochondrial respiration. Nitazoxanide mitigates experimental parkinsonism Nitazoxanide mitigates experimental parkinsonism i h th th i i OCR b NTZ i T i h th NTZ iti t MPP i d d bl k i t of NTZ on overall mitochondrial OCR and at individual respiratory complexes. A and B, human SK-N-SH neuroblastoma cells were different concentrations of NTZ (1, 5, and 10 M) on (A) pyruvate and malate-driven respiration and (B) OCR at individual respiratory complexes red by electron flow assay in permeabilized cells as mentioned in “Experimental Procedures.” Data are representative of the mean S.D. of d in triplicates (n 3) from three independent experiments. g p p Figure 2. Effect of NTZ on overall mitochondrial OCR and at individual respiratory complexes. A and B, human SK-N-SH neuroblastoma cells were incubated with different concentrations of NTZ (1, 5, and 10 M) on (A) pyruvate and malate-driven respiration and (B) OCR at individual respiratory complexes and was monitored by electron flow assay in permeabilized cells as mentioned in “Experimental Procedures.” Data are representative of the mean S.D. of samples analyzed in triplicates (n 3) from three independent experiments. Figure 2. Effect of NTZ on overall mitochondrial OCR and at individual respiratory complexes. A and B, human SK-N-SH neuroblastoma cells were incubated with different concentrations of NTZ (1, 5, and 10 M) on (A) pyruvate and malate-driven respiration and (B) OCR at individual respiratory complexes and was monitored by electron flow assay in permeabilized cells as mentioned in “Experimental Procedures.” Data are representative of the mean S.D. of samples analyzed in triplicates (n 3) from three independent experiments. Next, to examine whether the increase in OCR by NTZ is because of mitochondrial respiration or not, we have added assay medium that is devoid of pyruvate and malate to dop- aminergic cells in a 24-well Seahorse assay plate in the presence or absence of NTZ (1–10 M) and found no increase in the OCR. However, addition of pyruvate and malate instanta- neously increased OCR in control and NTZ-treated cells (Fig. 2A). There was a dose-dependent increase in OCR in cells incu- bated with increasing concentrations of NTZ (Fig. 2A). Addi- tion of rotenone (1.5 M) almost completely inhibited the OCR in control as well as NTZ-treated cells to similar extents, indi- cating that NTZ-induced OCR is derived from mitochondrial respiration (Fig. 2A). To examine this, we added either buffer or different concentra- tions of NTZ (0.25–10 M) directly to the human dopaminergic cells seeded in 24-well plate through one of the ports of the Seahorse flux analyzer and real-time OCRs were measured. Results indicate that NTZ dose-dependently increased cellular OCR as soon as it was added to the cells (Fig. 1B). A substantial increase in OCR was evident starting from 1 M NTZ wherein an increase in the OCR from 220 to 250 pmol/min was evident, whereas at 5 M concentration there was a steep and significant increase from 220 to 369 pmol/min, and the values remained nearly constant until 2 h. At 10 M NTZ, although there was a steep increase in OCR during the initial 20 min, it gradually decreased until 2 h. Because NTZ consists of two basic chemical scaffolds/moieties, i.e. 2-amino-5-nitrothiazole (ANT) and O-acetyl-salicylamide (OAS), we next examined whether the effect of NTZ is because of any of these scaffolds. Addition of either ANT (1–10 M) or OAS (1–10 M) did not induce any increase in cellular OCR unlike NTZ (Fig. 1C). 15732 J. Biol. Chem. (2017) 292(38) 15731–15743 Nitazoxanide mitigates experimental parkinsonism To examine whether NTZ affects mito- chondrial respiration at specific mitochondrial complexes, we have performed electron flow assay using permeabilized human dopaminergic cells in the presence and absence of different concentrations of NTZ (1–10 M) (Fig. 2B). Results indicate that there was a dose-dependent increase in OCR by NTZ in the pyruvate and malate (complex-I)–, succinate (complex-II)–, as well as ascorbate and TMPD (complex-IV)–induced respira- tion (Fig. 2B). To examine whether NTZ mitigates MPP-induced block in the mitochondrial respiration, dopaminergic cells were seeded in 24-well assay plates and Mito Stress assay was performed with MPP in the presence or absence of NTZ, whereas control cells were treated with assay medium in place of MPP or NTZ under similar experimental conditions. Addition of MPP (100 M) gradually decreased the mitochondrial respiration as indi- cated by the decrease in OCR and virtually no respiration was evident at 2 h following addition of MPP. At this stage addi- tion of oligomycin had negligible effect on the OCR, indicating that ATP generation was almost completely ceased in cells treated with MPP (Fig. 3A). However, when MPP was added along with NTZ (1 M), there was a significant increase in the respiration (OCR) as compared with MPP treatment alone or controls which persisted until 2 h (Fig. 3A). Addition of oligo- mycin to MPP NTZ–treated cells exhibited a decline in OCR, indicating that cells were capable of generating ATP (Fig. 3, A and B). Under similar conditions, NTZ alone enhanced mitochondrial respiration and NTZ-treated cells could gener- ate ATP nearly to the same extent as control cells (Fig. 3, A and B). However, at higher concentrations of NTZ (5 M) cells Nitazoxanide mitigates experimental parkinsonism Nitazoxanide mitigates experimental parkinsonism mitigates MPP-induced mitochondrial dysfunction. A, SK-N-SH cells were treated with MPP in the presence and absence of NTZ (1 tress assay was performed as described in “Experimental Procedures.” B and C, bar diagrams representing (B) ATP production and (C) m the data obtained from (A). D, SK-N-SH cells were incubated with NTZ (1 M and 5 M) for 60 min before exposure to MPP for 4, 8, and ar ATP content was analyzed by luciferase-based kit as mentioned in the “Experimental Procedures” section. Data shown are the of mean S.E. from triplicate measurements per each condition (n 3) for (A) from three independent experiments. B–D, values e mean S.E. of triplicate measurements per each condition from two different experiments (n 6). *, p 0.001; , p 0.01; , p ed with controls. ###, p 0.001; ##, p 0.01; #, p 0.05 as compared with MPP group as analyzed by two-way ANOVA with Tukey’s risons test. de mitigates experimental parkinsonism Figure 3. NTZ mitigates MPP-induced mitochondrial dysfunction. A, SK-N-SH cells were treated with MPP in the presence and absence of NTZ (1 M) and Mito Stress assay was performed as described in “Experimental Procedures.” B and C, bar diagrams representing (B) ATP production and (C) protein leak from the data obtained from (A). D, SK-N-SH cells were incubated with NTZ (1 M and 5 M) for 60 min before exposure to MPP for 4, 8, and 24 h and cellular ATP content was analyzed by luciferase-based kit as mentioned in the “Experimental Procedures” section. Data shown are the representative of mean S.E. from triplicate measurements per each condition (n 3) for (A) from three independent experiments. B–D, values indicated are the mean S.E. of triplicate measurements per each condition from two different experiments (n 6). , p 0.001; , p 0.01; , p 0.05 as compared with controls. ###, p 0.001; ##, p 0.01; #, p 0.05 as compared with MPP group as analyzed by two-way ANOVA with Tukey’s multiple comparisons test. Nitazoxanide mitigates experimental parkinsonism To revalidate the obtained results and to examine the long- term effects of NTZ on MPP-induced block in ATP produc- tion, human dopaminergic cells were treated with MPP in the presence and absence of NTZ (1 and 5 M) for 4, 8, and 24 h and cellular ATP was estimated by luciferase-based assay method (Fig. 3D). Results demonstrate that treatment with MPP for 4, 8, and 24 h inhibited cellular ATP levels to nearly 40, 56, and exhibited a dose-dependent decrease in ATP production, pos- sibly because of more uncoupling (data not shown). Neverthe- less, there was a significant increase in proton leak in both NTZ alone and NTZ MPP–treated conditions (Fig. 3, A and C). Addition of FCCP to cells resulted in a steep increase in OCR under all the treatment conditions, indicating the presence of intact functional mitochondria (Fig. 3A). exhibited a dose-dependent decrease in ATP production, pos- sibly because of more uncoupling (data not shown). Neverthe- less, there was a significant increase in proton leak in both NTZ alone and NTZ MPP–treated conditions (Fig. 3, A and C). Addition of FCCP to cells resulted in a steep increase in OCR under all the treatment conditions, indicating the presence of intact functional mitochondria (Fig. 3A). J. Biol. Chem. (2017) 292(38) 15731–15743 15733 exhibited a dose-dependent decrease in ATP production, pos- sibly because of more uncoupling (data not shown). Neverthe- less, there was a significant increase in proton leak in both NTZ alone and NTZ MPP–treated conditions (Fig. 3, A and C). Addition of FCCP to cells resulted in a steep increase in OCR To revalidate the obtained results and to examin term effects of NTZ on MPP-induced block in AT tion, human dopaminergic cells were treated with M presence and absence of NTZ (1 and 5 M) for 4, 8, a cellular ATP was estimated by luciferase-based ass Figure 3. NTZ mitigates MPP-induced mitochondrial dysfunction. A, SK-N-SH cells were treated with MPP in the presence and abse M) and Mito Stress assay was performed as described in “Experimental Procedures.” B and C, bar diagrams representing (B) ATP produ protein leak from the data obtained from (A). D, SK-N-SH cells were incubated with NTZ (1 M and 5 M) for 60 min before exposure to MPP 24 h and cellular ATP content was analyzed by luciferase-based kit as mentioned in the “Experimental Procedures” section. Data sh representative of mean S.E. from triplicate measurements per each condition (n 3) for (A) from three independent experiments indicated are the mean S.E. of triplicate measurements per each condition from two different experiments (n 6). , p 0.001; , p 0.05 as compared with controls. ###, p 0.001; ##, p 0.01; #, p 0.05 as compared with MPP group as analyzed by two-way ANOVA multiple comparisons test. Nitazoxanide mitigates experimental parkinsonism Nitazoxanide mitigates experimental parkinsonism 60%, respectively, as compared with controls. At the 8 h time period, NTZ could significantly improve cellular ATP levels as compared with MPP treatment alone, but not at the 4 h time point. However, following 24 h incubation, NTZ significantly protected from MPP-induced loss in cellular ATP levels (Fig. 3D). NTZ per se decreased ATP levels as compared with con- trols at 4 and 8 h time periods, possibly because of its uncou- pling effect. However, ATP levels were found to be nearly the same as controls at 24 h time period, indicating that the uncou- pling effect of NTZ could be a transient event (Fig. 3D). MPP (500 M) resulted in a significant decrease in the caspase-3 activation from 6.8-fold to 5.3-, 3.8-, and 3.3-fold, respectively. Under similar experimental conditions, NTZ per se at these concentrations did not induce any significant apo- ptosis in cells (Fig. 6C). 60%, respectively, as compared with controls. At the 8 h time period, NTZ could significantly improve cellular ATP levels as compared with MPP treatment alone, but not at the 4 h time point. However, following 24 h incubation, NTZ significantly protected from MPP-induced loss in cellular ATP levels (Fig. 3D). NTZ per se decreased ATP levels as compared with con- trols at 4 and 8 h time periods, possibly because of its uncou- pling effect. However, ATP levels were found to be nearly the same as controls at 24 h time period, indicating that the uncou- pling effect of NTZ could be a transient event (Fig. 3D). To know whether the observed effects of NTZ could be because of any of its antioxidant activity, in vitro DPPH radical scavenging activity of NTZ (1–25 M) was performed and vita- min C was used as a positive control. As shown in Fig. 6D, NTZ did not exhibit any antioxidant activity (DPPH radical scaveng- ing activity) up to 25 M concentration. However, under similar conditions, vitamin C dose-dependently inhibited DPPH radi- cal production (Fig. 6D). The observed effects of NTZ were found to be reversible as cells treated with NTZ (1 M in assay medium) for 1 h exhibited a significant increase in basal respiration and proton leak with a concomitant decrease in ATP production (Fig. 4, A and B). However, replacement of NTZ-containing medium with fresh assay medium restored the basal respiration, proton leak, and ATP levels significantly (Fig. 4, A and B). Nitazoxanide mitigates experimental parkinsonism The observed uncou- pling effects of NTZ may not be operative through uncoupling proteins (UCPs) or adenine nucleotide translocase as NTZ could induce OCR in the presence of GTP (UCP inhibitor) and carboxyatractyloside, a specific inhibitor of adenine nucleotide transferase (Fig. 4, C and D). NTZ prevents MPTP-induced loss of TH-positive neurons in vivo To evaluate the in vivo effects in a preventive model, NTZ (50 mg/kg body weight) was administered orally for 7 days follow- ing treatment with MPTP (20 mg/kg, four injections at 2-h intervals) in an acute mouse model of PD (Fig. 7, A–C). For therapeutic model, NTZ (50 mg/kg body weight) was adminis- tered orally starting from 8th day post MPTP treatment (20 mg/kg, four injections at 2-h intervals) and continued the same until 14th day. Results demonstrate that although there was an intense staining of TH-positive neurons in control animals, staining of the same was significantly reduced on days 7 and 14 following MPTP treatment (Fig. 7B). However, a significant recovery in TH staining was evident in NTZ MPTP–treated group as compared with MPTP treatment alone. Under similar experimental conditions, administration of NTZ alone did not induce any significant loss in TH staining (Fig. 7, B–F). In the therapeutic model, administration of NTZ post MPTP treat- ment could not significantly restore the TH staining of SNPc or striatum to that of control values or 7th day post MPTP treat- ment. However, as compared with day 14 MPTP treatment, the TH staining in the SNPc and striatum was significantly restored in MPTP NTZ group. The above data clearly suggest that NTZ could not reverse the loss of TH-positive neurons but has the ability in delaying the death of existing dopamine neurons of SNPc (Fig. 7, B–E). The locomotor activity of the animals as measured by open field test reflected the above findings (Fig. 7F). MPTP-treated animals exhibited a time-dependent loss in the locomotor activity as compared with controls. Pretreat- ment with NTZ (NTZMPTP 7D group) significantly allevi- ated MPTP-induced (MPTP 7D group) loss in motor activity. However, in the therapeutic model, administration of NTZ 7 days post MPTP treatment for 7 consecutive days significantly restored motor activity of mice as compared with 14 days post MPTP treatment (MPTP 14D group), but the values were not significant as compared with 7 days post MPTP treatment (MPTP 7D group). Mice treated with NTZ alone exhibited motor activity similar to that of control group (Fig. 7F). NTZ restores respiration in partially damaged mitochondria by MPP 15734 J. Biol. Chem. (2017) 292(38) 15731–15743 15734 J. Biol. Chem. (2017) 292(38) 15731–15743 NTZ restores respiration in partially damaged mitochondria by MPP As the above results suggest that NTZ mitigates MPP-in- duced block in mitochondrial respiration, we next examined whether NTZ has the potential to restore respiration in mito- chondria that were preincubated with MPP in which the res- piration is completely ceased. To examine this, human neuro- blastoma cells were incubated with MPP (100 M) for 2 h because at this time point there was no or negligible OCR as compared with control cells (Fig. 5A). Interestingly, addition of NTZ (1 and 5 M) to MPP-treated cells immediately reversed the block in mitochondrial respiration at both the concentra- tions examined, and the values were found to be substantially higher than control cells (Fig. 5A). Similarly, dopaminergic cells treated with MPP (1 mM) for 12 h exhibited a 2.5-fold increase in mitochondrial ROS as mea- sured by MitoSOX staining (Fig. 5, B and C). Either pretreat- ment of cells for 1 h with NTZ followed by 12 h incubation with MPP or posttreatment of NTZ for 1 h to cells that were pre- incubated with MPP for 12 h significantly reduced MitoSOX staining to nearly the control values (Fig. 5, B and C). NTZ ameliorates MPP-induced loss of mitochondrial membrane potential, ROS, and apoptosis Addition of MPP (500 M) to N27 cells (rat midbrain-de- rived dopaminergic cell line) for 24 h resulted in a substantial increase in depolarized mitochondria as indicated by an increase in the net green fluorescence (JC-1 staining) as well as cellular ROS (DCF staining) (Fig. 6, A and B). However, pre- treatment of cells with NTZ (1 M) prevented MPP-induced loss in mitochondrial membrane potential as well as oxidative stress (Fig. 6, A and B). Also, treatment of N27 cells with MPP (0.5 mM) for 24 h resulted in a 6.8-fold increase in apoptosis as indicated by caspase-3 activity (Fig. 6C). However, pretreat- ment of cells with 1, 5, or 10 M NTZ prior to the addition of Discussion Mitochondrial dysfunction was found to be associated with several neurodegenerative diseases, and defects in various mitochondrial respiratory chain complexes have been detected in Alzheimer’s, Parkinson’s, and Huntington’s diseases (18, 19). J. Biol. Chem. (2017) 292(38) 15731–15743 15735 B, values indicated are the mean S.E. of triplicate measurements per each condition from two independent experiments (n 6). *, p 0.001; , p 0.01 as compared with controls. ###, p 0.001; ##, p 0.01 as compared with NTZ (medium) group as analyzed by two-way ANOVA with Tukey’s multiple comparisons test. Figure 4. NTZ-induced uncoupling effects are reversible and do not involve UCPs or adenine nucleotide translocase. A, SK-N-SH cells were treated with NTZ (1 M) for 1 h in a 24-well Seahorse assay plate. In few wells the NTZ containing medium was replaced with fresh assay medium after 1 h incubation and Mito Stress assay was performed as described in “Experimental Procedures.” B, bar diagrams representing basal respiration, proton leak, and ATP production from the data obtained from (A). C, cells treated with the UCP inhibitor GTP (4 mM) for 20 min, and NTZ (5 M) or FCCP (3 M) was added and OCR was recorded further for 25 min. Control cells were treated with equal volumes of buffer under similar conditions. D, cells were treated with the adenine nucleotide translocase inhibitor CAT (3 g/ml) for 20 min, and later NTZ (1 and 5 M) or FCCP (3 M) was added and OCR was recorded further for 25 min. Control cells were treated with equal volumes of buffer under similar conditions. A, C, and D, data are the representative of mean S.E. of triplicate measurements per each condition (n 3) from two independent experiments. B, values indicated are the mean S.E. of triplicate measurements per each condition from two independent experiments (n 6). *, p 0.001; , p 0.01 as compared with controls. ###, p 0.001; ##, p 0.01 as compared with NTZ (medium) group as analyzed by two-way ANOVA with Tukey’s multiple comparisons test. Figure 4. NTZ-induced uncoupling effects are reversible and do not involve UCPs or adenine nucleotide tra Figure 4. NTZ-induced uncoupling effects are reversible and do not involve UCP Figure 4. NTZ-induced uncoupling effects are reversible and do not involve UCPs or adenine nucleotide translocase. A, SK-N-SH cells were treated with NTZ (1 M) for 1 h in a 24-well Seahorse assay plate. Nitazoxanide mitigates experimental parkinsonism ed uncoupling effects are reversible and do not involve UCPs or adenine nucleotide translocase. A, SK-N-SH a 24-well Seahorse assay plate. In few wells the NTZ containing medium was replaced with fresh assay medium a s performed as described in “Experimental Procedures.” B, bar diagrams representing basal respiration, proton le ed from (A). C, cells treated with the UCP inhibitor GTP (4 mM) for 20 min, and NTZ (5 M) or FCCP (3 M) was added Control cells were treated with equal volumes of buffer under similar conditions. D, cells were treated with t CAT (3 g/ml) for 20 min, and later NTZ (1 and 5 M) or FCCP (3 M) was added and OCR was recorded further for 25 olumes of buffer under similar conditions. A, C, and D, data are the representative of mean S.E. of triplicate m om two independent experiments. B, values indicated are the mean S.E. of triplicate measurements per ea ments (n 6). *, p 0.001; , p 0.01 as compared with controls. ###, p 0.001; ##, p 0.01 as compared wit way ANOVA with Tukey’s multiple comparisons test. g p p Figure 4. NTZ-induced uncoupling effects are reversible and do not involve UCPs or adenine nucleotide translocase. A, SK-N-SH cells were treated with NTZ (1 M) for 1 h in a 24-well Seahorse assay plate. In few wells the NTZ containing medium was replaced with fresh assay medium after 1 h incubation and Mito Stress assay was performed as described in “Experimental Procedures.” B, bar diagrams representing basal respiration, proton leak, and ATP production from the data obtained from (A). C, cells treated with the UCP inhibitor GTP (4 mM) for 20 min, and NTZ (5 M) or FCCP (3 M) was added and OCR was recorded further for 25 min. Control cells were treated with equal volumes of buffer under similar conditions. D, cells were treated with the adenine nucleotide translocase inhibitor CAT (3 g/ml) for 20 min, and later NTZ (1 and 5 M) or FCCP (3 M) was added and OCR was recorded further for 25 min. Control cells were treated with equal volumes of buffer under similar conditions. A, C, and D, data are the representative of mean S.E. of triplicate measurements per each condition (n 3) from two independent experiments. Nitazoxanide mitigates experimental parkinsonism Nitazoxanide mitigates experimental parkinsonism Nitazoxanide mitigates experimental parkinsonism Nitazoxanide mitigates experime e 5. Reversal of MPP-induced block in mitochondrial respiration by NTZ. A, following 20 min of recording basal OCR in SK-N-SH cells, MPP was d through port A and OCR was recorded further for 2 h. Later, NTZ (1 and 5 M) was added through port B and OCR was measured. Buffer was added in of MPP in controls, whereas, buffer was added in place of NTZ for MPP-treated samples. Values indicated are the mean S.E. of triplicate measure- s (n 3) and the experiment was repeated two times. B, SK-N-SH cells were either pretreated with NTZ (1 M) for 1 h before incubation with MPP (1 mM) 2 h or posttreated for 1 h following 12 h incubation with MPP (1 mM). At the end of incubation MitoSOX staining was performed as described in rimental Procedures” and fluorescence images were captured from five different fields of view C the average intensity units from five different fields of Figure 5. Reversal of MPP-induced block in mitochondrial respiration by NTZ. A, following 20 min of recording basal OCR in SK-N-SH cells, MPP was added through port A and OCR was recorded further for 2 h. Later, NTZ (1 and 5 M) was added through port B and OCR was measured. Buffer was added in place of MPP in controls, whereas, buffer was added in place of NTZ for MPP-treated samples. Values indicated are the mean S.E. of triplicate measure- ments (n 3) and the experiment was repeated two times. B, SK-N-SH cells were either pretreated with NTZ (1 M) for 1 h before incubation with MPP (1 mM) for 12 h or posttreated for 1 h following 12 h incubation with MPP (1 mM). At the end of incubation MitoSOX staining was performed as described in “Experimental Procedures” and fluorescence images were captured from five different fields of view. C, the average intensity units from five different fields of view from two independent experiments (n 8–10) were represented as mean S.D. , p 0.05 as compared with controls. ##, p 0.01; #, p 0.05 as compared with MPP group by two-way ANOVA with Sidak’s multiple comparisons test. In few wells the NTZ containing medium was replaced with fresh assay medium after 1 h incubation and Mito Stress assay was performed as described in “Experimental Procedures.” B, bar diagrams representing basal respiration, proton leak, and ATP production from the data obtained from (A). C, cells treated with the UCP inhibitor GTP (4 mM) for 20 min, and NTZ (5 M) or FCCP (3 M) was added and OCR was recorded further for 25 min. Control cells were treated with equal volumes of buffer under similar conditions. D, cells were treated with the adenine nucleotide translocase inhibitor CAT (3 g/ml) for 20 min, and later NTZ (1 and 5 M) or FCCP (3 M) was added and OCR was recorded further for 25 min. Control cells were treated with equal volumes of buffer under similar conditions. A, C, and D, data are the representative of mean S.E. of triplicate measurements per each condition (n 3) from two independent experiments. B, values indicated are the mean S.E. of triplicate measurements per each condition from two independent experiments (n 6). , p 0.001; , p 0.01 as compared with controls. ###, p 0.001; ##, p 0.01 as compared with NTZ (medium) group as analyzed by two-way ANOVA with Tukey’s multiple comparisons test. 15736 J. Biol. Chem. (2017) 292(38) 15731–15743 f evidence implicate PD as a free radical dis- mitochondrial dysfunction leading to the pro- ee radicals and energy failure (10, 20, 21). chelators, AAV2-neurturin, creatine, and pioglitazo they were found to have no significant effect on disease sion (24–27). In fact, we reported earlier that the m l of MPP-induced block in mitochondrial respiration by NTZ. A, following 20 min of recording basal OCR in SK-N-SH cells, rt A and OCR was recorded further for 2 h. Later, NTZ (1 and 5 M) was added through port B and OCR was measured. Buffer wa ontrols, whereas, buffer was added in place of NTZ for MPP-treated samples. Values indicated are the mean S.E. of triplicate the experiment was repeated two times. B, SK-N-SH cells were either pretreated with NTZ (1 M) for 1 h before incubation with MP eated for 1 h following 12 h incubation with MPP (1 mM). At the end of incubation MitoSOX staining was performed as de cedures” and fluorescence images were captured from five different fields of view. Nitazoxanide mitigates experimental parkinsonism Several lines of evidence implicate PD as a free radical dis- ease involving mitochondrial dysfunction leading to the pro- duction of free radicals and energy failure (10, 20, 21). Increased oxidative damage and aggregation of the protein -synuclein (-syn) were found to be characteristic hall- marks of PD (22). Several lines of evidence implicate PD as a free radical dis- ease involving mitochondrial dysfunction leading to the pro- duction of free radicals and energy failure (10, 20, 21). Increased oxidative damage and aggregation of the protein -synuclein (-syn) were found to be characteristic hall- marks of PD (22). chelators, AAV2-neurturin, creatine, and pioglitazone, but they were found to have no significant effect on disease progres- sion (24–27). In fact, we reported earlier that the mitochon- dria-targeted antioxidant MitoQ, as well as some of the lipo- philic iron chelators, confer significant protection in cellular or animal models of PD (13, 14). However, several of these obser- vations could not reach clinics because they are either toxic (26) or by the time the disease is evident, it was found to be too late to rescue the remaining dopamine neurons (28, 29). Currently, isradipine, caffeine, nicotine, inosine, glial cell- line derived neurotrophic factor, and -syn immunotherapy are being focused on or under clinical trials for the treatment of PD (27). In fact, various attempts have been made to understand the pathways leading to the aggregation of -syn, and inhibitors of -syn aggregation, active and passive immunization against -syn, are under focus (23). As oxidative stress resulting from mitochondrial damage happens to play a pivotal role in the pathophysiology of PD, numerous attempts have been made over the decades to find a cure employing antioxidants and iron J. Biol. Chem. (2017) 292(38) 15731–15743 15737 C, the average intensity units from five differe ependent experiments (n 8–10) were represented as mean S.D. , p 0.05 as compared with controls. ##, p 0.01; #, p PP group by two-way ANOVA with Sidak’s multiple comparisons test. Nitazoxanide mitigates experimental parkin Nitazoxanide mitigates experimental parkinsonism Nitazoxanide mitigates experimental parkinsonism igure 6. NTZ suppressed MPP-induced loss of mitochondrial membrane potential, ROS generation, and cellular apoptosis. Rat mesencephalic N27 ells were treated with NTZ (1 M) for 1 h before exposure to MPP (500 M) for 24 h. A, mitochondrial membrane potential was analyzed by JC-1 staining. B, OS production was determined by H2DCF-DA as described in “Experimental Procedures.” C, caspase-3 activity in cells treated with MPP in the presence and bsence of NTZ (1, 5, and 10 M). D, DPPH radical scavenging activity of NTZ and vitamin C was employed as a positive control. A and B, data are the epresentative of three independent experiments. C and D, values are the mean S.E. of triplicate measurements from two independent experiments (n 6). , p 0.001; , p 0.01 as compared with controls. ###, p 0.001 as compared with MPP group. For (C) two-way ANOVA with Sidak’s test and for (D) ne-way ANOVA with Dunnett’s test was performed. Nitazoxanide mitigates experimental parkinsonism Figure 6. NTZ suppressed MPP-induced loss of mitochondrial membrane potential, ROS generation, and cellular apoptosis. Rat mesencephalic N27 cells were treated with NTZ (1 M) for 1 h before exposure to MPP (500 M) for 24 h. A, mitochondrial membrane potential was analyzed by JC-1 staining. B, ROS production was determined by H2DCF-DA as described in “Experimental Procedures.” C, caspase-3 activity in cells treated with MPP in the presence and absence of NTZ (1, 5, and 10 M). D, DPPH radical scavenging activity of NTZ and vitamin C was employed as a positive control. A and B, data are the representative of three independent experiments. C and D, values are the mean S.E. of triplicate measurements from two independent experiments (n 6). , p 0.001; , p 0.01 as compared with controls. ###, p 0.001 as compared with MPP group. For (C) two-way ANOVA with Sidak’s test and for (D) one-way ANOVA with Dunnett’s test was performed. Recently, more attention has been paid to understanding the role of mitochondrial uncoupling proteins (UCPs) as their activity was found to have a profound influence on mitochon- drial biogenesis, calcium flux, free radical production, and neu- ronal function. In fact, UCP-4A was found to play a protective role in PD models (30–32). F, locomotor activity of animals as measured by open of experiment (n 4). D and E, data are the mean S.E. (n 3). F, values indicated are the mean S.E. from four animals (n 4). *, p as compared with controls. ###, p 0.001 as compared with MPTP 7D group. $$, p 0.01; $, p 0.05 as compared with MPTP 14D group -way ANOVA with Tukey’s multiple comparisons test. Figure 7. NTZ mitigates MPTP-induced loss of TH neurons and locomotor activity in vivo. Effects of NTZ on MPTP-induced loss of dopamine neurons as measured by TH-positive staining in the SNPc and striatum of mouse brain in an acute mouse model of PD. A, schematic representation of in vivo experiment depicting the therapeutic and preventive models employed. B and C, TH-positive staining and TH-positive fiber density in the SNPc and striatum of mice. D and E, densitometric analysis of TH-positive staining from SNPc and striatum from three different mice (n 3). F, locomotor activity of animals as measured by open field test at the end of experiment (n 4). D and E, data are the mean S.E. (n 3). F, values indicated are the mean S.E. from four animals (n 4). , p 0.001; *, p 0.01 as compared with controls. ###, p 0.001 as compared with MPTP 7D group. $$, p 0.01; $, p 0.05 as compared with MPTP 14D group as analyzed by two-way ANOVA with Tukey’s multiple comparisons test. case as treatment of cells with GTP or carboxyatractyloside (CAT) could not prevent NTZ-induced OCR (Fig. 4, C and D). Treatment of cells with the parkinsonian mimetic MPP resulted in a gradual decrease in the mitochondrial respiration and by the end of 2 h there was hardly any noticeable OCR in MPP-treated cells. However, the mitochondria were in a highly coupled state at this stage as addition of FCCP induced a rapid increase in OCR in MPP-treated cells. It has been well established that higher levels of ROS accumulate in mitochon- dria following perturbation of electron transport chain in a OCR and noticed that NTZ dose-dependently enhanced OCR as measured by Seahorse flux analyzer in real time, but not the individual scaffolds (Fig. 1). Based on the above observations, attempts have been made to examine the protective role of pharmacological small molecule uncouplers in neurodegenera- tive disease processes. Earlier reports indicate that uncouplers, such as FCCP, 2,4-dinitrophenol (DNP), were found to be toxic and this was attributed to their plausible off-target effects and narrow therapeutic index (33, 34). However, the liver-targeted form of DNP viz. DNP methyl ether was found to protect against fatty liver and hyperglycemia because of its wide thera- peutic index (35). Also, low concentrations of DNP were found to protect from cerebral ischemia as well as amyloid- peptide– induced toxicity by scavenging mitochondrial free radicals (36, 37). Because of the increasing importance of uncouplers in therapeutic research, new molecules are being synthesized that are less toxic (34, 35). In line with the above observations, repurposing of FDA- approved drugs appears to be a safer option for PD patients in terms of both safety as well as cost effectiveness, provided they can confer significant protection. Recently, the respiration-en- hancing properties of FDA-approved drugs have been assessed and nitazoxanide was among them (15). It was interesting to find that NTZ happens to be one of the safest and most bioavail- able drugs that can reach the CNS. Oral doses of 4 g in healthy adults did not induce any significant adverse effects, and in animals the LD50 was found to be more than 10 g/kg (oral) (16, 17). Although NTZ was designed to inhibit pyruvate-ferre- doxin oxidoreductase (PFOR) under anaerobic conditions, it was also found to enhance mitochondrial respiration in mam- malian cells in recent studies (15, 38). Prompted by these initial observations, we have examined the effect of NTZ and some of its core functional groups, such as ANT and OAS, on cellular 15738 J. Biol. Chem. (2017) 292(38) 15731–15743 Nitazoxanide mitigates experimental parkinsonism Nitazoxanide mitigates experimental parkinsonism gates MPTP-induced loss of TH neurons and locomotor activity in vivo. Effects of NTZ on MPTP-induced loss of dopamine neurons as ositive staining in the SNPc and striatum of mouse brain in an acute mouse model of PD. A, schematic representation of in vivo experiment peutic and preventive models employed. B and C, TH-positive staining and TH-positive fiber density in the SNPc and striatum of mice. D and alysis of TH-positive staining from SNPc and striatum from three different mice (n 3). NTZ-induced OCR is not specific to individual respiratory complexes, but appears to exhibit this effect on the overall mitochondrial respiration (Fig. 2). Also, NTZ-induced OCR was associated with an increase in the pro- ton leak with a concomitant decrease in ATP production and these effects were found to be reversible (Figs. 3D and 4, A and B). The observed uncoupling effects of NTZ do not appear to be mediated through either UCPs or adenine nucleotide translo- J. Biol. Chem. (2017) 292(38) 15731–15743 15739 Mito Stress assay Mitochondrialbioenergeticsemployingwholecellswasmea- sured in a Seahorse XF24 extracellular flux analyzer. SK-N-SH cells (30,000/well) were plated in 24-well Seahorse assay plate a day before the experiment. Before taking the readings, media were replaced with assay medium and cells in 24-well assay plate were incubated in a non-CO2 incubator for 1 h as per the manufacturer’s protocol (Seahorse Bioscience). The four ports of the plate were filled with either NTZ (1–5 M, final concen- tration) or MPP (100 M, final concentration), oligomycin (1 M, final concentration), FCCP (1 M, final concentration), rotenone antimycin A (2 M each, final concentration), respectively. Oxygen consumption rate (OCR) was recorded after the addition of contents from each port for a period of 20 min to 2 h as per the design of experiment. At the end of exper- iment, cells were lysed using 0.1 NaOH and the protein content was measured by Bradford assay (Sigma). The recordings were normalized to the total cellular protein content in each well. A survey of the literature at this juncture indicates that uncouplers of mitochondrial respiration not only mitigate mitochondrial free radicals but, during this process, also acti- vate cellular autophagy (39, 40). These properties appear to be the most sought after ones for any anti-parkinsonism drug as they might clear the buildup of abnormal protein aggregates and improve the cellular respiration. In line with the above observations, it has been reported recently that NTZ enhances autophagy (41), and we also observed similar results (data not shown). Although a recent study indicated that preformed -synuclein aggregates could not be cleared merely by inducing autophagy, it was surmised that enhanced autophagic flux could prevent accumulation of protein aggregates in cells (42). Hence, the mild uncoupling effects of NTZ in conjunction with its autophagy-enhancing property might confer potential ther- apeutic benefit to counter PD. In other experiments, basal OCR was monitored for 20 min and then NTZ or its individual chemical moieties, such as O-acetyl salicylamide (OAS) and 2-amino-5-nitrothiazole (ANT), were added directly through port A, whereas control cells were treated with equal volumes of buffer and OCR was monitored further for 2 h. Overall, the present study demonstrates the ability of NTZ in mitigating MPP-induced biochemical abnormalities under both in vitro and in vivo conditions by preventing the buildup of toxic mitochondrial ROS. Cell culture The human SK-N-SH and rat N27 dopaminergic cells were grown in Dulbecco’s modified Eagle’s medium and RPMI 1640 (containing 10% fetal bovine serum, 2 mmol/liter L-glutamine, 100 units/ml penicillin, and 100 units/ml streptomycin), respectively, at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. For cell culture experiments, after attaining 70–80% confluence and before 12 h of treatments, the cell cul- ture media were replaced with 2% serum-containing media. Unless otherwise mentioned, cells were pretreated with NTZ 60 min before exposure to MPP for indicated time intervals. Materials Nitazoxanide; MPP iodide; MPTP hydrochloride; oligomy- cin; FCCP; rotenone; antimycin A (AA); pyruvate; malate; suc- cinate; ascorbate; TMPD; ATP; and the dyes, JC-1, carboxya- tractyloside (CAT), GTP, and carboxyl H2-DCF-DA, were purchased from Sigma. 2-Amino-5-nitrothiazole (ANT) was from HiMedia (India). O-acetyl-salicylamide was a generous gift from Dr. Chinaraju Bhimapaka from CSIR–Indian Institute of Chemical Technology (India). Luciferase-based assay kit for ATP measurement was from Sigma. Nitazoxanide mitigates experimental parkinsonism tightly coupled mitochondria (33). However, by allowing pro- tons into the mitochondrial matrix uncouplers play a pivotal role in neutralizing mitochondrial ROS and essentially we found the same in cells treated with NTZ and MPP. NTZ per se increased OCR because of its mild uncoupling effect with a negligible reduction in ATP levels at 1 M concentration; how- ever, it could significantly relieve the block in OCR and increase ATP production in MPP-treated cells (Fig. 3). Also, treatment of cells with NTZ significantly reversed MPP-induced loss in cellular ATP levels by 24 h (Fig. 3D). Nevertheless, no signifi- cant cell death was observed even at the highest concentrations of NTZ employed for up to 24 h (data not shown). NTZ instan- taneously enhanced the mitochondrial OCR in cells that were pretreated with MPP for 2 h, wherein, there was a complete block in respiration implying that NTZ could restore respira- tion in partially damaged mitochondria (Fig. 5A). In fact, the mitochondrial ROS induced by MPP over a period of time could be nullified soon after the addition of NTZ (Fig. 5, B and C), indicating that it has good therapeutic potential. As symp- toms of PD are apparent following a significant loss of dop- amine neurons of SNPc, compounds that can energize the existing cellular mitochondria and quench ROS might hold greater therapeutic potential in terms of delaying the disease progression. Also, NTZ prevented the loss of mitochondrial membrane potential, accumulation of ROS, as well as apoptosis induced by MPP over a 24-h time period (Fig. 6, A–C) possibly because of its mild uncoupling effect as it did not possess any antioxidant activity per se (Fig. 6D). Oral administration of NTZ conferred significant protection in an acute MPTP mouse model of PD against the loss of TH-positive neurons of SNPc and locomotor activity in both preventive and therapeutic models (Fig. 7, A–F). effects of NTZ, if any, needs to be carefully examined to take the present findings further. Nitazoxanide mitigates experimental parkinsonism concentration) was added to MPP treated cells. In parallel, control and MPP treatments alone were added with equal volumes of buffer and OCR was monitored further for 50 min. To examine the reversible nature of NTZ-mediated effects, cells were pretreated with NTZ (1 M) for 1 h. Before placing the plate into the flux analyzer, medium containing NTZ was replaced with fresh assay medium in some wells and the plate was incubated in a non-CO2 incubator for additional 1 h and Mito Stress assay was performed as described above. concentration) was added to MPP treated cells. In parallel, control and MPP treatments alone were added with equal volumes of buffer and OCR was monitored further for 50 min. JC-1 staining The membrane-permeable dye JC-1 was used to assess the changes in mitochondrial membrane potential in N27 cells as described earlier (14). Briefly, following the termination of incubation, cells were washed twice with Dulbecco’s phos- phate-buffered saline (DPBS) and treated with JC-1 at a final concentration of 5 g/ml in fresh serum-free culture medium and then incubated at 37 °C for 30 min. Later, cells were gently washed with DPBS and fixed with 4% paraformaldehyde and the fluorescence images were captured using Olympus fluores- cence microscope under FITC and rhodamine filter settings. To examine the reversible nature of NTZ-mediated effects, cells were pretreated with NTZ (1 M) for 1 h. Before placing the plate into the flux analyzer, medium containing NTZ was replaced with fresh assay medium in some wells and the plate was incubated in a non-CO2 incubator for additional 1 h and Mito Stress assay was performed as described above. Mito Stress assay NTZ also improved mitochondrial OCR and nullified ROS in cells that were preexposed to MPP, indicating that it may have disease-delaying effects. Because NTZ is an FDA-approved drug with a very good safety profile and has the ability to reach the CNS, it appears to be a promis- ing candidate for the treatment of PD. However, the chronic To examine the ability of NTZ in reversing MPP-induced block in mitochondrial respiration, cells in 24-well Seahorse plate were treated directly with MPP (100 M final concentra- tion) through port A, whereas control cells received equal vol- umes of buffer in place of MPP. Following 2 h of treatment with continuous monitoring of OCR, NTZ (1 and 5 M final 15740 15740 J. Biol. Chem. (2017) 292(38) 15731–15743 ATP measurement by luminescence Cellular ATP levels were measured using ATP biolumines- cent somatic cell assay kit according to the manufacturer’s pro- tocol (Sigma). Briefly, following the termination of incubation, cells in 6-well plates were collected by trypsinization and 50 l of cell suspension was added to 150 l of 1.5 ATP releasing reagent (Sigma). The samples were mixed thoroughly and the emitted luminescence was measured immediately in a micro- plate reader (EnSpire, PerkinElmer). Electron flow assay SK-N-SH cells (30,000/well) were plated in 24-well Seahorse assay plate. Before initiating the experiment, the complete media was replaced with Mitochondrial Assay Solution (1: 70 mM sucrose, 220 mM mannitol, 10 mM KH2PO4, 5 mM MgCl2, 2 mM HEPES, 1.0 mM EGTA, and 0.2% (w/v) fatty acid–free BSA, pH 7.4 at 37 °C) and XF PMP reagent as recommended by the manufacturer (Seahorse Bioscience). Briefly, pyruvate (10 mM) and malate (1 mM) were used as substrates along with ADP (4 mM). Final concentrations of the compounds that were injected through respective ports into each well of 24-well plate are as follows: (a) rotenone (2 M), (b) succinate (10 mM), (c) antimy- cin A (2 M), (d) ascorbate (10 mM) TMPD (0.5 mM). Initially, pyruvate- and malate-induced OCR was measured for 10 min, rotenone was added through port A and the drop in OCR was recorded for 10 min. Later, succinate was added through port B and OCR was recorded for 10 min for succinate-induced OCR. Similarly, antimycin A was added through port C and the drop in OCR was recorded for 10 min. Lastly, ascorbate and TMPD were added through port D to measure the complex-IV (cyto- chrome C oxidase)–induced OCR. ROS detection Intracellular ROS generation was monitored using the fluo- rogenic probe 6-carboxy-2,7-dichlorofluorescein diacetate (carboxy H2DCF-DA) (Sigma) as described previously (13). The mitochondrial superoxide was measured using MitoSOX dye as per the manufacturer’s protocol (Invitrogen). Briefly, following the termination of incubation, medium was aspirated and N27 cells were washed twice with DPBS. Fresh serum-free culture medium containing 10 M carboxy H2DCF-DA or MitoSOX was added and the cells were incubated further for 30 min at 37 °C in a humidified cell culture incubator. Later, the medium was aspirated, cells were washed three times with ice cold DPBS and fixed using 4% paraformaldehyde. The presence of ROS in cells was measured by the increase in the intensity of DCF fluo- rescence and MitoSOX red fluorescence under FITC and rho- damine filter settings, respectively, using an Olympus fluores- cence microscope from at least five different fields of view. ANT and UCP inhibition assay SK-N-SH cells (30,000/well) were plated and grown in 24-well Seahorse analyzer plate for 24 h. Just before the initia- tion of assay, cells were washed twice with 1 Mitochondrial Assay Solution (MAS) buffer and permeabilized with 1.0 nM XF PMP reagent (Seahorse Bioscience) and simultaneously cells were supplemented with pyruvate (10 mM), malate (1 mM), and ADP (4 mM). OCR was recorded for 20 min before the addition of GTP (4 mM) or CAT (3 g/ml) through port A, whereas control cells were treated with equal volumes of buffer through port A. OCR was recorded further for 20 min and cells were treated with either NTZ or FCCP through port B (control cells were treated with equal volumes of buffer) and the changes in OCR were recorded for an additional 25 min before terminat- ing the assay. DPPH-antioxidant assay The antioxidant activity of NTZ was analyzed using DPPH (2, 2-diphenyl-1-picrylhydrazyl) assay as described earlier (43). Briefly, different concentrations of compounds in 2 l were added to the reaction mixture containing methanolic solution of DPPH (100 M, 98 l) in 96-well plate. The plate was incu- bated in dark for 30 min and absorbance was measured at 520 nm using a microplate reader (EnSpire, PerkinElmer). Caspase-3 activity assay The increase in fluorescence because of the release of 7- amino-4-trifluoromethylcoumarin (AFC) from the tetra pep- tide substrate, acetyl-DEVD-AFC was considered as a measure of caspase-3 activity and was performed as described previously using N27 cells (13). Increase in fluorescence because of the release of AFC (excitation 400 nm and emission 505 nm) was monitored every 5 min for up to 1 h at 37 °C in a microplate reader (EnSpire, PerkinElmer). The obtained fluorescence val- ues were normalized to the total protein content. In vivo experiments Male C57BL-6J mice (6–8 weeks), weighing 22–25 g were divided into six groups of six animals in each group: (a) control, (b) MPTP (7 days), (c) MPTP (14 days), (d) NTZ, (e) NTZ MPTP (pretreatment with NTZ, preventive model), and (f) MPTP NTZ (posttreatment with NTZ, therapeutic model). Nitazoxanide mitigates experimental parkinsonism Acute dosage of MPTP was given to mice (20 mg/kg, four injec- tions at 2-h intervals) intraperitoneally as described earlier (44). For preventive model, a day before MPTP injection, NTZ was administered orally (50 mg/kg) and continued daily for 7 days post MPTP treatment. To study the therapeutic effects, NTZ (50 mg/kg) was administered on the 8th day following MPTP treatment and the dosage was continued up to the 14th day. Following the termination of experiment, animals in each group were perfused (whole body perfusion) using 4% parafor- maldehyde in phosphate buffered saline. 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https://openalex.org/W4295777544	https://javs.journals.ekb.eg/article_258579_04909724bc81b9a754cd3c844e9e1965.pdf	English	null	Assessment of the Knowledge, Attitude and Practices of Dog Owners on Rabies in Ilorin, Kwara State, Nigeria	Journal of Applied Veterinary Sciences/Journal of Applied Veterinary Sciences	2,022	cc-by	5,612	Journal of Applied Veterinary Sciences, 7 (4): 81-87 (October, 2022) ISSN: Online: 2090-3308, Print: 1687-4072 Journal of Applied Veterinary Sciences, 7 (4): 81-87 (October, 2022) ISSN: Online: 2090-3308, Print: 1687-4072 Journal of Applied Veterinary Sciences, 7 (4): 81-87 (October, 2022) ISSN: Online: 2090-3308, Print: 1687-4072 Journal homepage : https://javs.journals.ekb.eg Assessment of the Knowledge, Attitude and Practices of Dog Owners on Rabies in Ilorin, Kwara State, Nigeria Aiyedun, J. O. 1, Darajat, A. T.1, Daodu, B. O.2, Oludairo, O. O.1, Olorunshola, I. D.2, Nwoha, R. I. O.3 and Oluboyede, I. S.1 1Department of Veterinary Public Health and Preventive Medicine, University of Ilorin, Nigeria 2Department of Veterinary Microbiology, University of Ilorin, Nigeria 3Department of Veterinary Medicine, Micheal Okpara University of Agriculture, Umudike, Nigeria Corresponding Author: Oludairo, O. O., E-Mail: oludairo@hotmail.com : +234 7032131111 Original Article: Sci., 7(4 ): 81-87. Keywords: Attitude, Dog owners, Knowledge, Practices, Rabies. Original Article: Rabies is an acute viral zoonotic disease that affects all warm-blooded animals. Its constant evaluation of the perception of the public is essential. This study assessed the knowledge, attitude and practices of dog owners on rabies in Ilorin, Kwara State, Nigeria. This was achieved by administering 150 structured questionnaires to dog owners, breeders and hunters in the study area using face to face interviews. The data collected were analysed using SPSS version 21. Dog owners with more than 3 dogs (13.3%) and those with one dog (50.7%) had the highest (80%) and least (47.4%) rabies vaccination rates respectively. Dogs kept for both breeding and security (18%) were vaccinated more than those kept for (74.1%) other reasons. Lhasa apso and local breeds were the highest (100%) and least (14.3%) vaccinated dogs in the study. Dogs < 1 year (30.7%) and those >6 years (0.7%) recorded (65.2%) and (0%) current rabies vaccination status respectively, while 90% and 72.6% of the respondents knew about rabies and its transmission to man respectively, only 58.7% had updated rabies vaccination for their dogs. Although 56% of the dog owners in this study rightly indicated how rabies is transmitted, 16% and 64.7% didn’t know how humans contract the disease and the clinical presentations in dogs respectively. Most (35.6%) respondents that didn’t have up to date vaccinations for their dogs didn’t have cogent reasons for failing to do so. Dog owners that were not knowledgeable about pre-exposure rabies vaccination were 58.7% while only 7.3 % of them had received previous shots of the vaccine. Dog bite experience was recorded in 78% of the respondents while only 20.7% of the 82.7% that claimed to know what to do when bitten by a dog identified the right course of action. Most (69.3%) respondents however indicated they would seek medical help at the government hospital (40%) if bitten by a rabid dog. The result of this study showed that there is some knowledge gap in the knowledge of rabies among stakeholders in the study area. Periodic mass rabies awareness campaigns to improve the perception of the disease in the study area is indicated. Received :04 August, 2022. Accepted :08 September, 2022. Published in October, 2022 s s r w e ) e e d % o ) t e e n J. Appl. Vet. Sci., 7(4 ): 81-87. J. Appl. Vet. Sci., 7(4 ): 81-87. J. Appl. Vet. ABSTRACT Original Article: DOI: HTTPS://DX.DOI.ORG/10.21608/JA VS.2022.154332.1168 Received :04 August, 2022. Accepted :08 September, 2022. Published in October, 2022 This is an open access article under the term of the Creative Commons Attribution 4.0 (CC-BY) International License . To view a copy of this license, visit: http://creativecommons.org/licenses/by/4.0/ Original Article: DOI: HTTPS://DX.DOI.ORG/10.21608/JA VS.2022.154332.1168 Received :04 August, 2022. Accepted :08 September, 2022. Published in October, 2022 This is an open access article under the term of the Creative Commons Attribution 4.0 (CC-BY) International License . To view a copy of this license, visit: http://creativecommons.org/licenses/by/4.0/ Prevalence of Salmonella in Suya in Ilorin: Prevalence of Salmonella in Suya in Ilorin: Out of 102 samples of Suya that were collected from the fifteen (15) locations in Ilorin, Kwara state, Nigeria, 5 (4.9%) were positive for Salmonella. The organism was isolated from four Suya stands and their prevalence were 1 (20%), 1 (20%), 1 (20%), and 2 (40%) for Tipper Garage/Unilorin PS, Zamfara Hostel, Sabo-Oke and Sabo-Oke/Maraba Junction respectively (Table 1) This neglected tropical disease is underfunded, underdiagnosed and underreported in Nigeria (Ehizibolo et al., 2011; Ojo et al., 2016; Elelu et al., 2019). To reduce the incidence of dog-bites and prevent dog-mediated human rabies, it is essential to assess the awareness level and further educate the general public. This has proved effective in changing the risk perception of the populace and resulted in attitudinal changes that were necessary to reduce the burden of rabies (Edukugho et al., 2018). Challenges such as poor awareness and knowledge of rabies, lack of political will, shortage of vaccines and other vaccine-related factors had led to the endemicity of rabies and significant under-reporting of rabies in Nigeria (Ajayi et al, 2006). This study evaluated the knowledge, attitude and practices of dog owners on rabies in Ilorin, Nigeria. Table 1: Socio-demographic characteristics of the dog owners: Feature Freq. (%) Age of respondents (years) 6-15 4 (2.7) 16-25 49 (32.7) 26-35 51 (34.0) 36-45 24 (16.0) 46-55 8 (5.3) >55 14 (9.3) Gender Male 109 (72.7) Female 41 (27.3) Educational level No formal education 1 (0.7) Primary 4 (2.7) Secondary 28 (18.7) Degree/ HND 95 (63.3) Post graduate 22 (14.7) Marital status Single 79 (52.7) Married 70 (46.7) Divorce 1 (0.7) Occupation Civil servant 48 (32.0) Private worker 51 (34.0) Hunter 19 (12.7) Student 32 (21.3) Religion Christianity 77 (51.3) Islam 71 (47.3) No religion 2 (1.3) Assessment of the Knowledge, Attitude and Practices of ………. veterinary hospitals/clinics, wildlife rehabilitation facilities and animal shelters (Aiyedun et al., 2017). According to the Advisory Committee on Immunization Practices, the importance of receiving this vaccination, particularly rabies pre-exposure vaccination, is more important in endemic nations (Daodu et al., 2017). veterinary hospitals/clinics, wildlife rehabilitation facilities and animal shelters (Aiyedun et al., 2017). According to the Advisory Committee on Immunization Practices, the importance of receiving this vaccination, particularly rabies pre-exposure vaccination, is more important in endemic nations (Daodu et al., 2017). Study location and participants selection The study was conducted in urban, semi urban and rural communities spread across four local government areas (Ilorin South, Ilorin west, Ilorin East and Moro Local government areas) of Kwara State. Only dog owners and managers were selected to participate in the study. Statistical Analysis Data obtained were manually entered into a Microsoft Excel and imported into Statistical Package for Social Science (SPSS) software v. 21 for analysis. Descriptive and inferential statistical analysis was performed. Descriptive analysis was presented in frequency and percentage while inferential test was conducted using Fisher’s exact test to compare association. Test of association was identified as significant when p value is <0.05. The disease occurs regularly in the dog accounting for about 94% of the confirmed diagnosed cases. Reported cases of rabies in humans in Nigeria are low; this could be attributed to poor reporting of cases, cultural beliefs, mis-diagnosis of the disease and poor knowledge on the mode of transmission and prevention of the disease (Chernet and Nejash, 2016, Awoyomi et al., 2021). INTRODUCTION Edukugho et al., 2018). The most prevalent mode of infection is through the bite of a rabid animal, however other modes of transmission such as organ transplantation and aerosol are also possible (WHO, 2013, OIE, 2017). Rabies is a vaccine-preventable, viral, zoonotic disease that affects mammals in various parts of the world (Daodu and Oluwayelu, 2016). Rabies is caused by rabies virus, a neurotropic organism belonging to the family Rhabdoviridae and genus Lyssavirus (WHO, 2016). Rabies causes fatal encephalomyelitis and results in death if not attended to (Lembo et al., 2006, Daodu and Oluwayelu, 2016). Mammals such as jackal, fox, wolf, skunk, raccoon, bat and dog act as reservoir-hosts of rabies virus and transmit it to other animals (Aiyedun et al., 2017, Preventive vaccination against rabies virus is a very effective method for preventing the disease (Adedeji et al., 2010; Aiyedun et al., 2021). Pre and post-exposure vaccinations against the virus have existed for a long period of time to protect humans, particularly those who are occupationally exposed, such as those who work in rabies laboratories, 81 Assessment of the Knowledge, Attitude and Practices of ………. Aiyedun, J. O., et al……… Aiyedun, J. O., et al……… The results showed that 50.7% (76/150) of the respondents owned a dog each while only 13.3% (20/150) had more than three dogs. (Table 2). Dog owners with more than three dogs had the highest rabies vaccination rate (80.0%, 16/20) while the least occurred among owners with one dog (47.4%, 36/76) (Table 2). People keeping more than one dog were two to four times more likely to have their dogs vaccinated with anti-rabies as compared with those who had just one (p≤ 0.033) (Table 2). Dogs were mainly kept for security (52%, 78/150), breeding/security (18.0%, 27/150) and breeding purposes only (15.3%, 23/150), although some respondents still kept them for hunting (6.7%, 10/150) and as pet animals (4.6%, 7/150). Table 2: Socio-demographic characteristics of dogs owned by respondents: Table 2: Socio-demographic characteristics of dogs owned by respondents: Features Freq (%) No. of vaccinated dogs (%) OR (95%CI) P-value No of dogs owned 1 76 (50.7) 36 (47.4) 1 2-3 54 (36) 36 (66.7) 2.2 (1.1 – 4.6) 0.0330 >3 20 (13.3) 16 (80.0) 4.4 (1.4 – 14.5) 0.0114* Purpose for keeping dog Security 78 (52.0) 50 (64.1) 16.1 (1.9 – 133.6) 0.0015* Breeding 23 (15.3) 17 (73.9) 25.5 (2.6 – 246.0) 0.0015* Breeding and security 27 (18) 20 (74.1) 25.7 (2.7 – 241.2) 0.0007* Hunting 10 (6.7) 1 (10.0) 1 Pet 7 (4.6) 0 (0.0) 0.4 (0.0 – 11.9) 1.0000 Others 5 (3.3) 0 (0.0) 0.6 (0.0 – 16.7) 1.0000 Breed of dog(s) German shepherd 60 (40.0) 38 (63.3) 10.4 (3.5 -30.6) <0.0001* Lhasa apso 6 (4.0) 6 (100.0) 72.1 (3.5 – 1473) 0.0001* Rottweiler 21 (14.0) 17 (81.0) 25.5 (6.0 – 108.0) < 0.0001* Local breed 35 (23.30) 5 (14.3) 1 Caucasian 8 (5.3) 6 (75.0) 18.0 (2.8 – 115.6) 0.0017* Boerboel 7 (4.7) 7 (100.0) 83.2 (4.1 – 1677) < 0.0001* Dobberman 2 (1.3) 1 (50.0) 6.0 (0.3 – 112.3) 0.3018 English eskimo 8 (5.3) 6 (75.0) 18.0 (2.8 – 115.6) 0.0017* Others (foreign breed) 3 (2.0) 2 (100.0) 12.0 (0.9 – 158.5) 0.0813 Age of dog <1 46 (30.7) 30 (65.2) 6.9 (1.7 – 23.3) 0.0056* 1-3 89 (59.3) 55 (61.8) 5.9 (1.5 – 22.8) 0.0075* 4-6 14 (9.3) 3 (21.4) 1 >6 1 (0.7) 0 (0.0) 1.1 (0.0 – 33.4) 1.0000 How did you get the dog(s)? Aiyedun, J. O., et al……… Gift 32 (21.3) 21 (65.6) 1.4 (0.6 – 3.2) 0.5424 Bought 116 (77.3) 67 (57.8) 1 Found 2 (1.3) 0 (0.0) 0.15 (0.0 – 3.1) 0.1467 Key: Freq- Frequency OR- Odds ratio 95%CI- 95% confidence interval *Significant at p<0.005 Freq (%) No. of vaccinated dogs (%) OR (95%CI) P-value However, dogs kept for breeding/security purpose (74.1%, 20/27) had the highest rabies vaccination rank followed by breeding dogs (73.9%, 17/150) and security dogs (64.1%, 50/150). The rabies vaccination of dogs kept for breeding, security or both had higher significant difference when compared with those kept for hunting purposes (p≤ 0.0015). Among the dog breeds kept by dog owners, German shepherd was highest (40.0, 60/150) followed by indigenous (local) breeds (23.3%, 35/150). However, it was observed that foreign breeds of dogs such as lhasa apso, boerboel, rottweiler, caucasian and english eskimo had ≥75% current rabies vaccination coverage while the least vaccinated breed are indigenous local dogs (14.3%, 5/35). Our analysis further indicated high significant differences when current rabies vaccination in lhasa apso, boerboel, rottweiler, caucasian and english eskimo and german shepherd breed of dogs were compared individually to that in Nigerian local dogs (p≤ 0.0017) (Table 2). Most (59.3%, 89/150) of the dogs kept by the dog owners were within 1- 3 years old. However, dogs <1 year old had the highest (65.2%, 30/46) current rabies vaccination while dogs >6 years old were not vaccinated. Univariate analysis further indicated high significant differences in rabies vaccination of dogs aged <1 year old and 1-3 years old as compared individually with those that were within 4-6 years of age (p≤0.0075) (Table 2). Most of the dogs kept by the study participants were mostly bought (77.3%, 116/150) or given as gift (21.3%, 32/150) but some were stray dogs that were adopted (1.3%, 2/150). Additionally, we However, dogs kept for breeding/security purpose (74.1%, 20/27) had the highest rabies vaccination rank followed by breeding dogs (73.9%, 17/150) and security dogs (64.1%, 50/150). The rabies vaccination of dogs kept for breeding, security or both had higher significant difference when compared with those kept for hunting purposes (p≤ 0.0015). Among the dog breeds kept by dog owners, German shepherd was highest (40.0, 60/150) followed by indigenous (local) breeds (23.3%, 35/150). Questionnaire A pre-tested structured questionnaire was administered to dog owners through face-to-face interview between October, 2021 to February, 2022. The interview was conducted at University of Ilorin Veterinary Teaching Hospital, Aromokeye Veterinary Clinic, Kwara State Zonal Veterinary Clinic and Sabo- Oke areas of Ilorin. Additionally, questionnaire was administered to hunters who use dogs for hunting in the semi urban and rural communities of Kwara State. The questionnaire assessed knowledge, attitude and practise of dog owners in relation to vaccination of their dogs against rabies and other vaccine preventable diseases 82 Assessment of the Knowledge, Attitude and Practices of ………. observed that dogs given as gift had highest current rabies vaccination rank (65.6%, 21/150) followed by those purchased (57.8%, 67/116) while none of the adopted dogs were vaccinated against rabies (Table 2). observed that dogs given as gift had highest current rabies vaccination rank (65.6%, 21/150) followed by those purchased (57.8%, 67/116) while none of the adopted dogs were vaccinated against rabies (Table 2). Although a large proportion of dog owners had heard about rabies (90.0%, 135/150) and its possible transmission to humans (72.6%, 109/150), only 58.7% (55/150) of the dog owners had updated rabies vaccination for their dogs (Table 3). Table 3: Assessment of knowledge on Rabies Features Freq. (%) Have you heard of rabies? Yes 135 (90.0) No 15 (10.0) Do you know rabies can be transmitted from dogs to humans? Yes 109 (72.6) No 41 (27.3) Do you know it can be transmitted by Bite 84 (56.0) Scratch 21 (14.0) Open wound 6 (4.0) Bite and scratch 2 (1.3) Bite and open wound 5 (3.3) Bite, scratch and open wound 8 (5.3) I don’t know 24 (16.0) Do you know how rabies is presented in an infected dog? Yes 53 (35.3) No 97 (64.7) Are you aware of rabies vaccination in dogs? Yes 119 (79.3) No 31 (20.7) Is your dog’s anti-rabies vaccination current (within one year)? Yes 88 (58.7) No 62 (41.3) a Why haven’t you vaccinated your dog (s)? I have no reason 21 (35.6) Did not know about it 19 (32.2) Expensive 6 (10.1) Vaccination point is too far 5 (8.5) I don’t know any veterinarian 4 (6.8) Couldn’t catch the dog 4 (6.8) b Are you aware of the zoonotic danger posed by a non-vaccinated dog? Yes 22 (37.3) No 37 (62.7) Do you know about human vaccination against rabies? Yes 62 (41.3) No 88 (58.7) Have you been vaccinated against rabies? Assessment of the Knowledge, Attitude and Practices of ………. Yes 11 (7.3) No 51 (92.7) Key: a This question was a follow up of preceding question b Calculation was based on only those who responded Although several dog owners were aware that rabies can be transmitted to humans from infected dogs through bite (56%, 84/150), scratch (14%, 21/150) and open wound (4%, 6/150) however, 16.0% (24/150) of the dog owners didn’t know how humans can contract it while 64.7% (97/150) of them didn’t know clinical t ti f bid d Additi ll th k l d f i ti f d i t bi hi h (79 3% g Features Freq. (%) Have you heard of rabies? Yes 135 (90.0) No 15 (10.0) Do you know rabies can be transmitted from dogs to humans? Yes 109 (72.6) No 41 (27.3) Do you know it can be transmitted by Bite 84 (56.0) Scratch 21 (14.0) Open wound 6 (4.0) Bite and scratch 2 (1.3) Bite and open wound 5 (3.3) Bite, scratch and open wound 8 (5.3) I don’t know 24 (16.0) Do you know how rabies is presented in an infected dog? Yes 53 (35.3) No 97 (64.7) Are you aware of rabies vaccination in dogs? Yes 119 (79.3) No 31 (20.7) Is your dog’s anti-rabies vaccination current (within one year)? Yes 88 (58.7) No 62 (41.3) a Why haven’t you vaccinated your dog (s)? I have no reason 21 (35.6) Did not know about it 19 (32.2) Expensive 6 (10.1) Vaccination point is too far 5 (8.5) I don’t know any veterinarian 4 (6.8) Couldn’t catch the dog 4 (6.8) b Are you aware of the zoonotic danger posed by a non-vaccinated dog? Yes 22 (37.3) No 37 (62.7) Do you know about human vaccination against rabies? Yes 62 (41.3) No 88 (58.7) Have you been vaccinated against rabies? Aiyedun, J. O., et al……… However, it was observed that foreign breeds of dogs such as lhasa apso, boerboel, rottweiler, caucasian and english eskimo had ≥75% current rabies vaccination coverage while the least vaccinated breed are indigenous local dogs (14.3%, 5/35). Our analysis further indicated high significant differences when current rabies vaccination in lhasa apso, boerboel, rottweiler, caucasian and english eskimo and german shepherd breed of dogs were compared individually to that in Nigerian local dogs (p≤ 0.0017) (Table 2). Most (59.3%, 89/150) of the dogs kept by the dog owners were within 1- 3 years old. However, dogs <1 year old had the highest (65.2%, 30/46) current rabies vaccination while dogs >6 years old were not vaccinated. Univariate analysis further indicated high significant differences in rabies vaccination of dogs aged <1 year old and 1-3 years old as compared individually with those that were within 4-6 years of age (p≤0.0075) (Table 2). Most of the dogs kept by the study participants were mostly bought (77.3%, 116/150) or given as gift (21.3%, 32/150) but some were stray dogs that were adopted (1.3%, 2/150). Additionally, we 83 Aiyedun, J. O., et al……… the respondents claimed they didn’t know about the vaccination. However, several other responses included high cost of vaccination (10.1%) and far distance of veterinary clinic (8.5%, 5/59). Generally, more than half of the dog owners (58.7%, 88/150) were not knowledgeable about human pre-exposure rabies vaccination while only 7.3% (11/62) of those who knew about it had received the vaccination shot (Table 3). the respondents claimed they didn’t know about the vaccination. However, several other responses included high cost of vaccination (10.1%) and far distance of veterinary clinic (8.5%, 5/59). Generally, more than half of the dog owners (58.7%, 88/150) were not knowledgeable about human pre-exposure rabies vaccination while only 7.3% (11/62) of those who knew about it had received the vaccination shot (Table 3). We recorded that, 22.0% (33/150) of the respondents had prior experience of dog bite (Table 4). Although 82.7% (124/150) of dog owners claimed to know what to do when bitten by dog, only few (20.7%, 21/150) identified the right course of action to take. However, 69.3% (104/150) would seek for medical help at the government hospital (40%, 60/150) if bitten by dogs infected with rabies (Table 4). Table 4: Attitude of dog owners towards dog bite Features Freq. (%) Have you ever been bitten by a dog? Yes 33 (22.0) No 117 (78.0) Do you know the steps to take if bitten by a dog? Yes 124 (82.7) No 26 (17.3) If you were bitten by dog, what would you do? Wash wound with soap and water 31 (20.7) Wash wound with soap and water and painkillers 3 (2.0) Wash wound with soap and water and antibiotics 10 (6.7) Wash wound with soap and water and traditional medicine 4 (2.3) Bandage the injury 10 (6.7) Use painkillers 10 (6.7) Traditional medicine 20 (13.3) Antibiotic 20 (13.3) Traditional med only 7 (4.7) Don’t do anything 35 (23.3) If you were bitten by dog, would you seek medical help? Yes 104 (69.3) No 46 (30.7) aIf yes, which facility would you seek help from? Private clinic 34 (22.7) Government hospital 60 (440.0) Chemist 3 (2.0) Pharmacy 3 (2.) Vet hospital I don’t know 4 (2.7) 46 (30.7) Key: a it is a follow up question to the preceding question Table 4: Attitude of dog owners towards dog bite Assessment of the Knowledge, Attitude and Practices of ………. Yes 11 (7.3) No 51 (92.7) Key: a This question was a follow up of preceding question b Calculation was based on only responded Although several dog owners were aware that rabies can be transmitted to humans from in through bite (56% 84/150) scratch (14% 21/150) and open wound (4% 6/150) however 16 0% (24/ Although several dog owners were aware that rabies can be transmitted to humans from infected dogs through bite (56%, 84/150), scratch (14%, 21/150) and open wound (4%, 6/150) however, 16.0% (24/150) of the dog owners didn’t know how humans can contract it while 64.7% (97/150) of them didn’t know clinical presentations of a rabid dog. Additionally, the knowledge of vaccination of dog against rabies was high (79.3%, 119/150), yet only 58.7% (88/150) had up to date vaccination for their dogs. Dog owners without up-to-date rabies vaccination of their dogs claimed no reason for not having done so (35.6%, 21/59) while 32.2% (19/59) of Although several dog owners were aware that rabies can be transmitted to humans from infected dogs through bite (56%, 84/150), scratch (14%, 21/150) and open wound (4%, 6/150) however, 16.0% (24/150) of the dog owners didn’t know how humans can contract it while 64.7% (97/150) of them didn’t know clinical presentations of a rabid dog. Additionally, the knowledge of vaccination of dog against rabies was high (79.3%, 119/150), yet only 58.7% (88/150) had up to date vaccination for their dogs. Dog owners without up-to-date rabies vaccination of their dogs claimed no reason for not having done so (35.6%, 21/59) while 32.2% (19/59) of 84 CONCLUSION vulnerable members of the society, especially members with little or no formal education (Lapiz, et al., 2012). Educated members of the society are may have better access to information on rabies on electronic/print media and internet. Similar findings were reported in studies conducted by Awosanya, et al., 2013 and Ameh, et al., (2014) in other cities in Nigeria who reported reasonable level of rabies awareness among their respondents because they were quite educated. vulnerable members of the society, especially members with little or no formal education (Lapiz, et al., 2012). Educated members of the society are may have better access to information on rabies on electronic/print media and internet. Similar findings were reported in studies conducted by Awosanya, et al., 2013 and Ameh, et al., (2014) in other cities in Nigeria who reported reasonable level of rabies awareness among their respondents because they were quite educated. From the demographic analysis of respondents in the study area, single, self-employed, youth/middle aged males were the major dog owners mostly raising a single dog. The highest compliance to rabies vaccination were recorded in respondents that raised more than 3 dogs, dogs kept for breeding/security purposes, those purchased by respondents and not given as gifts , dogs < 1 year of age and exotic breeds like lhasa apso. Good general knowledge was recorded among respondents in knowing about the name of the disease, its transmission to man and route of transmission, a good number of them did not know how humans contract the disease and its clinical presentation in dogs. Many respondents did not have up to date vaccination for their dogs and were not able to give very cogent reasons for failure of vaccination of their dogs considering the deadly nature of the disease. Most of the respondents were also not knowledgeable about human pre-exposure rabies vaccination therefore very few had received previous shots. Majority of the respondents had never had dog bite experience prior to the time of this study. Only about one fifth of those that claimed to know what to do in case of dog bite actually identified the right course of action although a good number of the interviewed dog owners indicated they will seek immediate medical help in government hospitals in case of dog bite. CONCLUSION The reason more dog owners in this study were single compared to those that are married could be the need for companionship (Jackson, 2016). This corroborates the study by Edukugbo, et al., (2018) who reported that the non-married were about three times more likely to raise dogs and have positive attitude towards rabies prevention compared to married dog owners. Dog owners that were private workers and self- employed are expected to have more time to cater for dogs and manage them better. The reasons Christians were the majority that kept dogs in the study area may be religious belief which may not encourage some of the Islamic religion to keep dogs. The low level of vaccination in hunting dogs when compared to dogs kept for security and breeding purposes could be because most hunters are not educated with little or no knowledge of rabies. Respondents that had more than one dog had higher vaccination coverage most likely due to the fact that people that had many dogs were mainly dog breeders who used the dogs for business and therefore, take better care of their dogs in addition to ensuring their vaccination status are up to date to prevent the dogs coming down with diseases including rabies. Local breeds had low level of rabies vaccination compared to exotic breeds of dogs because local dogs are usually kept for hunting purposes in the study area and were owned by dog owners who had low level of rabies vaccination awareness. Those that bought their dogs and also with higher number of vaccinated dogs compared to respondents who got their dogs as gifts because they paid money in getting the dogs which may make them to value the dogs and take greater precautions in other not to lose the them. This study had identified gap in the knowledge about rabies among dog owners, breeders and hunters in the study area. Government and other stakeholders should make provisions for annual rabies awareness campaigns through relevant agencies and professionals and mass media in order to educate dog owners on the transmission, prevention and steps to take when bitten by a dog. Free mass anti-rabies vaccination should be done regularly by government and non-governmental organisations aimed at achieving at least 70% of the dog population (vaccination coverage) necessary for maintaining the required level of herd immunity in the study area. DISCUSSION that male respondents were more likely to admit owning dog than female respondents (Alfayez et al., 2003). This could be because the responsibility of provision of security in most communities in Nigeria lies more with males than females in addition to the fact that males are also involved in game hunting than the females (Awoyomi, et al., 2019). The varying degree of knowledge of respondents/dog owners in this study shows that the age had influence on the level of knowledge of rabies; this was corroborated by Ameh, et al, (2014) who reported that owners within the age group 20-30 years were more likely to have better knowledge of rabies compared to older respondents. This may be because this age group are youthful and may be more likely to show more interest in dogs and by extension their management. The level of education of dog owners/respondents that is directly related to their knowledge of rabies may imply that the risk and burden of the development rabies in man and animals may likely be among the least educated/most Having more male respondent dog owners in the study is in agreement with other studies that found 85 Assessment of the Knowledge, Attitude and Practices of ………. Conflict of interest The authors acknowledge that there is no conflict of interest regarding the research idea and tools, actual, potential and financial, directly or indirectly. Greater number of respondents that had heard of rabies had higher number of vaccinated dogs as a result of their knowledge on rabies. Dog owners who had heard of rabies were more likely to vaccinate their dogs against the disease than those who did not. This is in agreement with Awuni, et al.,(2019). Majority of the dog owners paid for vaccination and quite a number of them paid more than three thousand naira (N3000=) per dog annually to ensure that their dogs are protected from rabies. Aiyedun, J. O., et al……… Aiyedun, J. O., et al……… EHIZIBOLO, O., EHIZIBOLO, P. O., EHIZIBOLO, E., SUGUN, M. Y., and IDACHABA, S. E., 2011. The control of neglected zoonotic diseases in Nigeria through animal intervention: an overview. African Journal of Biomedical Research, 14(2):81–88. Advanced Veterinary and Animal Research, 4 (2); 117- 124. http://doi.org/10.5455/javar.2017.d198. AIYEDUN, J. O., OLUBOYEDE, I. S., DAODU, O. B., OLUDAIRO O. O., OLORUNSHOLA, I. D., and DARAJAT, A. T., 2021. Rabies immune status of vaccinated and unvaccinated dogs in Kwara State, Nigeria. Journal of Sustainable Veterinary and Allied Sciences, 2 (1): 14- 19.https://doi.org/10.54328/covm.josvas.2022.066. ELELU, N., AIYEDUN, J. O., ODETOKUN, I. A., MOHAMMED, I. G., OLUDAIRO, O. O., MOHAMMED, K. M., BALE, J. O., and NURU, S., 2019. Neglected zoonotic diseases in Nigeria: role of public health veterinarian. Pan Africa Medical Journal, 32 (36); 1-12. DOI: 10.11604/pamj.2019.32.36.15659. AIYEDUN, J. O., OLUDAIRO, O. O., OLORUNSHOLA, I. D., and OKOLI, C. P., 2017. Strategies for control, prevention and elimination of urban rabies in West-Africa. Assiut Veterinary Medical Journal, 63 (153); 237-241. DOI: 10.21608/AVMJ.2017.170676. JACKSON, A. C. 2016. Rabies virus. The Curated Reference Collection in Neuroscience and Biobehavioral Psychology, 1027–1030. https://doi.org/10.1016/B978-0-12-809324-5.03909-2 AJAYI, B. B., RABO, J. S., and BABA, S. S., 2006. Rabies in apparently healthy dogs histological and immunohistochemical studies. The Nigerian Postgraduate Medical Journal, 13(2):128-134. LAPIZ, S. M., MIRANDIA, M. E., GARCIA, R. G., DAGURO, L .I., PAMEN, M. D., MADRINAN, F. P., RANCES, P. A., and BRIGGS, D. J., 2012. Implementation of an intersectoral programme to eliminate human and canine rabies: The Bohol rabies prevention and elimination project. PLos Neglected Tropical Diseases, 6(12): e1891 doi: 10.1371/journal.pntd.0001891. AL-FAYEZ, G., AWADALLA, A. TEMPLER, D. I. AND ARIKAWA, H. (2003). Companion animal attitude and its family pattern in Kuwait. Society and Animals, 11(1):17-28 AMEH, V. O., DZIKWI, A. A., and UMOH, J. U., 2014. Assessment of knowledge, attitude and practice of dog owners to canine rabies in Wukari metropolis, Taraba State, Nigeria. Global Journal of Health Science, 6(5), 226–240. https://doi.org/10.5539/gjhs.v6n5p226 LEMBO, T., NIEZGODA, M., VELASCO-VILLA, A., CLEAVELAND, S., ERNEST, E., and RUPPRECHT, C. E., 2006. Evaluation of a direct, rapid immunohistochemical test for rabies diagnosis. Emerging Infectious Diseases, 12: 310-313. doi: 10.3201/eid1202.050812. AWOSANYA, A. E., and ADEBIMPE, A. P., 2013. Factors associated with rabies awareness and attitude to dog bite in a university community. Bulletin of Animal Health and Production, Afr 2013;6:559-70. OFFICE INTERNATIONAL DES EPIZOOTIES (OIE), 2017. Rabies portal. http://www.oie.int/en/animal- health- in-the-world/rabies-portal/. REFERENCES ADEDEJI, A. O., OKONKO, I. O., EYAREFE, O. D., ADEDEJI, O. B., BABALOLA, E. T., and OJEZELE, M. O., 2010. An overview of rabies - History, epidemiology, control and possible elimination. African Journal of Microbiology Research, 4(22), 2327–2338. AIYEDUN J. O., OLUDAIRO, O. O., and OLORUNSHOLA, I. D., 2017. Roles of wildlife in epidemiology of Rabies: A review. Journal of 86 Aiyedun, J. O., et al……… Retrieved on 27-07-2022. OJO, D., NWADIKE, V., ONYEDIBE, K., KALU, I., and OJIDE, K., 2016. Rabies in Nigeria: A review of literature. African Journal of Clinical and Experimental Microbiology, 17(2), 159.https://doi.org/10.4314/ajcem.v17i2.12. AWOYOMI, J., BANKOLE, N. O., OLUGBENGA, K., and ADEBOWALE, O., 2021. Assessment of Dog Owners’ Knowledge on Dog Rabies Vaccination in Rural Communities in Ogun State, Nigeria. Nigerian Veterinary Journal. 40. 278-286. 10.4314/nvj.v40i4.3. WORLD HEALTH ORGANIZATION (WHO), 2013. Rabies, countries or areas at risk. WHO Rabnet/CDC (Data source) and WHO Public Health Information and Geographic Information Systems (Map production). World Health Organization, Avenue Appia 20, 1211 Geneva 27, Switzerland. AWUNI, B., TARKANG, E., MANU, E., AMU, H., AYANORE, M. A., AKU, F. Y., and KWEKU, M., 2019. Dog Owners’ Knowledge about Rabies and Other Factors That Influence Canine Anti-Rabies Vaccination in the Upper East Region of Ghana. Tropical Medicine and Infectious Disease, 4(3), 115. https://doi.org/10.3390/TROPICALMED4030115 WORLD HEALTH ORGANIZATION (WHO), 2016. Rabies Factsheet. Geneva, Switzerland: WHO; 2016. Available from:http://www.who.int/mediacentre/factsheets/ fs099/en/. p g CHERNET, B., and NEJASH, A., 2016. Review of rabies preventions and control. International Journal of Life Science, 4(2): 293-301. DAODU, O. B., and OLUWAYELU, D., 2016. Rabies knowledge and pre-exposure vaccination status of clinical veterinary medicine students in Nigeria. Vom Journal of Veterinary Science, 11: 117-125. How to cite this article: Aiyedun, J. O. , Darajat, A. T., Daodu, B. O., Oludairo, O. O., Olorunshola, I. D., Nwoha, R. I. O., and Oluboyede, I. S., 2022. Assessment of the Knowledge, Attitude and Practices of Dog Owners on Rabies in Ilorin, Kwara State, Nigeria. Journal of Applied Veterinary Sciences, 7 (4): 81– 86. DOI: HTTPS://DX.DOI.ORG/10.21608/JAVS.2022.154332.1168 DAODU, O. B., DAODU, O. C., and OLORUNSHOLA, I. D., 2017. Rabies Vaccination Status among Occupationally Exposed Humans in Nigeria. Nigerian Veterinary Journal, 38. 272-279. EDUKUGHO, A. A., UMOH, J. U., DIEM, M., AJANI, O., UBA, B., OKEKE, L., ADEDIRE, E., ADEFISOYE, A., EDUKUGHO, C., and NGUKU, P., 2018. Knowledge, attitudes and practices towards rabies prevention among residents of Abuja municipal area council, Federal Capital Territory, Nigeria. The Pan African Medical Journal, 31, 21. https://doi.org/10.11604/pamj.2018.31.21.15120. 87
https://openalex.org/W2594496604	https://zenodo.org/records/1129868/files/10006809.pdf	English	null	Application Procedure for Optimized Placement of Buckling Restrained Braces in Reinforced Concrete Building Structures	Zenodo (CERN European Organization for Nuclear Research)	2,017	cc-by	3,704	I. INTRODUCTION C o R C building structures are designed to satisfy a certain level of earthquake resistant requirement. However, it is concerned that, for quite severe earthquake columns, beams or slabs are damaged, and seismic story drift responses exceed the allowable limits [1]. To solve this problem, a variety of vibration control systems have been developed such as shear wall, base isolation, or dampers. Considering the case of seismic retrofitting, BRBs is a superior system to the other shear resisting devices from the point of view of construction and cost. BRB is expected to dissipate a great amount of energy and to reduce the seismic responses. R A lot of research works have been conducted about using BRB in newly-built or existing building structures. As for the fabrication of BRB, optimal length of BRB steel core is formulated analytically and tested experimentally [2]. On the other hand, there are quite few research works about practical BRB design method compared to the other types of damper systems. A simple and practical optimal placement procedure was proposed to find the optimal stiffness for BRBs to minimize the seismic story responses (drift) of the structure [11]. This procedure continuously traces the most effective placements of BRB by increasing total BRB stiffness from zero to a specified value based on nonlinear time-history analysis considering nonlinearity of BRB directly. This method provides stiffness of BRBs of each story as continuous value depending on the step length of optimization process. A lot of methods have been proposed to determine linear viscous damper placement for building structures and these methods can be directly extended to linear brace placement. A steel brace optimization procedure is proposed based on transfer function of linear model and two different rehabilitation techniques, steel brace and viscous damper, are compared [3]. A topology optimization method is applied to obtain optimal topology of the bracing system of structures with linear material under harmonic loads [4]. In order to construct a non-linear damper design method, most of the In a practical design procedure, it is required to select steel members from specified standard section lists. Therefore, in this paper, a simple rule is derived to convert continuous horizontal stiffness of BRBs obtained from the previous method into standard sectional sizes of BRB. World Academy of Science, Engineering and Technology International Journal of Civil and Environmental Engineering Vol:11, No:4, 2017 World Academy of Science, Engineering and Technology International Journal of Civil and Environmental Engineering Vol:11, No:4, 2017 I. INTRODUCTION In this paper, a new practical method is proposed to determine the sectional sizes of BRBs from the result of the previously proposed method for BRB placement optimization in RC building structures. International Science Index, Civil and Environmental Engineering Vol:11, No:4, 2017 waset.org/Public Keywords—Buckling restrained brace, building engineering, optimal damper placement, structural engineering. S. A. Faizi, Master Student, is with the Ritsumeikan University, Shiga, CO 525-8577 Japan (phone: +8170-1749-4391; e-mail: shabirfaizi@gmail.com/ gr0258ei@fc.ritsumei.ac.jp). S. Yoshitomi Ph.D., Professor, is with the Ritsumeikan University, Shiga, 525-8577 Japan (e-mail: yositomi@fc.ritsumei.ac.jp). S. A. Faizi, Master Student, is with the Ritsumeikan University, Shiga, CO 525-8577 Japan (phone: +8170-1749-4391; e-mail: shabirfaizi@gmail.com/ gr0258ei@fc.ritsumei.ac.jp). S. Yoshitomi Ph.D., Professor, is with the Ritsumeikan University, Shiga, 525-8577 Japan (e-mail: yositomi@fc.ritsumei.ac.jp). Application Procedure for Optimized Placement of Buckling Restrained Braces in Reinforced Concrete Building Structures S. A. Faizi, S. Yoshitomi previous methods use linearization or simplification. An efficient procedure to determine optimal placement of nonlinear hysteretic dampers has been proposed based on design response spectrum and equivalent linearization of hysteretic model [5]. Abstract—The optimal design procedure of buckling restrained braces (BRBs) in reinforced concrete (RC) building structures can provide the distribution of horizontal stiffness of BRBs at each story, which minimizes story drift response of the structure under the constraint of specified total stiffness of BRBs. In this paper, a simple rule is proposed to convert continuous horizontal stiffness of BRBs into sectional sizes of BRB which are available from standardized section list assuming realistic structural design stage. 11, No:4, 2017 waset.org/Publication/10006809 As for the design method of BRBs, a seismic retrofit design method is considered for RC buildings with elastic steel frame and BRBs in order to evaluate the amount of BRB using a simplified equivalent linearization method [6]. A simplified formulation is suggested to determine BRB ductility and strength for steel frames equipped with BRBs considering design spectra of Eurocode 8 [7]. Sensitivity analysis method has been proposed to investigate the relation between seismic performances and brace over-strength distributions as a tool for safer design of BRBs [8]. On the other hand, several methods have been suggested to consider the nonlinearity of dampers directly. A practical method is recommended for optimum design of the non-linear oil dampers with relief mechanism installed in multi-story framed building structures based on a sensitivity analysis by using nonlinear time-history response analysis [9]. A systematic methodology was proposed for determining the optimal cross sectional area of BRBs on the seismic upgrading of existing structures using genetic algorithm [10]. II. TARGET MODEL WITHOUT BRB The target model is the same as the model used in [11]. The model, as shown in Fig. 1, has been modeled in ETABS. This scholar.waset.org/1307-6892/10006809 International Scholarly and Scientific Research & Innovation 11(4) 2017 414 World Academy of Science, Engineering and Technology International Journal of Civil and Environmental Engineering Vol:11, No:4, 2017 World Academy of Science, Engineering and Technology International Journal of Civil and Environmental Engineering Vol:11, No:4, 2017 (a) Linear direct integration, time history analysis) (a) Linear direct integration, time history analysis) (b) Nonlinear direct integration, time history analysis) Fig 2 Story responses before optimization by MATLAB model is containing five typical stories, and the structure material is RC. The height of each story is typically 3.6 m. Young’s modulus of concrete for the RC structure is 2.094174x104 MPa. The damping ratio of the structure is assumed to be as ξ (h) =0.05 which means 5% damping [10]. A. Optimal BRB Placement Problem In this chapter, the previously proposed BRB optimization procedure is introduced. The optimal BRB placement problem is how to determine the adequate BRB stiffness in order to minimize the maximum response of the structure. BRB optimization problem is defined in three equations. onal Science Index, Civil and Environmental Engineering Vol:11, No:4, 2017 waset.org/Publication/10006809 Find X=(k1 BRB, ….kN BRB) (1) To minimize f(X)=max௜ߜ௜௠௔௫ (2) Subject to ∑݇௜ ஻ோ஻ ௜ =ܭഥ (3) (1) (2) (3) (1) International Science Index, Civil and Environmental Engineering Vol:11, No:4, 2017 waset.org/Publication/10006809 (a) Linear direct integration, time history analysis) (a) Linear direct integration, time history analysis) (b) Nonlinear direct integration, time history analysis) (2) (3) International Science Index, Civil and Environmental Engineering Vol:11, No:4, 2017 waset.org/Publicat where ܭഥ and ݇௜ ஻ோ஻ indicate total BRB stiffness and BRB stiffness of i-th story, and ߜ௜௠௔௫ indicates the maximum story drift response of the i-th story [10]. Fig. 1 RC Building 3D-view TABLE I STORY MASS AND STORY STIFFNESS Story Mass (kg) Stiffness (N) Stiffness (N/m) 1 626430 6143179.77 226315592 2 624705 6126263.31 148560662 3 624705 6126263.31 138974375 4 624705 6126263.31 131338900 5 632842 6206060.02 130239362 International Science Index, Civil and Environmental Engineering Vol:11, No:4, 2017 (b) Nonlinear direct integration, time history analysis) 415 International Scholarly and Scientific Research & Innovation 11(4) 2017 B. Optimization Procedure In order to solve the optimization problem described in the previous section, a simple algorithm is proposed in this section. This procedure continuously traces the most effective placements of BRB by increasing total BRB stiffness from zero. The proposed method is summarized in five steps. (1) Consider N-story shear building model and set the yield displacements of the structure and BRB and the incremental stiffness of the BRB ∆k. Fig. 1 RC Building 3D-view (2) Consider N candidate models. As for the i-th candidate model, BRB with stiffness ∆k is added to the i-th story of the model with optimal BRBs obtained in the previous optimization step. (3) Compute the time-history responses of the N candidate models in (2) for input ground acceleration and evaluate the maximum response as an objective function. the maximum response as an objective function. (4) Find the candidate model with the lowest objective function among the N candidate models. (5) Update the stiffness of BRBs and return to (2) until the total stiffness of BRBs reaches to the specified value. The most characteristic point of the proposed method is that 415 International Scholarly and Scientific Research & Innovation 11(4) 2017 scholar.waset.org/1307-6892/10006809 415 World Academy of Science, Engineering and Technology International Journal of Civil and Environmental Engineering Vol:11, No:4, 2017 World Academy of Science, Engineering and Technology International Journal of Civil and Environmental Engineering Vol:11, No:4, 2017 sectional area, we have to follow the procedures according to Fig. 4. this procedure needs only N candidate models at every optimization step which is equal to the number of the story. Fig. 3 shows the detailed sample of three optimization steps. At every optimization step, only five candidate models are compared with respect to the maximum drift response. In the first optimization step, each of the five candidate models has BRB with stiffness ∆݇ in the different story. In this case, candidate model 1 is selected as an optimal BRB placement at the first optimization step. In the second optimization step, each of the five candidate models has additive BRB with stiffness ∆݇ in the different story to the optimal BRB placement at 1st optimization step. In this case, candidate model 3 is selected as an optimal BRB placement among in the second optimization step. These steps are continued until we find the optimal placement [11]. B. Optimization Procedure (a) Normal model without Earthquake force) (b) Displaced model by earthquake force (c) BRB absorbing earthquake force Fig. 4 State of model of the structure before and during earthquake with BRB (a) Normal model without Earthquake force) (a) Normal model without Earthquake force) (a) Normal model without Earthquake force) ce Index, Civil and Environmental Engineering Vol:11, No:4, 2017 waset.org/Publication/10006809 (b) Displaced model by earthquake force Fig. 3 Schematic model for BRB placement onal Science Index, Civil and Environmental Engineering Vol:11, No:4, 2017 waset.org/Publication/100068 (b) Displaced model by earthquake force (c) BRB absorbing earthquake force (c) BRB absorbing earthquake force Fig. 4 State of model of the structure before and during earthquake with BRB Fig. 4 State of model of the structure before and during earthquake with BRB Fig. 4 State of model of the structure before and during earthquake with BRB Form Fig. 4 we can get K. Ԫ ൌ ௱௟ ௅ Ԫ ൌ ఋ௖௢௦ሺఏሻ ௅ ܰൌܧܣԪ ൌܧܣ ఋ௖௢௦ ሺఏሻ ௅ ܲൌܰܿ݋ݏሺߠሻൌ ா஺ఋ௖௢௦²ሺఏሻ ௅ ܭൌ ௉ ఋൌ ா஺௖௢௦²ሺఏሻ ௅ ൌ ா௔௕௖௢௦²ሺఏሻ ௅ Ԫ ൌ ఋ௖௢௦ሺఏሻ ௅ International Scholarly and Scientific Research & Innovation 11(4) 2017 C. Application of the Optimized Stiffness of the BRBs In a practical structural design stage, steel members are selected from standard section lists with specified section sizes. In a practical structural design stage, steel members are selected from standard section lists with specified section sizes. Therefore, in this section, a simple rule is derived to convert continuous horizontal stiffness of BRBs obtained by the previous method into standard sectional sizes of BRB. Therefore, in this section, a simple rule is derived to convert continuous horizontal stiffness of BRBs obtained by the previous method into standard sectional sizes of BRB. (1) Change the optimized stiffness of BRBs into the form of sectional area. The optimized stiffness of BRBs from the last step of the selected candidate has continuous value depending on the step length of optimization process. To change the optimized stiffness of BRBs into the form of where, Ԫ: Strain; ߂݈: Elongation of BRB; L: real length of BRB in normal model; ߜ: Displacement of the structure as drift; ߠ: Angle between BRB and beam; N: axial force of BRB; E=29000ksi: modulus of elasticity of the steel for BRB; scholar.waset.org/1307-6892/10006809 International Scholarly and Scientific Research & Innovation 11(4) 2017 416 World Academy of Science, Engineering and Technology International Journal of Civil and Environmental Engineering Vol:11, No:4, 2017 ܮൌඥሺ3.6ሻଶ൅ሺ6ሻଶൌ6.997݉ A=ab: cross sectional area of the steel for BRB with thickness a and width b; P: horizontal force (earthquake force); K: stiffness of a brace with specified sectional sizes. The stiffness of one BRB can be calculated as shown in Table III for a desired cross sectional area (A=1 cm x 1.2 cm=0.0012 m²). Here, Es=29000 ksi = 1.9995760x 1011 N/m². The optimized number of BRBs in each floor can be obtained as shown in Table IV by dividing optimized BRB stiffness in Table II by KBRB. These real numbers are rounded into integer numbers. The modified BRBs stiffness corresponding to the integer numbers is shown in Table IV. The stiffness of one BRB can be calculated as shown in Table III for a desired cross sectional area (A=1 cm x 1.2 cm=0.0012 m²). Here, Es=29000 ksi = 1.9995760x 1011 N/m². When the sectional sizes of BRB, i.e. a and b, are specified, the horizontal stiffness of one BRB (KBRB) can be evaluated from the formulation resulted in Fig. 4 as KBRB=Eൈaൈbൈcos2 (θ)/L. International Science Index, Civil and Environmental Engineering Vol:11, No:4, 2017 waset.org/Publication/10006809 mental Engineering Vol:11, No:4, 2017 waset.org/Publication/10006809 In the case that the number of BRBs does not have integer value, it should be rounded to integer number. In general, it is desirable to select the nearest integer number. (2) Compare the response of the structure into three cases that the second and third cases should have almost the same result. (2) Compare the response of the structure into three cases that the second and third cases should have almost the same result. Fig. 6 Dimensions of the braced frame  Case 1: model without BRBs.  Case 1: model without BRBs.  Case 1: model without BRBs.  Case 2: model with the optimized stiffness of the BRBs.  Case 3: model with the modified optimized stiffness of the BRBs corresponding to the integer number of the BRBs Case 3: model with the modified optimized stiffness of the BRBs corresponding to the integer number of the BRBs. BRBs corresponding to the integer number of the BRBs. C. Application of the Optimized Stiffness of the BRBs The necessary number of BRB of each story can be obtained by dividing the optimized BRB stiffness of each story by KBRB The optimized number of BRBs in each floor can be obtained as shown in Table IV by dividing optimized BRB stiffness in Table II by KBRB. These real numbers are rounded into integer numbers. The modified BRBs stiffness corresponding to the integer numbers is shown in Table IV. NBRBi=KBRBi/KBRB TABLE II OPTIMIZED STIFFNESS OF BRBS (LAST OPTIMIZATION STEP OF 250 STEPS) Story Optimized stiffness of BRBs (N/m) Stiffness of structure according to Table I (N/m) 1 35429824.62 226315592.45 2 116131091.81 148560662.30 3 90542885.14 138974375.92 4 53144736.93 131338900.74 5 196832359.00 130239362.10 Fig. 6 Dimensions of the braced frame TABLE II OPTIMIZED STIFFNESS OF BRBS (LAST OPTIMIZATION STEP OF 250 STEPS) Story Optimized stiffness of BRBs (N/m) Stiffness of structure according to Table I (N/m) 1 35429824.62 226315592.45 2 116131091.81 148560662.30 3 90542885.14 138974375.92 4 53144736.93 131338900.74 5 196832359.00 130239362.10 TABLE II OPTIMIZED STIFFNESS OF BRBS (LAST OPTIMIZATION STEP OF 250 STEPS) where, NBRBi is the number of BRB in i-th floor, KBRBi is the total stiffness needed for BRB in i-th floor from the last optimization step. e Index, Civil and Environmental Engineering Vol:11, No:4, 2017 waset.org/Publication/10006809 REFERENCES [1] Minimum Design Loads for Buildings and Other Structures, ASCE 7-05, chapter.12 [2] Mirtaheri. M., Gheidi, A., Zandi, A. P., Alanjari, P., and Samani, H. R.: Experimental optimization studies on steel core lengths in buckling restrained braces, Journal of Constructional Steel Research, ELSEVIER, Vol.67, 2011, pp.1244-1253. TABLE V SEISMIC STORY DRIFT RESPONSES OF THE STRUCTURE INTO THREE CASES Story Drift (deltamax) with BRB stiffness=0 Drift (deltamax) with BRB stiffness =Optimized Drift (deltamax) with BRB stiffness =Modified optimized M m m 1 0.014 0.024 0.022 2 0.029 0.025 0.025 3 0.028 0.025 0.026 4 0.026 0.024 0.024 5 0.012 0.005 0.005 Fig. 7 Seismic story drift responses of the structure into three cases (nonlinear direct integration, time history analysis by MATLAB) 0 2 4 6 0.000 0.010 0.020 0.030 Story Number Drift (m) Drift (deltamax) with BRB stiffness=0 Drift (deltamax) with BRB stiffness =Optimized Drift (deltamax) with BRB stiffness=Modified optimized nvironmental Engineering Vol:11, No:4, 2017 waset.org/Publication/10006809 TABLE V SEISMIC STORY DRIFT RESPONSES OF THE STRUCTURE INTO THREE CASES TABLE V [3] Aydin, E. and Boduroglu, M. H.: Optimal placement of steel diagonal braces for upgrading the seismic capacity of existing structures and its comparison with optimal dampers, Journal of Constructional Steel Research, ELSEVIER, Vol.64, 2008, pp.72-86. [4] Allahdadian, S., Boroomand, B., and Barekatein, A. R.: Towards optimal design of bracing system of multi-story structures under harmonic base excitation a topology optimization scheme, Finite Elements in Analysis and Design, ELSEVIER, Vol.61, 2012, pp.60-74. nce Index, Civil and Environmental Engineering Vol:11, No:4, 2017 waset.org/Publication/10006809 International Science Index, Civil and Environmental Engineering Vol:11, No:4, 2017 waset.org/Publication/10006809 [5] Martinez, C. A., Curadelli, O., and Compagnoni, M. E.: Optimal placement of nonlinear hysteretic dampers on planar structures under seismic excitation, Engineering Structures, ELSEVIER, Vol. 65, 2014, pp.89-98. [6] Sutcu, F., Takeuchi, T., and Matsui, R.: Seismic retrofit design method for RC buildings using buckling-restrained braces and steel frames, Journal of Constructional Steel Research, ELSEVIER, Vol.101, 2014, pp.304-313. [7] Bosco, M., Marino, E.M., and Rossi, P.P.: Design of steel frames equipped with BRBs in the framework of Eurocode8, Journal of Constructional Steel Research, ELSEVIER, Vol.113, 2015, pp.43-57. International Science Index, Civil and Environmental Engineering Vol:11, No:4, 2017 wa [8] Zona, A., Ragni, L., and Dall’Asta, A.: Sensitivity-based study of the influence of brace over-strength distributions on the seismic response of steel frames with BRBs, Engineering Structures, ELSEVIER, Vol.37, 2012, pp.179-192. IV. NUMERICAL EXAMPLES In this section, we are calculating the exact number of BRBs. Fig. 5 and Table II show the optimized placement of BRBs with their stiffness considering the last optimization step of 250 steps. Yielding displacements of building structure and BRB are set as ߜ௬௦=0.01m and ߜ௬ௗ=0.007m, respectively [11]. Fig. 6 Dimensions of the braced frame Fig. 5 Optimized stiffness of BRBs (last optimization step of 250 steps) International Science Index, Civil and Environmental TABLE III 1 KBRB CALCULATIONS, BRB AREA= (112) CM² Items Units Values One BRB area (A) m² 0.0012 Modulus of elasticity of the steel for BRB(E) N/m² 199957597569.9800 θ degree 30.9640 Length of BRB (L) m 6.9970 KBRB (one BRB’s stiffness) =AEcos²(θ)/L N/m 25205459.6337 TABLE IV MODIFIED OPTIMIZED STIFFNESS OF BRBS Story Total optimized number of BRBs =optimized stiffness of BRBs/1KBRB Selected exact number of BRBs Modified optimized stiffness of BRBs (N/m) =selected exact number of BRBs1KBRB 1 1.39 2.00 50821710.64 2 4.57 5.00 127054276.60 3 3.56 4.00 101643421.28 4 2.09 2.00 50821710.64 5 7.75 8.00 203286842.56 V. RESULT Table V and Fig. 7 show the comparisons of the maximum TABLE III 1 KBRB CALCULATIONS, BRB AREA= (112) CM² Items Units Values One BRB area (A) m² 0.0012 Modulus of elasticity of the steel for BRB(E) N/m² 199957597569.9800 θ degree 30.9640 Length of BRB (L) m 6.9970 KBRB (one BRB’s stiffness) =AEcos²(θ)/L N/m 25205459.6337 TABLE III 1 KBRB CALCULATIONS, BRB AREA= (112) CM² TABLE IV MODIFIED OPTIMIZED STIFFNESS OF BRBS MODIFIED OPTIMIZED STIFFNESS OF BRBS Story Total optimized number of BRBs =optimized stiffness of BRBs/1KBRB Selected exact number of BRBs Modified optimized stiffness of BRBs (N/m) =selected exact number of BRBs1KBRB 1 1.39 2.00 50821710.64 2 4.57 5.00 127054276.60 3 3.56 4.00 101643421.28 4 2.09 2.00 50821710.64 5 7.75 8.00 203286842.56 The θ and L can be known from the dimensions of Fig. 6 as: ߠൌܶܽ݊¯ ଵቀ ଷ.଺௠ ଺௠ቁൌ30.964° ܿ݋ݏଶൌ ଵ ଶ൅ ଵ ଶܿ݋ݏሺ2ߠሻൌ ଵ ଶ൅ ଵ ଶܿ݋ݏሺ61.928°ሻൌ ଵ ଶ൅ ଵ ଶ∗ 0.471 ൌ0.735 Table V and Fig. 7 show the comparisons of the maximum 41 International Scholarly and Scientific Research & Innovation 11(4) 2017 scholar.waset.org/1307-6892/10006809 417 World Academy of Science, Engineering and Technology International Journal of Civil and Environmental Engineering Vol:11, No:4, 2017 almost the same as responses of optimized stiffness of BRBs, and both of them can decrease the maximum responses effectively. story drifts among the three models, i.e. REFERENCES [9] Adachi, F., Yoshitomi, S., Tsuji, M., and Takewaki, I.: Nonlinear optimal oil damper in seismically controlled multi-story building frame, Soil Dynamics and Earthquake Engineering, ELSEVIER, Vol.44, 2013, pp.1-13. International Science Index, Civil and Environmental Engine [10] Farhat, F., Nakamura, S., and Takahashi, K.: Application of genetic algorithm to optimization of buckling restrained braces for seismic upgrading of existing structures, Computers and Structures, ELSEVIER, Vol.87, 2009, pp.110-119. Fig. 7 Seismic story drift responses of the structure into three cases (nonlinear direct integration, time history analysis by MATLAB) [11] S., A. Faizi. and S. Yoshitomi: Optimal placement of buckling restrained braces in reinforced concrete building structures, proceedings of JAEE annual meeting 2016. International Science Index, Civil and En IV. NUMERICAL EXAMPLES model without BRB, model with optimized BRB stiffness in Table II, and model with modified optimized BRB stiffness in Table IV. From Fig. 7 it can be understood that the responses due to modified optimized stiffness of BRBs are almost the same as responses of optimized stiffness of BRBs, and both of them can decrease the maximum responses effectively. scholar.waset.org/1307-6892/10006809 International Scholarly and Scientific Research & Innovation 11(4) 2017 VI. CONCLUSION A new practical procedure is proposed to determine the sectional sizes of BRBs from the result of the previously proposed method for BRB placement optimization in RC building structures, which minimizes the seismic response drifts, so three conclusions have been derived. (1) Considering the practical structural design stage, a formulation is derived to convert the optimized BRB stiffness with real number into the integer number of BRB with sectional sizes selected from standardized section list. (2) The validity of the proposed method is demonstrated through numerical examples of five storied building structures. (3) The comparisons of the maximum story drifts have been conducted among three models, i.e. model without BRB, model with optimized number of BRBs as real number and model with modified optimized number of BRBs as integer number. From the results, it is demonstrated that the responses due to modified optimized stiffness of BRBs are scholar.waset.org/1307-6892/10006809 International Scholarly and Scientific Research & Innovation 11(4) 2017 418
https://openalex.org/W2152838366	https://diposit.ub.edu/dspace/bitstream/2445/178136/1/620541.pdf	English	null	Processing of primary and secondary rewards: A quantitative meta-analysis and review of human functional neuroimaging studies	Neuroscience & biobehavioral reviews/Neuroscience and biobehavioral reviews	2,013	public-domain	17,481	Processing of primary and secondary rewards: a quantitative meta-analysis and review of human functional neuroimaging studies Guillaume Sescousse1,2, Xavier Caldú1,3, Bàrbara Segura1,4 and Jean-Claude Dreher1 1 Reward and decision making team, Cognitive Neuroscience Centre, CNRS, 67 Bd Pinel, 69675 Bron, France 2 Current address: Radboud University Nijmegen, Donders Institute for Brain, Cognition, and Behaviour, Centre for Cognitive Neuroimaging, 6500 HB, Nijmegen, The Netherlands 3 Current address: Department of Psychiatry and Clinical Psychobiology, Faculty of Psychology, University of Barcelona, Barcelona, Spain 4 Current address: Department of Psychiatry and Clinical Psychobiology, Faculty of Medicine, University of Barcelona, Barcelona, Spain 1 Reward and decision making team, Cognitive Neuroscience Centre, CNRS, 67 Bd Pinel, 69675 Bron, France 2 Current address: Radboud University Nijmegen, Donders Institute for Brain, Cognition, and Behaviour, Centre for Cognitive Neuroimaging, 6500 HB, Nijmegen, The Netherlands 3 Current address: Department of Psychiatry and Clinical Psychobiology, Faculty of Psychology, University of Barcelona, Barcelona, Spain 4 Current address: Department of Psychiatry and Clinical Psychobiology, Faculty of Medicine, University of Barcelona, Barcelona, Spain Corresponding author: Jean-Claude Dreher, PhD CNRS, Reward and decision-making team Cognitive Neuroscience Center 67 Bd Pinel, 69675 Bron France Email: dreher@isc.cnrs.fr Tel: +33 (0)4 37 91 12 38 Corresponding author: Jean-Claude Dreher, PhD CNRS, Reward and decision-making team Cognitive Neuroscience Center 67 Bd Pinel, 69675 Bron France Email: dreher@isc.cnrs.fr Tel: +33 (0)4 37 91 12 38 Corresponding author: Jean-Claude Dreher, PhD CNRS, Reward and decision-making team Cognitive Neuroscience Center 67 Bd Pinel, 69675 Bron France Email: dreher@isc.cnrs.fr Tel: +33 (0)4 37 91 12 38 1 Abstract One fundamental question concerning brain reward mechanisms is to determine how reward-related activity is influenced by the nature of rewards. Here, we review the neuroimaging literature and explicitly assess to what extent the representations of primary and secondary rewards overlap in the human brain. To achieve this goal, we performed an Activation Likelihood Estimation meta-analysis of 87 studies (1452 subjects) comparing the brain responses to monetary, erotic and food reward outcomes. Those three rewards robustly engaged a common brain network including the ventromedial prefrontal cortex, ventral striatum, amygdala, anterior insula and mediodorsal thalamus, although with some variations in the intensity and location of peak activity. Money-specific responses were further observed in the most anterior portion of the orbitofrontal cortex, supporting the idea that abstract secondary rewards are represented in evolutionary more recent brain regions. In contrast, food and erotic (i.e. primary) rewards were more strongly represented in the anterior insula, while erotic stimuli elicited particularly robust responses in the amygdala. Together, these results indicate that the computation of experienced reward value does not only recruit a core “reward system” but also reward type-dependent brain structures. Keywords: reward processing; meta-analysis; experienced value; reward outcome; monetary reward; food reward; erotic reward; fMRI; neuroimaging; ventral striatum; orbitofrontal cortex; anterior insula 2 2 1. Introduction Much of our daily life is driven by the prospect of rewards. A classical distinction concerns primary rewards -i.e. food, sex and shelter- and secondary rewards -such as money or power. In contrast to primary rewards which have an innate value and are essential for the maintenance of homeostasis and reproduction, secondary rewards are not directly related to survival and only gain value through learned association with lower-level rewards. A long-standing question is whether primary and secondary rewards are processed in common and/or distinct brain structures (Schultz, 2000). Due to their evolutionary differences, it is tempting to speculate that primary and secondary rewards may be represented in phylogenetically distinct brain regions (Knutson and Bossaerts, 2007), but the evidence remains scarce. In contrast, animal research provides empirical evidence for a centralized processing of reward in the brain, based on a wealth of studies showing that a core set of brain regions -including the ventral tegmental area, nucleus accumbens, amygdala and ventromedial prefrontal cortex- are sensitive to various types of rewards (Berridge, 2003; Hikosaka et al., 2008; Schultz, 2006). However, studies in animals use a very limited number of primary rewards (e.g. food and juice), which hinders the generalizability of the results to more abstract secondary rewards. Furthermore, investigation techniques such as electrophysiology and focal brain lesions used in animals typically focus on specific brain structures and do not offer a “full picture” at the brain system level. Neuroimaging appears as an ideal tool to overcome these limitations, since it allows to visualize cerebral activity throughout the whole brain, and can be easily used in humans in order to compare secondary versus primary rewards. Building on this opportunity, recent studies in decision neuroscience and neuroeconomics have investigated how the brain represents reward value while deciding between different goods. Such a choice is assumed to rely on the computation of a “decision value”, allowing the comparison of various reward prospects on a common scale. Several studies have demonstrated that this computation is performed in the ventromedial prefrontal 3 cortex (vmPFC) and ventral striatum regardless of the goods at stake, providing compelling evidence in favour of a “common reward currency” (Chib et al., 2009; Hare et al., 2008; Knutson et al., 2007; FitzGerald et al., 2009; Plassmann et al., 2007; see also Peters and Buchel, 2010 for a review). 1. Introduction Yet, these neuroeconomic approaches focus on subjective value as inferred from choices, and only a handful of neuroimaging studies have made similar direct comparisons concerning the “experienced value” of rewards, computed at the time of outcome (Izuma et al., 2008; Rademacher et al., 2010; Sescousse et al., 2010; Smith et al., 2010; Grabenhorst et al., 2010a). Moreover, although the ventral striatum and vmPFC tend to emerge as prime candidates for computing experienced values, the results of these studies are rather heterogeneous and general conclusions are difficult to draw. Alternatively, insights can be gleaned from reviews synthesizing the results of single reward studies. Several of them have been published in the past years, and concur on including the striatum, dopaminergic midbrain, amygdala and orbitofrontal cortex in a “common reward circuit”. However, most of these reviews are qualitative and hence suffer from some degree of subjectivity: only evidence supporting the involvement of the above regions is presented, and is not critically weighed against all the studies not reporting similar evidence (Haber and Knutson, 2010; O'Doherty, 2004; McClure et al., 2004b). Addressing this issue, a few quantitative meta-analyses published in recent years have provided a more objective overview of the human neuroimaging literature. However, those meta-analyses were either pooling different reward types together (Liu et al., 2011; Cauda et al., 2011; Kuhn and Gallinat, 2012) or focusing on a single reward type (e.g. monetary rewards: Knutson and Greer, 2008; erotic rewards: Kuhn and Gallinat, 2011; Stoleru et al., 2012). To deepen our understanding of the functional architecture of reward processing in the brain, it is crucial to evaluate the consistency and specificity of reward-related activations across different types of rewards. To achieve this goal, we propose to perform an activation likelihood estimation (ALE) meta-analysis of the neuroimaging literature on reward processing. This approach has several features which make it well-suited to address some of the previous concerns (Laird et al., 2005; Wager et al., 2009). First, ALE provides a 4 quantitative measure of cross-study consistency. In contrast to label-based reviews which start from anatomical labels and draw conclusions based on the clustering of these labels, ALE is a voxel-based approach which uses stereotaxic coordinates as input, performs analyses on their spatial distribution, and then only derives anatomical labels based on the resultant clusters. 1. Introduction This confers high objectivity to this method, which is immune to risks of incongruent or erroneous labelling among the selected studies. Moreover, ALE produces statistically defensible conclusions, by using numerical estimates (ALE statistics) and significance thresholds to measure the degree of agreement between studies. Importantly, ALE is able to make comparisons between meta-analyses, similar to contrasts between conditions in individual studies. This feature can be used to investigate specificity, e.g. by subtracting activations related to different reward types. Besides providing a synthetic overview of the literature, the use of a quantitative meta-analytic approach also has practical benefits. For instance, many studies restrict their analyses to regions of interest (ROIs), based on the alleged role of these regions in reward processing. However, this kind of assumption concerning structure-function relationships should be ideally based on an objective meta-analysis. Moreover, for functionally defined brain regions such as the ventral striatum or medial OFC, current label-based reviews (e.g. Delgado, 2007; Haber and Knutson, 2010; Noonan et al., 2012) do not provide average stereotaxic coordinates from which to derive ROIs. As a consequence, a common practice for anatomically constrained analyses is to build ROIs based on the coordinates extracted from a particular study, which by definition is not representative of the literature. A quantitative meta-analysis would bring a simple solution to these issues. To investigate the consistency and specificity of reward-related responses in the brain, we compared three types of reward outcomes: monetary gains, pleasant foods and visual erotic stimuli. These rewards were chosen because they are widely used by humans and are also the most studied in the neuroimaging literature, therefore ensuring a sufficient number of activation foci to conduct our meta-analyses. We first assessed the consistency of brain activations elicited by each reward independently, and we then investigated the 5 consistency and specificity of these meta-analytic results across the three selected rewards by performing conjunction and contrast analyses. Specific details regarding study selection and analytic procedures are provided below. 2.1. Selection of studies We conducted three independent searches in PubMed in order to identify fMRI and Positron Emission Tomography (PET) studies dealing with the processing of monetary, erotic and food reward outcomes. Each search used a combination of key words referring to the type of reward (i.e. [(”money” OR “monetary” OR “financial”) AND (“reward”)]; [(“food” OR “taste” OR “juice”) AND (“reward” OR “pleasant”)]; [(“erotic” OR “sexual”) AND (“stimuli”)]) and to the investigation technique (i.e. [“fMRI” OR “PET” OR “neuroimaging”]). These searches retrieved 242, 138 and 66 studies, respectively (July 2010). The lists of references cited in these studies were also scrutinized and relevant studies incorporated to our pool. One unpublished study from our laboratory, manipulating food and erotic rewards, was further added (Domenech and Dreher, 2008). Each article was then carefully read by at least two of the authors to make sure that it fulfilled the following selection criteria: 1) Only studies reporting whole-brain results were included. Indeed, in order to provide an objective view of reward processing in the brain, it is important to make sure that all cerebral regions have an equal chance of being represented, by specifically excluding studies reporting partial (and inherently biased) results. In particular, we excluded studies entirely based on ROI analyses, studies without a full-brain coverage, and PET studies using selective radiotracers other than H2 15O. 1) Only studies reporting whole-brain results were included. Indeed, in order to provide an objective view of reward processing in the brain, it is important to make sure that all cerebral regions have an equal chance of being represented, by specifically excluding studies reporting partial (and inherently biased) results. In particular, we excluded studies entirely based on ROI analyses, studies without a full-brain coverage, and PET studies using selective radiotracers other than H2 15O. 2) Because the ALE approach is based on activation foci, only studies reporting spatial coordinates in a standardized stereotaxic space were included. Talairach coordinates were transformed to MNI space using the Lancaster transform implemented in the 2) Because the ALE approach is based on activation foci, only studies reporting spatial coordinates in a standardized stereotaxic space were included. Talairach coordinates were transformed to MNI space using the Lancaster transform implemented in the 6 GingerALE software (see below). In case of doubt about the coordinate system used, ambiguity was resolved by writing to the authors. GingerALE software (see below). 2.1. Selection of studies In case of doubt about the coordinate system used, ambiguity was resolved by writing to the authors. GingerALE software (see below). In case of doubt about the coordinate system used, ambiguity was resolved by writing to the authors. 3) Since brain activity in reward-related regions is known to be sensitive to age (Dreher et al., 2008b), as well as to various types of pharmacological manipulations (Nestler, 2005), we only included results obtained in healthy, drug-free, adult subjects. Moreover, we excluded studies with a particularly low sensitivity, i.e. based on fewer than six subjects. 3) Since brain activity in reward-related regions is known to be sensitive to age (Dreher et al., 2008b), as well as to various types of pharmacological manipulations (Nestler, 2005), we only included results obtained in healthy, drug-free, adult subjects. Moreover, we excluded studies with a particularly low sensitivity, i.e. based on fewer than six subjects. 4) Regardless of the protocol used, included studies had to involve the delivery of a pleasant stimulus. Moreover, the results had to unambiguously reflect reward processing at the time of outcome, i.e. they had to be based on contrasts such as “reward > control condition”, “reward > omission of reward”, “reward > punishment” or “correlation with reward intensity”. In particular, studies focusing on reward anticipation or the computation of decision values were excluded, as well as contrasts investigating specific questions such as prediction errors, sexual preference or sensory-specific satiation. Food reward studies systematically involved the delivery of real food/juice in the mouth, while all erotic reward studies involved the presentation of pictures or movies featuring sexually explicit content. All these studies further reported behavioural ratings (assessing pleasantness, arousal or motivation) showing that the stimuli used were indeed rewarding to the participants. 5) In case of multiple studies based on the same dataset, only one of them was included. Moreover, only one contrast per study was selected. Almost all included foci survived a statistical threshold corrected for multiple comparisons or an uncorrected p-value threshold of p<0.001 (see Tables 1, 2 and 3 for details). Following the implementation of these criteria, there remained 33 experiments (394 foci / 565 subjects) on monetary rewards, 26 experiments (469 foci / 443 subjects) on erotic rewards and 28 experiments (318 foci / 444 subjects) on food rewards (Tables 1, 2 and 3). 7 2.2. Data analysis Analyses were performed using the revised ALE method as implemented in the latest version of the GingerALE software (version 2.2, http://brainmap.org/ale), (Turkeltaub et al., 2002; Eickhoff et al., 2009). While the original implementation of the ALE method used a fixed-effects procedure testing for a clustering between foci (Laird et al., 2005), it was recently modified so as to offer a random-effects approach assessing clustering between experiments (Eickhoff et al., 2009; Eickhoff et al., 2012). The ALE method starts by modelling each activation focus reported for a given experiment as the centre of a 3D Gaussian probability distribution. The width of this distribution, reflecting spatial uncertainty, is derived from an empirical model and weighted by the sample size of each experiment (Eickhoff et al., 2009). Then, a modelled activation (MA) map is computed for each experiment by combining the probability distributions of all foci. The union of these MA maps across experiments yields voxel-wise ALE scores, describing the convergence of results at each particular location of the brain. This convergence between experiments is then compared to a random convergence (i.e. noise) in order to make spatial inferences. Specifically, experimental ALE scores are compared to an analytically derived null-distribution reﬂecting a random spatial association between experiments (Eickhoff et al., 2012). Importantly, this procedure is akin to a random-effects analysis. The statistical significance of the resulting p-values is determined using a false discovery rate (FDR) corrected threshold, which is applied to the ALE map along with a minimum cluster size. This procedure was first employed to perform three independent meta-analyses aimed to identify the brain regions consistently activated by monetary, food and erotic rewards. We employed an FDR-corrected threshold of p<0.01 and a minimum cluster size of 600 mm3. We then performed contrast meta-analyses between these rewards. First, the ALE maps corresponding to two rewards were subtracted on a voxel-by-voxel basis. Then, the studies associated with these two rewards were pooled together and randomly divided into two groups of the same size as the two original sets of studies reﬂecting the contrast ALE 8 analysis. ALE scores for these two randomly assembled groups were calculated and the difference between these ALE scores was recorded for each voxel. This procedure was then repeated for 5000 permutations of randomly assembled studies, yielding a null-distribution of ALE score differences in each voxel. 2.2. Data analysis Random-effects inference was achieved by comparing this null-distribution voxel-by-voxel with the experimental ALE score differences. Based on this approach, we compared monetary versus erotic rewards, monetary versus food rewards, and erotic versus food rewards. We employed an FDR-corrected threshold of p<0.05, along with a minimum cluster size of 600 mm3 (a less stringent p-value was used because contrast analyses are more conservative). This resulted in six contrast maps, which were further binarized and combined to identify “reward type-specific” regions. “Money-specific” regions were defined as those stemming from the conjunction of money>erotic and money>food maps, “erotic-specific” regions were defined as those stemming from the conjunction of erotic>money and erotic>food maps, and “food-specific” regions were defined as those stemming from the conjunction of food>erotic and food>money maps. In addition, “primary reward-specific” regions were defined as those stemming from the conjunction of erotic>money and food>money maps. Anatomical localization of functional clusters was performed with the Talairach Daemon application (Lancaster et al., 2000) and a probabilistic atlas (Hammers et al., 2003). For visualization purposes, statistical output maps were overlaid on the Colin brain provided with GingerALE, using the Mango software (www.ric.uthscsa.edu/mango). 3. Results The first goal of this study was to identify a “common reward circuit” as defined by the regions of overlap between monetary, food and erotic reward outcomes. The results of the meta-analyses conducted separately for each reward are illustrated in Figure 1, as well as the resulting overlaps. They show that a set of brain regions was consistently recruited by all 9 three rewards, although with varying levels of significance and spatial extent. These regions included the bilateral striatum, mostly in its ventral part, the bilateral anterior insula/frontal operculum, the mediodorsal thalamus, the bilateral amygdala and the ventromedial prefrontal cortex (vmPFC) extending into the pregenual anterior cingulate cortex (pgACC). Fully detailed results are reported in Supplementary Tables 1, 2 and 3. Moreover, we performed statistical comparisons between studies, in order to identify brain regions responding more robustly to one reward relative to the other two (Fig. 2). The results showed that the bilateral ventral striatum and the right anterior OFC were more likely to be activated by monetary compared to food and erotic rewards. In contrast, the dorsal anterior insula and the somatosensory cortex appeared more likely to be activated by food compared to monetary and erotic rewards. Finally, the bilateral amygdala, the ventral anterior insula and the extrastriate body area were more robustly activated by erotic than by monetary and food rewards. The only brain area more reliably activated by primary (i.e. erotic and food) compared to secondary (i.e. monetary) rewards was located in the middle insula. Perhaps surprisingly at first sight, one may note that among ”reward type-specific” regions, defined as those more reliably activated by one reward compared to the other two, some of them were also part of the “common reward circuit”. This result simply illustrates the fact that, despite being recruited by several rewards, some regions were still more reliably activated by one of them in particular. Moreover, in order to ensure that our results were not biased by potential false positives resulting from the most liberal statistical thresholds used in some studies, we ran the above analyses again only including the most conservative studies (i.e. those in which the statistical threshold was explicitly corrected for multiple comparisons or was the combination of an uncorrected voxel-level threshold and a cluster-extent threshold; see Tables 1, 2 and 3). All the results presented in Figure 1 survived this more stringent procedure, confirming the robustness of our findings. 3. Results 10 10 4. Discussion This meta-analysis provides a synthetic and objective overview of reward processing in the human brain, as provided by the paradigmatic examples of monetary, food and erotic rewards. As expected, the results confirmed the existence of a core set of brain regions processing reward outcomes in an indiscriminate fashion, in line with the idea of a centralized “reward circuit”. In addition, comparative analyses between rewards revealed that some regions were more specifically recruited by one type of reward compared to the others. Below we discuss those results in the light of current views on the putative functional role of these regions, and offer some tentative explanations to account for the observed differences between rewards. The discussion is organized by cerebral region, so that the reader can easily navigate from one sub-section to another. 4.1.1. Modality-independent activations The striatum, essentially in its ventral part, was found to be consistently activated by monetary, food and erotic outcomes in our meta-analysis. Many other rewards were found to elicit similar responses in the striatum, including beautiful faces (Aharon et al., 2001; Kampe et al., 2001), desirable objects (Erk et al., 2002), pleasant music (Menon and Levitin, 2005; Blood and Zatorre, 2001) or reputation and social hierarchy (Izuma et al., 2008; Zink et al., 2008). Importantly, recent studies using large-scale reverse inferences have suggested that the ventral striatum has a relatively specific role in reward processing, as compared to other cognitive processes (Cauda et al., 2011; Yarkoni et al., 2011). Anatomically, the striatum is at the crossroads of several cortico-basal ganglia loops involved in limbic, associative and sensorimotor functions (Haber and Knutson, 2010). The ventral part, centred on the nucleus accumbens, is part of the limbic loop and receives many projections from the OFC, ACC, amygdala and midbrain. It is hence in an ideal place to 11 integrate cognitive, motor and affective information and influence goal-directed behaviour independently of reward modality (Haber and Knutson, 2010; Delgado, 2007). Nonetheless, the precise computational function of the ventral striatum is the subject of a lively debate, which indirectly reflects the long-standing argument over the role of mesolimbic dopamine in reward processing (Berridge, 2007; Berridge and Robinson, 2003). A wealth of studies has shown that ventral striatal activity correlates with the intensity of received rewards, supporting a role in hedonic value representation (Smith et al., 2010; Izuma et al., 2008; Blood and Zatorre, 2001). However, reward value is intrinsically correlated with prediction error, a learning signal which measures the difference between received and expected rewards, and is used to update future predictions (Niv and Schoenbaum, 2008). Many fMRI studies have found brain responses consistent with such a reward prediction error in the ventral striatum (O'Doherty et al., 2004; Bray and O'Doherty, 2007; D'Ardenne et al., 2008). Besides, two studies that have explicitly tried to disentangle reward value from prediction error have reported a better correlation with the latter (Hare et al., 2008; Rohe et al., 2012). 4.1.1. Modality-independent activations Interestingly, the ventral striatum has also been involved in the computation of aversive prediction errors (Delgado et al., 2008), and a recent study manipulating both rewards and punishments has proposed a more general account in terms of salient prediction error coding, regardless of reinforcer type or valence (Météreau and Dreher, 2012). Taken together, these studies suggest that the ventral striatal responses observed in our meta- analysis might reflect prediction error rather than reward value computation. This ambiguity in the interpretation illustrates the importance of distinguishing learning versus hedonic processes at the brain level, and points to the need for more sophisticated fMRI protocols able to separate them. 4.1.2. Modality-dependent activations Our results indicate that monetary rewards activate the ventral striatum more reliably than do erotic and food rewards. However, we believe that this is unlikely to be related to the very nature of monetary rewards. Instead, we think that the present result stems from at least 12 two important differences in how monetary and non-monetary rewards are usually delivered. First, the protocols used in monetary studies often involve learning of probabilistic stimulus- reward associations, whereas most erotic and food studies use passive stimulation tasks with fully predictable rewards (see Table 1). Hence, following the hypothesis expressed in the previous paragraph, a possibility is that the differential activation observed in the ventral striatum results from a difference in prediction error computation. Supporting this idea, several studies show that erotic and food rewards elicit higher ventral striatal activity when they are unexpected compared to when they are expected (Sescousse et al., 2010; McClure et al., 2003; D'Ardenne et al., 2008; Veldhuizen et al., 2011). Another typical feature of monetary studies is the contingency between reward outcomes and motor action: in over 85% of the studies included in our meta-analysis, the delivery of monetary rewards was dependent on the participants’ performance or decision, whereas such contingency was observed in very few (i.e. less than 10%) of the food and erotic studies. Importantly, this form of instrumental conditioning was shown to elicit more robust striatal activations than passive reward delivery, supposedly by increasing the salience of pleasant outcomes (Zink et al., 2004; Tricomi et al., 2004; Elliott et al., 2004). This suggests that the differential activity presently observed in the ventral striatum might simply reflect a difference in terms of motor demands between monetary and non-monetary studies. Supporting this view, a previous study comparing monetary gains with erotic pictures delivered in the exact same context (i.e. involving identical motor demands) found virtually identical striatal activations in response to these two rewards (Sescousse et al., 2010). Our results did not confirm a previous hypothesis suggesting that primary rewards such as juice might recruit more lateral portions of the striatum (i.e. the putamen) compared to secondary rewards such as money (Delgado, 2007). In contrast, we found that food- and erotic-related activations tended to cluster in the medial portion of the striatum and were less extended than money-related activations (see Fig. 1). 4.1.2. Modality-dependent activations This could be the sign of a territorialisation of reward processing in the striatum, and is in agreement with primate data 13 showing enhanced sexual behaviour following local microinjection of a GABA receptor antagonist specifically in the medial part of the ventral striatum (Worbe et al., 2009). 4.2. Ventromedial prefrontal / orbitofrontal cortex The orbitofrontal cortex is a vast and heterogeneous region, which can be broadly divided into three main sections based on anatomical and cytoarchitectonic considerations: a posterior OFC region, an anterior OFC region and a vmPFC region (Haber and Knutson, 2010). Our meta-analysis revealed different patterns of activation in these regions depending on reward type. 4.2.1. Modality-independent activations in the vmPFC In concert with the ventral striatum, the vmPFC responded to all three tested rewards. Strongly connected to limbic regions including the amygdala, ventral striatum and hippocampus, this region is considered to play a central role in reward valuation. During reward anticipation, the vmPFC has been shown to be sensitive to various generic properties of rewards such as magnitude, probability or delay (Haber and Knutson, 2010). It has also found to be crucial in comparing the “goal values” of different rewards during decision- making (Rangel and Hare, 2010; Hare et al., 2009; Padoa-Schioppa and Assad, 2008). The present meta-analysis confirms that the vmPFC is equally important for the computation of experienced reward values. In fact, many studies using primary rewards have shown that brain activity in this region correlates with ratings of pleasantness, as confirmed in two recent meta-analyses (Peters and Buchel, 2010; Kuhn and Gallinat, 2012). Studies using monetary rewards have further demonstrated that vmPFC activity tracks the value of financial payoffs (O'Doherty et al., 2001a; Knutson et al., 2003). Importantly, the vmPFC seems to be sensitive to the subjective value of rewards rather than to their mere intensity. For instance, vmPFC activity decreases with satiety (O'Doherty et al., 2000; Small et al., 2001) and increases with personal preferences based on brand or price (Plassmann et al., 2008; 14 McClure et al., 2004a). This indicates that the vmPFC is able to integrate value across different stimulus dimensions and different stimuli. One might further note that the activations observed in our meta-analysis spread over the pregenual ACC, which has strong connections with both the ventral striatum and medial OFC, and has been shown to be involved in reward and emotion processing in two other meta-analyses (Beckmann et al., 2009; Fujiwara et al., 2009). 4.2.2. Modality-dependent activations in the lateral OFC Our results further revealed money-specific activations in the right anterior OFC. This finding supports the view that secondary (i.e. evolutionary recent) rewards such as money might recruit more anterior OFC regions than primary (i.e. evolutionary ancient) rewards such as food and sex (Kringelbach and Rolls, 2004; Sescousse et al., 2010). Such a dissociation is in line with the cytoarchitectonic properties of the OFC, showing that the anterior part, characterized by a granular cell layer, is phylogenetically more recent than the posterior part consisting in agranular and dysgranular cortices (Ongür and Price, 2000; Wise, 2008). A similar antero-posterior gradient has also been revealed using a functional parcellation of the OFC based on resting-state connectivity patterns with other brain regions (Kahnt et al., 2012). Furthermore, this hypothesis can be integrated in a broader perspective on frontal lobe organization, suggesting a trend in complexity and abstraction along a posterior-anterior axis with the frontopolar cortex at the apex (Badre and D'Esposito, 2009). For instance the rostro-caudal axis of the lateral prefrontal cortex (dlPFC) was shown to support a control hierarchy whereby posterior-to-anterior lateral PFC mediates progressively abstract, higher- order cognitive control (Dreher et al., 2008a; Koechlin et al., 2003). Along the same lines, patients with lesions in the anterior OFC have been reported to be specifically impaired in making decisions entailing abstract, i.e. distant, consequences, and not in making decisions leading to concrete, i.e. immediate, consequences, further supporting the idea of a postero- anterior trend in the representation of abstractness in the OFC (Bechara and Damasio, 2005). 15 In accordance with the above hypothesis and previous results from our group (Sescousse et al., 2010), one would have expected primary rewards (i.e. food and erotic stimuli) to specifically recruit the posterior portion of the OFC in comparison to monetary rewards. Unfortunately we were not able to fully substantiate this claim, as the lateral posterior OFC was seemingly recruited by all three rewards in our meta-analysis (see Supplementary Materials - Fig. S1). Note however that erotic and food rewards did elicit activity in the lateral posterior OFC as expected. This is consistent with a recent report showing that aesthetic appraisal across diverse sensory modalities primarily recruits the OFC in its posterior part (Brown et al., 2011). 4.2.2. Modality-dependent activations in the lateral OFC Note also that the localization of reward-related activity in the lateral portion of the OFC challenges the hypothesis of a medio-lateral dissociation between rewards and punishments in the OFC (Noonan et al., 2012; O'Doherty, 2007). 4.3.1. Modality-independent activations The amygdala receives projections from a number of cortical regions, but is most strongly connected to the ventral striatum and OFC (Haber and Knutson, 2010; Murray, 2007). Our meta-analysis showed that, in concert with these two regions, the amygdala responded to all rewards regardless of their type. These results shed light on the debate opposing valence and salience coding in the amygdala. Early studies in humans and animals were mostly in favor of the valence hypothesis, suggesting that the amygdala is specialized in the processing of negative emotions such as fear or anger (LeDoux, 2000; Calder et al., 2001), or aversive stimuli such as unpleasant odors or monetary losses (Zald, 2003; Yacubian et al., 2006). Yet, the results from the present meta-analysis provide strong evidence that the amygdala is equally sensitive to rewarding stimuli, as confirmed by a wealth of animal studies (Sugase-Miyamoto and Richmond, 2005; Tye and Janak, 2007; Bermudez et al., 2012). This suggests that the amygdala might be better regarded as coding the salience, and not the valence, of affectively 16 laden stimuli, consistent with the recent proposal of a salient prediction error computation in this region (Météreau and Dreher, 2012). Interestingly, several fMRI studies have manipulated both these dimensions while comparing positive and negative reinforcers of varying intensity in humans. In line with the salience hypothesis, they have shown that amygdala activity is linked to the level of arousal induced by these reinforcers, regardless of their valence (Small et al., 2003; Anderson and Sobel, 2003). Note however that the amygdala is a heterogeneous structure whose various nuclei are hardly distinguishable with fMRI, and that positive versus negative valence might still be encoded by distinct neuronal populations or distinct sub-regions (Prevost et al., 2011; Paton et al., 2006). Despite the consistent response of the amygdala to rewarding stimuli, it is nevertheless unclear whether these responses reflect reward processing per se or assignment of emotional value. Based on seemingly overlapping brain mechanisms, emotion and reward have often been closely related and described together in a model where emotions are a by-product of reinforcement (Rolls, 2000). However, this view has been challenged by animal studies demonstrating that amygdala lesions may reduce emotional reactions without disturbing reward processing (Murray, 2007). As a consequence, it was proposed that, rather than a direct role in reward processing, the amygdala was in charge of providing an “affective tag” to stimuli. 4.3.2. Modality-dependent activations 4.3.1. Modality-independent activations This is in line with several findings linking amygdala activity with skin conductance responses (SCR), thought to reflect autonomic responses (Bechara et al., 1999; Petrovic et al., 2008a). This emotional tagging would further participate in the updating of current reward value and the flexible adaptation of behavior, as illustrated by the decrease in amygdala activity following reinforcer devaluation (Baxter and Murray, 2002; Gottfried et al., 2003). Therefore, the activity observed in the amygdala in response to money, food and erotic pictures in the present meta-analysis is more likely to reflect the emotional impact of these stimuli rather than their intrinsic reward value. 4.3.2. Modality-dependent activations 17 17 Our meta-analysis also revealed that the amygdala, and possibly its centro-medial nucleus, was more reliably activated by erotic than by monetary and food rewards. This observation supports a role of the amygdala in the emotional appraisal of affectively laden reinforcers such as erotic pictures, known to elicit particularly strong affective and visceral reactions (Lang et al., 1993). In line with this interpretation, two recent studies showed enhanced amygdala activation in response to smiley faces and erotic pictures compared to monetary gains (Rademacher et al., 2010; Sescousse et al., 2010). The present result may also stem from the fact that, in contrast to monetary and food outcomes often delivered in a repetitive fashion in reward experiments, erotic stimuli are more salient and offer more variety, therefore limiting habituation effects commonly observed in the amygdala (Breiter et al., 1996). Moreover, one should note that most neuroimaging studies manipulating erotic rewards were conducted in men, who show significantly higher amygdala responses than women when presented with visual sexual stimuli (Hamann et al., 2004). Overall, our results are mostly compatible with an emotional account of amygdala function. 4.4.1. Modality-independent activations Our meta-analysis showed that the anterior insula bordering the frontal operculum was consistently activated by monetary, food and erotic rewards. Surprisingly, this structure has been relatively overlooked in the reward literature, and most often associated with aversive events such as monetary losses (Knutson and Bossaerts, 2007; Knutson and Greer, 2008; Petrovic et al., 2008b). We discuss below what might be its function in the processing of appetitive reward outcomes. Whereas the posterior insula receives its main inputs from associative cortical regions, the anterior insula is mostly connected to limbic regions such as the vmPFC, amygdala and ventral striatum, and is essentially innervated by dopaminergic neurons. This situation enables the anterior insula to integrate autonomic and visceral information with emotional and motivational processes, in line with its proposed role in interoception (Naqvi 18 and Bechara, 2009). Interoception consists in the neural mapping of bodily states which are meaningful for the maintenance of homeostasis, such as pain, thirst or autonomic arousal (Craig, 2002). These bodily states arise as a consequence of external emotional stimulation, but do not reach consciousness before they are mapped at the insular level. This mapping then leads to an explicit emotional feeling, after being integrated with the events originally eliciting those bodily states (Bechara and Damasio, 2005; Critchley, 2005). Importantly, the classic view of the anterior insula as a purely visceral sensory cortex was recently extended. Indeed, a number of neuroimaging studies have shown that it is involved in the representation of a wide variety of subjective feelings, and not just those arising from bodily states, as well as in many other cognitive processes such as attention, time perception or perceptual decision-making (Craig, 2009). Based on such evidence, the role of the anterior insula was reframed and associated more broadly with awareness. This generic account of anterior insula function is compatible with the bilateral activation observed across monetary, erotic and food outcomes, and suggests a role in the subjective affective experience of rewards. Moreover, the recruitment of the anterior insula by monetary rewards confirms that this region does not deal exclusively with bodily states relevant for homeostasis, but more generally with positive feelings elicited by pleasant outcomes. Another function assigned to the anterior insula is the processing of risk and uncertainty (Knutson and Bossaerts, 2007; Craig, 2009; Singer et al., 2009). 4.4.1. Modality-independent activations A wealth of fMRI studies using monetary (Huettel et al., 2006) and non-monetary stimuli (Huettel et al., 2005; Grinband et al., 2006) have demonstrated increased anterior insula activity when faced with risk or risky decisions. The consistency of this finding for risky monetary rewards was established in a recent meta-analysis, which further suggested that insular engagement might be even stronger when potential losses are involved (Mohr et al., 2010). Along the same lines, it has been proposed that the anterior insula computes a risk prediction and a “risk prediction error”, and thus participates in risk learning (Preuschoff et al., 2008). This bears similarity to the concept of an “interoceptive prediction error”, signaling mismatch between actual and anticipated bodily arousal, and suggested to play a role in anxiety 19 (Paulus and Stein, 2006). It is further consistent with the idea that the anterior insula tracks the salience of outcomes, regardless of their valence (Rutledge et al., 2010) Thus, in the context of reward processing, the anterior insula might be in charge of tracking both expected and experienced risk, a mechanism that would participate more broadly in emotional appraisal. 4.4.2. Modality-dependent activations 4.4.2. Modality-dependent activations Finally, although our results unambiguously support a role of the anterior insula in the processing of both primary and secondary rewards, they suggest a stronger involvement in the processing of primary rewards. Indeed, we found that the middle insula was more reliably activated by both erotic and food rewards as compared to money, while the ventral and dorsal parts of the anterior insula were more specifically recruited by erotic and food rewards, respectively. This is in agreement with a recent meta-analysis showing that aesthetic appraisal across various sensory modalities (i.e. primary rewards) produces very robust activations in the anterior insula (Brown et al., 2011). This could reflect the higher autonomic arousal induced by primary rewards: in line with their prominent role in homeostasis and survival, erotic and food rewards are known to generate acute changes in bodily states and autonomic arousal, as evidenced by changes in heart beat, skin conductance, sexual drive or satiety levels. Such changes are in turn often correlated with activity in the anterior insula, shown to be critically involved in autonomic conditioning (Critchley et al., 2002; Kuhn and Gallinat, 2011). The apparent segregation between erotic-specific and food-specific activations along a ventro-dorsal axis is difficult to interpret at this stage. Based on the role of the ventral insula in emotional appraisal and its frequent coactivation with the amygdala (Deen et al., 2010; Mutschler et al., 2009), the present result might reflect the particularly strong emotional impact of erotic rewards. The activation observed in the anterior insula for food rewards is consistent with the location of the primary gustatory cortex (Naqvi and 20 Bechara, 2009). This is further consistent with the food-specific activation observed in the somatosensory cortex and adjacent middle insula, known to be involved in the processing of the physical properties of food and the mapping of bodily states (Bechara and Damasio, 2005). Therefore, it is possible that those food-specific activations reflect the sensory properties of food rewards, rather than their specificity as a primary reward. 4.5. Mediodorsal thalamus Our meta-analysis revealed that the mediodorsal thalamus, a structure which is rarely discussed in the reward literature, was consistently activated by monetary, erotic and food rewards. This brain region is an important relay between the basal ganglia and the prefrontal cortex: it receives inputs from the ventral striatum via the ventral pallidum, and projects in turn to various regions of the prefrontal cortex, especially the ventromedial part, which projects back to the ventral striatum (Haber and Knutson, 2010; Ongür and Price, 2000; Garcia-Cabezas et al., 2007). Embedded within this striatal–thalamo–cortical loop, the mediodorsal thalamus is thought to bridge basic reward signals with higher cognitive processes such as motivation and goal-directed behaviour (Elliott et al., 2000; Galvan et al., 2005). The role of the mediodorsal thalamus in reward prediction is supported by a wealth of studies in both animals and humans. In rats, thalamic neurons were found to fire in anticipation of pleasant food rewards (Komura et al., 2001), while lesions in the mediodorsal thalamus were found to impair instrumental conditioning (Corbit et al., 2003). In humans, reward predictive cues were found to elicit robust thalamic activity, often increasing with reward probability (Galvan et al., 2005; Roiser et al., 2010). A meta-analysis further revealed that this activity was stronger than the one observed at the time of reward outcome (Knutson 21 and Greer, 2008). These anticipatory responses have generally been interpreted as reflecting increased attention towards motivationally salient stimuli. Importantly, the present results demonstrate that the mediodorsal thalamus also plays an important role in processing the experienced value of rewards. Interestingly, activity in the thalamus was found to scale with reward intensity in a number of studies manipulating monetary gains (Martin-Soelch et al., 2003; Elliott et al., 2000), erotic stimuli (Redouté et al., 2000) or pleasant music (Blood and Zatorre, 2001). These results can be interpreted within the previous framework, i.e. as reflecting increased arousal, but could alternatively be seen as reflecting reward value coding. However, if the mediodorsal thalamus reflected reward value, we should expect positive reinforcement to produce much higher thalamic activity than negative reinforcement. This does not seem to be the case in our meta-analysis: out of the nine monetary studies using contrasts between gains and losses, only one reported activity in the thalamus (Cox et al., 2005). 4.5. Mediodorsal thalamus This is consistent with a meta-analysis showing that anticipation of reward versus punishment does not elicit differential activation in the thalamus (Knutson and Greer, 2008). Hence, although more research is needed to confirm this hypothesis, it is possible that the currently observed mediodorsal thalamus activity reflects the general arousal induced by behaviorally salient rewards. This is in line with a recent study in which monetary rewards, found to be motivationally more salient than social rewards (as objectivised by faster reaction times), also elicited higher thalamic activation (Rademacher et al., 2010). It is also consistent with a study investigating sexual orientation, and showing that despite similar attractiveness judgments, faces of the preferred sex elicited higher activity in the mediodorsal thalamus than less salient faces of the non-preferred sex (Kranz and Ishai, 2006). 4.6. The hypothalamus and extrastriate body area 22 Finally, our meta-analysis revealed that the hypothalamus and extrastriate body area, two brain regions not usually associated with reward processing, were robustly and specifically activated by erotic rewards. The hypothalamus is part of the limbic system and plays a general role in homeostatic control and autonomic responses, essentially by means of its neuroendocrine function. In particular, it is known to be important for feeding behaviour, and its activity was shown to be modulated by food cues as well as by hunger and satiety feelings (Grabenhorst et al., 2010b; Hikosaka et al., 2008). However, this role seems to be distinct from the coding of food hedonics, as illustrated by the absence of hypothalamic response to pleasant foods in our meta-analysis. In contrast, the hypothalamus appeared to be particularly sensitive to the presentation of visual erotic stimuli, consistent with the rich animal and human literature demonstrating the pivotal role of this region in sexual behaviour (Karama et al., 2002; Walter et al., 2008). Several neuroimaging studies have found a correlation of hypothalamic activity with subjective sexual arousal, as well as with physiological arousal responses in men, such as erection (Arnow et al., 2002; Karama et al., 2002; Redouté et al., 2000). Overall these results confirm the central role of the hypothalamus in human sexual motivation, although this role might be more likely related to the regulation of autonomic responses than to the appraisal process through which erotic stimuli are evaluated as sexual incentives (Redouté et al., 2000). Erotic stimuli also elicited large activations in the extrastriate body area, a higher order visual area of the lateral occipitotemporal cortex, known to selectively respond to images of the human body or parts of it (Downing et al., 2001). This result is consistent with the nature of erotic stimuli typically representing naked bodies. Moreover, several studies found that these activations appeared to be independent of the gender of the bodies displayed and the individuals’ sexual preference (Paul et al., 2008; Ponseti et al., 2006). This suggests that the extrastriate body area is mainly sensitive to the visual features of erotic stimuli, rather than to their rewarding properties. 23 4.7. Limitations and strengths The present meta-analysis is not free of limitations, demanding to treat the results with some caution. First, our ALE approach ignored information such as peak statistical values or spatial extent of functional clusters reported in individual studies. Yet, it would be desirable to weigh included studies depending on these criteria, as they often reflect the underlying quality of the imaging results (Yarkoni et al., 2010). Note however, that this ideal procedure is difficult to implement and seldom used in practice, because of the need to possess full datasets processed in a homogeneous fashion. An additional limitation might come from the heterogeneity of the tasks used in the reward literature and included in our meta-analysis. First, variations in reward delivery may act as a confounding effect. As mentioned previously, monetary studies often involve probabilistic or performance-dependent rewards, whereas food and erotic rewards are usually delivered in a passive and predictable way. Moreover, a number of food and erotic studies used block designs, whereas most monetary studies used event-related designs. These methodological discrepancies may confound effects related to reward type with effects related to how rewards are delivered. Note however that the difference in block versus event- related designs does not seem to impact reward-related activations, at least for erotic stimuli (Buhler et al., 2008). Furthermore, within each reward category, we combined foci resulting from a wide variety of protocols and contrasts. As a consequence, we might have pooled rather heterogeneous results: for instance, a contrast between gains and losses in a guessing task is likely to produce different activations from a contrast between gains and non-gains in a classical conditioning task. This has the advantage of revealing the most robust and replicable effects across paradigms, but the disadvantage of limiting our ability to ascribe specific brain regions to discrete reward processes. Finally, it could be argued that the differential activation patterns presently observed between rewards are confounded by differences in reward intensity. Disentangling these two hypotheses is intrinsically difficult within a meta-analysis. Indeed, in contrast to single studies in which the matching of different conditions is under the control of the experimenter, such 24 matching cannot be easily achieved when comparing different groups of studies. 4.7. Limitations and strengths However, it should be noted that since this is a coordinate-based meta-analysis, we are not comparing the amplitude of brain activity between conditions (as would be done by contrasting Betas within a regular GLM analysis), but the spatial consistency of reported peaks of activity between groups of studies (regardless of peak t-values). Yet, this consistency is not influenced by the intensity of the reward stimuli, but rather by their ability to elicit reliable and detectable activity in the brain. Finally, one might be surprised not to observe midbrain activity emerging from our meta-analysis. In fact, all three rewards did elicit activity in the midbrain, but the location of functional clusters was not overlapping between rewards and was not entirely consistent with the dopaminergic portion of the midbrain (see Supplementary Materials - Fig. S2). This variability likely results from the high susceptibility of fMRI signals in the midbrain to noise and artifacts, due to local field inhomogeneity, motion and partial volume effects (Haber and Knutson, 2010; D'Ardenne et al., 2008). This loss of reliability of fMRI signals in deeper brain structures warrants a word of caution in interpreting the present results as a truly whole-brain picture of reward-related activity, and calls in turn for adapted data acquisition protocols for those regions in the future (Krebs et al., 2011; D'Ardenne et al., 2008). Despite these limitations, the present work has several important strengths. To the best of our knowledge, this is the first study to systematically compare the neuroanatomical substrates of multiple rewards in order to distinguish common from specialized reward- related regions. Moreover, these comparisons were performed in a fully objective manner, since we used a voxel-based meta-analytic approach combined with whole-brain results only. In particular, by excluding ROI-based studies from our meta-analysis, we avoided the confirmation bias inherent to the ROI approach, and were able to reveal the most consistent findings in the absence of any a priori assumptions. In fact, we had to exclude a surprisingly high number of ROI-based studies, which is somehow paradoxical considering the only recent availability of objective reference (such as the present meta-analysis) for these studies in the literature. Perhaps as a consequence, our findings showed that regions such 25 as the thalamus and anterior insula were more robustly activated by reward outcomes than previously thought. Finally, we used a quantitative, random-effect, approach based on the estimated convergence of results across studies. 4.7. Limitations and strengths This provides valuable information in terms of average peak location and spatial extent of reward-related brain regions, which should prove useful for future ROI-based studies. 4.8. Conclusions This meta-analysis first reveals that there is ample support in the neuroimaging literature for a “common reward circuit” in the brain (Fig. 3). Although this ﬁnding was to be expected, our results offer an objective and quantitative demonstration. Within this circuit, the vmPFC appears to be directly responsible for computing the experienced value of rewards on a common scale. Strongly connected to the vmPFC, the ventral striatum is thought to primarily reflect prediction error and to contribute to learning and motivation, although its pattern of activation is also compatible with the computation of experienced value. Interestingly, both the vmPFC and ventral striatum have been involved in the valuation phase of a number of decision-making paradigms involving primary and secondary rewards (Kable et al., 2007; Prévost et al., 2010). Further meta-analyses are needed to investigate whether the computation of experienced value and decision value also engages distinct brain structures. As for the amygdala, even though its involvement in reward learning is still a matter of debate, considerable evidence shows that it plays a major role in assigning emotional value to rewards. Less often included in the canonical definition of the reward circuit, the anterior insula emerged as a key region in our meta-analysis. Its role in reward processing is likely related to the conscious awareness of emotions triggered by rewards, or to risk and salience monitoring. The mediodorsal thalamus, strongly activated by all rewards, is thought to play a role in the increased arousal induced by rewards. Importantly, in all these regions except the thalamus, our analyses revealed variations in peaks or levels of activity depending on reward type. These discrepancies are presumably attributable to intrinsic 26 differences between rewards or between the contexts in which these rewards were delivered. Note that these discrepancies might be informative and help elucidate the functional roles of specific brain regions in reward processing. For instance, the stronger activation observed in the ventral striatum for monetary rewards can be related to the frequent delivery of these rewards in a learning context, and therefore tends to support a role for this brain region in prediction error computation. Complementing this common reward circuit, our meta-analysis revealed specialized reward areas in the brain. 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Berridge, K.C., 2007. 4.8. Conclusions In addition, the anterior OFC was specifically recruited by monetary rewards, a result that we interpret in the broader perspective of a dissociation between primary and secondary rewards in the postero-anterior axis of the OFC (Sescousse et al., 2010). The question of one versus multiple reward circuits in the brain is complex and requires future development. First, it will be instructive to extend the present work in the future, when enough data has been accumulated to run meta-analyses on other types of rewards, such as beautiful faces, pleasant odors or positive social feedback. Addressing previously stated methodological limitations will be another challenge. Image-based meta- analyses, which make use of crucial information such as activation magnitude and spatial extent of clusters, but require access to the original data, seem like a promising avenue (Salimi-Khorshidi et al., 2009; Poldrack, 2008). 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Zald, D.H., Pardo, J.V., 2000. Cortical activation induced by intraoral stimulation with water in humans. Chem Senses 25, 267-275. Zink, C.F., Pagnoni, G., Martin-Skurski, M.E., Chappelow, J.C., Berns, G.S., 2004. References Human striatal responses to monetary reward depend on saliency. Neuron 42, 509-517. Zink, C.F., Tong, Y., Chen, Q., Bassett, D.S., Stein, J.L., Meyer-Lindenberg, A., 2008. Know your place: neural processing of social hierarchy in humans. Neuron 58, 273-283. 38 Figure captions Fig. 1. “Common reward circuit” as defined by the overlap of ALE maps produced by monetary, food and erotic reward studies. The ALE maps in the three leftmost columns show the brain regions consistently and commonly activated by monetary, erotic and food reward outcomes. The colour scale indicates the magnitude of ALE values, i.e. the degree of consistency across studies. The maps on the right illustrate the overlap of activation clusters across rewards (green = monetary rewards, red = erotic rewards, blue = food rewards). The ALE maps are overlaid on the Colin brain provided with GingerALE (p<0.01 FDR whole-brain corrected and cluster size > 600 mm3). Fig. 2. Brain regions more reliably activated by one reward compared to the other two (i e “reward type-specific”) These regions result from contrasts between the different Fig. 2. Brain regions more reliably activated by one reward compared to the other two (i.e. “reward type-specific”). These regions result from contrasts between the different meta-analyses: “money-specific” regions (green) result from the conjunction of money>erotic and money>food maps, “erotic-specific” regions (red) result from the conjunction of erotic>money and erotic>food maps, and “food-specific” regions (blue) result from the conjunction of food>money and food>erotic maps. “Primary reward-specific” regions (purple) result from the conjunction of erotic>money and food>money maps. The ALE maps are overlaid on the Colin brain provided with GingerALE (p<0.05 FDR whole-brain corrected and cluster size > 600 mm3; except for the primary reward-specific activation in the left insula: p<0.05 FDR whole-brain corrected and cluster size = 350 mm3). Fig. 3. Overview of the brain regions involved in reward outcome processing as a function of reward type. (A) “Common reward circuit”, i.e. the brain responding to monetary, erotic and food rewards. The putative main functional role assigned to each region is mentioned in italics. Rewards eliciting a particularly robust activation in certain regions are mentioned in square brackets. (B) Reward type-specific regions, i.e. more reliably activated by one reward compared to the other two. The putative main functional role assigned to each region is mentioned in italics. Green: “money-specific” regions, Red: “erotic-specific” regions, Blue: “food-specific” regions, Fuchsia: “primary reward-specific”. 39 Tables Table 1. Overview of the monetary reward studies included in our meta-analysis. For each study, the column “n” indicates the number of participants, the column “Foci” indicates the number of foci included in our meta-analysis, and the column “Task” provides a description of the type of paradigm used. In the column “Contrast”, “Omission” refers to a null outcome when a potential gain was expected, “Non-gain” refers to a null outcome when such a null outcome was expected, “Loss” refers to a monetary loss when either a gain or a loss was expected, and “Baseline” refers to any low-level condition such as a fixation cross. When multiple thresholds are reported for one study, “&” means that these thresholds were applied simultaneously to every foci, while “\|” means that these thresholds were applied to different foci. Table 2. Overview of the erotic reward studies included in our meta-analysis. For each study, the column “n” indicates the number of participants, the column “Foci” indicates the number of foci included in our meta-analysis, and the column “Task” provides a description of the type of paradigm used. In the column “Contrast”, “Omission” refers to a null outcome when a potential erotic stimulus was expected, and “Baseline” refers to any low-level condition such as a fixation cross. When multiple thresholds are reported for one study, “&” means that these thresholds were applied simultaneously to every foci, while “\|” means that these thresholds were applied to different foci. Tables Modality n Foci Task Contrast Statistical threshold fMRI 8 9 Monetary Incentive Delay task (modified) Monetary gain > Omission p<0.001 voxel-level uncorr & k≥10 fMRI 12 15 Monetary Incentive Delay task Monetary gain > Omission p<0.0001 \| p<0.00001 voxel-level uncorr fMRI 23 2 Monetary Incentive Delay task Monetary gain > Omission p<0.001 voxel-level uncorr & p<0.05 cluster-level corr fMRI 24 6 Monetary Incentive Delay task Monetary gain > Omission p<0.05 voxel-level FDR corr fMRI 17 18 Number guessing task Monetary gain > Baseline p<0.0001 voxel-level uncorr & p<0.05 cluster-level corr fMRI 15 18 Slot machine gambling task Monetary gain > Omission p<0.05 voxel-level FWE corr fMRI 22 28 Card guessing task Monetary gain > Loss p<0.05 voxel-level corr (permutation testing) fMRI 9 2 Card guessing task Positive correlation with cumulative reward level p<0.001 voxel-level uncorr \| p<0.05 corr fMRI 12 11 Target detection task Monetary gain > Baseline p<0.001 voxel-level uncorr \| p<0.05 corr fMRI 12 10 Target detection task Monetary gain > Non-gain p<0.001 voxel-level uncorr \| p<0.05 corr fMRI 14 16 Wheel of fortune gambling task Monetary gain > Omission p<0.001 voxel-level uncorr ) fMRI 17 18 Card guessing task Positive correlation with gain > Positive correlation with loss p<0.001 \| p<0.0001 voxel-level uncorr fMRI 18 7 Wheel of fortune gambling task (Gain > Gain omission) > (Loss omission > Loss) p<0.00001 voxel-level uncorr fMRI 19 23 Card guessing task High monetary gain > Non-gain p<0.005 voxel-level uncorr & p<0.05 cluster-level corr ) fMRI 9 6 Monetary Incentive Delay task Monetary gain > Omission p<0.0001 voxel-level uncorr ) fMRI 12 4 Monetary Incentive Delay task Monetary gain > Omission p<0.0001 voxel-level uncorr ) fMRI 12 15 Monetary Incentive Delay task Monetary gain > Omission p<0.0001 voxel-level uncorr & k≥4 PET 13 9 Pattern recognition task Monetary gain > Omission p<0.001 voxel-level uncorr fMRI 33 16 Reversal learning task Monetary gain > Loss p<0.05 voxel-level FDR corr fMRI 20 2 Classical conditioning task Monetary gain > Omission p<0.003 voxel-level uncorr & p<0.05 cluster-level corr 2001) PET 12 41 Pattern recognition task Monetary gain > Omission p<0.001 voxel-level uncorr r, 2012) fMRI 20 13 Classical conditioning task Monetary gain > Omission p<0.01 voxel-level FDR corr & k≥5 2005) fMRI 14 10 Card guessing task Monetary gain > Loss p<0.0005 voxel-level uncorr & k≥60 3) fMRI 10 7 Reversal learning task Monetary gain > Loss p<0.001 voxel-level uncorr b) fMRI 13 11 Wheel of fortune gambling task Monetary gain > Omission p<0.001 voxel-level uncorr \| p<0.05 cluster-level corr 4) fMRI 6 6 Classical conditioning task Monetary gain (unexpected) > Omission (expected) p<0.001 voxel-level uncorr fMRI 12 4 Card guessing task Monetary gain > Loss p<0.001 voxel-level uncorr fMRI 14 11 Wheel of fortune type gambling task Monetary gain > Loss Z>2.3 voxel-level uncorr & p<0.05 cluster-level corr al., 2010) fMRI 54 6 Behavioral Investment Allocation Strategy task Monetary gain > Loss p<0.0001 voxel-level uncorr & k≥8 10) fMRI 18 18 Monetary Incentive Delay task (modified) Monetary gain > Non-gain p<0.01 voxel-level FDR corr & k≥15 fMRI 23 10 Passive viewing task Monetary gain > Loss Z>2.3 voxel-level uncorr & p<0.05 cluster-level corr ., 2010) fMRI 15 3 Slot machine gambling task Monetary gain > Loss p<0.001 voxel-level uncorr fMRI 14 19 Target detection task Monetary gain > Non-gain p<0.001 voxel-level uncorr & k≥10 40 41 Study Modality n Foci Task Contrast Statistical threshold ., 2010) fMRI 26 15 Passive viewing during a letter discrimination task Erotic pictures > Neutral pictures p<0.05 voxel-level FWE corr & k≥20 ertales et al., 2010) fMRI 45 19 Passive viewing during a letter discrimination task Erotic pictures > Neutral pictures p<0.001 voxel-level uncorr & k≥20 et al., 2001) fMRI 10 7 Passive viewing task Erotic films > Neutral films p<0.005 corr , 2001) PET 10 8 Passive viewing task Erotic films > Baseline p<0.001 voxel-level uncorr & k≥50 ., 2008) fMRI 18 26 Passive viewing task Erotic films > Sport films p<0.001 voxel-level uncorr 2008) fMRI 10 14 Passive viewing task Erotic pictures > Neutral pictures p<0.05 voxel-level FDR corr & k≥10 nd Dreher, 2008) fMRI 14 25 Choice preference task Erotic pictures > Omission p<0.0001 voxel-level uncorr & k≥15 , 2005) fMRI 10 17 Passive viewing task Erotic films > Sport films p<0.05 Bonferroni corr al., 2004) fMRI 14 9 Passive viewing task Erotic pictures > Neutral pictures p<0.001 voxel-level uncorr & k≥5 08) fMRI 10 28 Passive viewing task Erotic films > Baseline p<0.001 voxel-level uncorr & k≥10 ., 2002) fMRI 20 18 Passive viewing task Erotic films > Neutral films p<0.001 voxel-level uncorr \| p<0.05 corr 006) fMRI 10 12 Passive viewing task Erotic films > Sport films p<0.001 voxel-level uncorr \| p<0.05 corr , 2006) fMRI 10 23 Passive viewing task (Nude > Dressed women) > (Nude > Dressed children) p<0.001 voxel-level uncorr \| p<0.05 corr ., 2008) fMRI 8 18 Passive viewing task Erotic films > Humorous films p<0.05 corr 008) fMRI 12 13 Passive viewing task Erotic films > Neutral films p<0.001 voxel-level uncorr & k≥5 ., 2006) fMRI 53 13 Passive viewing task Erotic pictures > Neutral pictures p<0.01 voxel-level uncorr & p<0.05 cluster-level corr ., 2010) fMRI 16 26 Delay and effort discounting task Erotic pictures > Baseline p<0.0001 voxel-level uncorr & k≥15 al., 2000) PET 9 14 Passive viewing task Erotic films > Neutral films p<0.001 voxel-level uncorr \| p<0.05 corr t al., 2007) fMRI 22 18 Passive viewing task Erotic pictures > Neutral pictures p<0.001 voxel-level uncorr 2007) fMRI 22 15 Passive viewing task Erotic pictures > Sport pictures p<0.001 voxel-level uncorr & p<0.05 cluster-level corr ., 2008) fMRI 12 39 Passive viewing task Erotic pictures > Neutral pictures p<0.05 voxel-level FDR corr & k≥10 010) fMRI 21 24 Passive viewing task Erotic pictures > Happy faces pictures Z>2.3 voxel-level uncorr & k≥10 et al., 2010) fMRI 18 30 Erotic Incentive Delay task Erotic pictures > Neutral pictures p<0.01 voxel-level FDR corr & k≥15 , 1999) PET 8 4 Passive viewing task Erotic films > Neutral films p<0.001 voxel-level uncorr \| p<0.05 corr t al., 2010) fMRI 14 19 Passive viewing task Erotic films > Baseline p<0.05 (unclear whether corrected or not) 2008) fMRI 21 15 Passive viewing task Erotic pictures > Neutral pictures p<0.001 voxel-level uncorr & k≥10 42 udy Modality n Foci Task Contrast Statistical threshold 001) fMRI 25 1 Passive delivery task Preferred > Nonpreferred (between juice and water) p<0.01 voxel-level uncorr & k=17 l., 2003) fMRI 11 8 Passive delivery task Sucrose > Tasteless drink p<0.001 \| p<0.0001 voxel-level uncorr \| p<0.05 corr al., 2002) PET 44 20 Passive delivery task Liquid meal formula > Water p<0.005 voxel-level uncorr d Dreher, 2008) fMRI 14 16 Choice preference task Juice > Omission p<0.01 voxel-level FDR corr 2010) fMRI 40 13 Passive delivery task Milkshake > Tasteless drink p<0.05 voxel-level FDR corr 1999) fMRI 6 8 Passive delivery task Glucose > Baseline p<0.005 corr et al., 2010b) fMRI 14 4 Passive delivery task Positive correlation with pleasantness of milkshake flavor p<0.001 voxel-level uncorr & k≥3 urphy, 2012) * fMRI 12 37 Passive delivery task Saccharin > Water p<0.001 voxel-level uncorr & p<0.05 cluster-level corr urphy, 2012) * fMRI 12 20 Passive delivery task Saccharin > Water p<0.001 voxel-level uncorr & p<0.05 cluster-level corr 2009) fMRI 18 36 Passive delivery task Sucrose > Water p<0.0005 voxel-level uncorr & p<0.05 cluster-level corr l., 2010) fMRI 19 17 Passive delivery task Sucrose > Water p<0.015 voxel-level uncorr & p<0.05 cluster-level corr t al., 2003) fMRI 9 5 Passive delivery task Liquid food > Tasteless drink p<0.05 corr Rolls, 2007) fMRI 12 5 Passive delivery task Umami food & vegetable odor > Tasteless drink p<0.001 voxel-level uncorr \| p<0.05 voxel-level FWE corr , 2011) fMRI 15 9 Passive delivery task Chocolate > Tasteless drink p<0.001 voxel-level uncorr & p<0.05 cluster-level corr , 2004a) fMRI 32 1 Classical conditioning task Positive correlation with preference for soda drinks p<0.05 voxel-level FDR corr Dreher, 2012) fMRI 20 20 Classical conditioning task Juice > Omission p<0.01 voxel-level FDR corr & k≥10 al., 2001b) fMRI 7 14 Passive delivery task Glucose > Tasteless drink p<0.01 voxel-level uncorr in a minimum of 6 of 7 subjects al., 2002) fMRI 8 2 Classical conditioning task Glucose > Tasteless drink p<0.001 voxel-level uncorr al., 2008) fMRI 20 10 Passive delivery task Wine > Tasteless drink p<0.001 voxel-level uncorr & k≥5 Cabe, 2007) fMRI 16 6 Passive delivery task Chocolate > Tasteless drink p<0.001 voxel-level uncorr \| p<0.05 voxel-level FWE corr 001) PET 9 18 Passive delivery task Positive correlation with pleasantness ratings for chocolate p<0.001 voxel-level uncorr \| p<0.025 voxel-level FWE corr 003) fMRI 9 12 Passive delivery task Sucrose > Tasteless drink p<0.005 voxel-level uncorr \| p<0.05 cluster-level corr 008) fMRI 12 4 Passive delivery task Juice > Tasteless drink p<0.001 voxel-level uncorr 06) fMRI 8 3 Passive delivery task Milkshake > Tasteless drink p<0.001 cluster-level corr (permutation testing) 009) PET 10 5 Passive delivery task with/without cognitive inhibition Food > Baseline unclear (p<0.01 cluster-level corr?) 98) PET 10 3 Passive delivery task Chocolate > Water p<0.0005 voxel-level uncorr o, 2000) PET 23 20 Passive delivery task Water > Baseline p<0.0001 voxel-level uncorr 02) PET 9 1 Passive delivery task Sucrose > Water p<0.0005 voxel-level uncorr Statistical threshold Table 3. Tables Overview of the food reward studies included in our meta-analysis. For each study, the column “n” indicates the number of participants, the column “Foci” indicates the number of foci included in our meta-analysis, and the column “Task” provides a description of the type of paradigm used. In the column “Contrast”, “Omission” refers to a null outcome when a potential food stimulus was expected, and “Baseline” refers to any low-level condition such as a fixation cross. * The study by Green and Murphy (2012) was split into two studies since results are reported separately for two distinct groups of healthy participants (diet soda drinkers and non-diet soda drinkers). When multiple thresholds are reported for one study, “&” means that these thresholds were applied simultaneously to every foci, while “\|” means that these thresholds were applied to different foci. 43 verlap of ALE maps produced by monetary, food and erotic reward studies. The ALE maps in consistently and commonly activated by monetary, erotic and food reward outcomes. The i.e. the degree of consistency across studies. The maps on the right illustrate the overlap of ary rewards, red = erotic rewards, blue = food rewards). The ALE maps are overlaid on the whole-brain corrected and cluster size > 600 mm3). Fig. 1. “Common reward circuit” as defined by the overlap of ALE maps produced by monetary, food and erotic reward studies. The ALE maps in he three leftmost columns show the brain regions consistently and commonly activated by monetary, erotic and food reward outcomes. The colour scale indicates the magnitude of ALE values, i.e. the degree of consistency across studies. The maps on the right illustrate the overlap of activation clusters across rewards (green = monetary rewards, red = erotic rewards, blue = food rewards). The ALE maps are overlaid on the Colin brain provided with GingerALE (p < 0.01 FDR whole-brain corrected and cluster size > 600 mm3). Fig. 1. “Common reward circuit” as defined by the overlap of ALE maps produced by monetary, food and erotic reward studies. The ALE maps in the three leftmost columns show the brain regions consistently and commonly activated by monetary, erotic and food reward outcomes. The colour scale indicates the magnitude of ALE values, i.e. the degree of consistency across studies. Tables The maps on the right illustrate the overlap of activation clusters across rewards (green = monetary rewards, red = erotic rewards, blue = food rewards). The ALE maps are overlaid on the Colin brain provided with GingerALE (p < 0.01 FDR whole-brain corrected and cluster size > 600 mm3). g. 1. “Common reward circuit” as defined by the overlap of ALE maps produced by monetary, food and erotic reward studies. The ALE maps in e three leftmost columns show the brain regions consistently and commonly activated by monetary, erotic and food reward outcomes. The olour scale indicates the magnitude of ALE values, i.e. the degree of consistency across studies. The maps on the right illustrate the overlap of ctivation clusters across rewards (green = monetary rewards, red = erotic rewards, blue = food rewards). The ALE maps are overlaid on the olin brain provided with GingerALE (p < 0.01 FDR whole-brain corrected and cluster size > 600 mm3). 44 Fig. 2. Brain regions more reliably activated by one reward comp contrasts between the different meta-analyses: “money-specific” maps, “erotic-specific” regions (red) result from the conjunction o from the conjunction of food > money and food > erotic maps. “P money and food > money maps. The ALE maps are overlaid on and cluster size > 600 mm3; except for the primary reward-speci size = 350 mm3). Fig. 2. Brain regions more reliably activated by one reward compared to the other two (i.e. “reward type-specific”). These regions result from contrasts between the different meta-analyses: “money-specific” regions (green) result from the conjunction of money > erotic and money > food maps, “erotic-specific” regions (red) result from the conjunction of erotic > money and erotic > food maps, and “foodspecific” regions (blue) result from the conjunction of food > money and food > erotic maps. “Primary reward-specific” regions (purple) result from the conjunction of erotic > money and food > money maps. The ALE maps are overlaid on the Colin brain provided with GingerALE (p < 0.05 FDR whole-brain corrected and cluster size > 600 mm3; except for the primary reward-specific activation in the left insula: p < 0.05 FDR whole-brain corrected and cluster size = 350 mm3). 45 Fig. 3. Overview of the brain regions involved in reward outcome processing as a function of reward type. (A) “Common reward circuit”, i.e. the brain responding to monetary, erotic and food rewards. Tables The putative main functional role assigned to each region is mentioned in italics. Rewards eliciting a particularly robust activation in certain regions are mentioned in square brackets. (B) Reward type-specific regions, i.e. more reliably activated by one reward compared to the other two. The putative main functional role assigned to each region is mentioned in italics. Green: “money-specific” regions, Red: “erotic-specific” regions, Blue: “food-specific” regions, Fuchsia: “primary reward-specific”. Fig. 3. Overview of the brain regions involved in reward outcome processing as a function of reward type. (A) “Common reward circuit”, i.e. the brain responding to monetary, erotic and food rewards. The putative main functional role assigned to each region is mentioned in italics. Rewards eliciting a particularly robust activation in certain regions are mentioned in square brackets. (B) Reward type-specific regions, i.e. more reliably activated by one reward compared to the other two. The putative main functional role assigned to each region is mentioned in italics. Green: “money-specific” regions, Red: “erotic-specific” regions, Blue: “food-specific” regions, Fuchsia: “primary reward-specific”. Fig. 3. Overview of the brain regions involved in reward outcome processing as a function of reward type. (A) “Common reward circuit”, i.e. the brain responding to monetary, erotic and food rewards. The putative main functional role assigned to each region is mentioned in italics. Rewards eliciting a particularly robust activation in certain regions are mentioned in square brackets. (B) Reward type-specific regions, i.e. more reliably activated by one reward compared to the other two. The putative main functional role assigned to each region is mentioned in italics. Green: “money-specific” regions, Red: “erotic-specific” regions, Blue: “food-specific” regions, Fuchsia: “primary reward-specific”. 46
https://openalex.org/W2612990438	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0177608&type=printable	English	null	Molecular characterization, expression of chicken TBK1 gene and its effect on IRF3 signaling pathway	PloS one	2,017	cc-by	9,155	RESEARCH ARTICLE Data Availability Statement: All the data are shown in the Figures and Tables. Funding: This work was financially supported by the Twelfth Five Year Plan for Breeding Program in Sichuan (No. 2011NZ0099-7), the thirteenth Five Year Plan for Breeding Program in Sichuan (No. 2016NYZ0050), the China Agriculture Research System (No. CARS-41) and National Natural Science Foundation of China (No. 31601936). Molecular characterization, expression of chicken TBK1 gene and its effect on IRF3 signaling pathway Yan Wang1☯, Yue Yin2☯, Xi Lan1☯, Fei Ye1☯, Kai Tian1, Xiaoling Zhao1, Huadong Yin1, Diyan Li1, Hengyong Xu1, Yiping Liu1, Qing Zhu1* Yan Wang1☯, Yue Yin2☯, Xi Lan1☯, Fei Ye1☯, Kai Tian1, Xiaoling Zhao1, Huadong Yin1, Diyan Li1, Hengyong Xu1, Yiping Liu1, Qing Zhu1* 1 Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu, China, 2 Institute of Laboratory Animal of Sichuan Academy of Medical Sciences & Sichuan Provincial People’s Hospital, Chengdu, China a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 ☯These authors contributed equally to this work. * zhuqing5959@163.com ☯These authors contributed equally to this work. * zhuqing5959@163.com Editor: Dong-Yan Jin, University of Hong Kong, HONG KONG Editor: Dong-Yan Jin, University of Hong Kong, HONG KONG Received: February 15, 2017 Accepted: April 28, 2017 Published: May 11, 2017 Copyright: © 2017 Wang 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: February 15, 2017 Accepted: April 28, 2017 Published: May 11, 2017 Copyright: © 2017 Wang 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. Abstract TRAF family member-associated NF-κB activator (TANK)-binding kinase1 (TBK1) is a ser- ine-threonine kinase at the crossroads of multiple interferon (IFN)-inducing signaling path- ways in innate immunity. The importance of TBK1 in antiviral immunity is well established in mammal models, but in chicken, the molecular characterization and potential function of TBK1 remain unclear. In the present study, the open-reading frame (ORF) of chicken TBK1 (chTBK1) was cloned and characterized. The sequencing results revealed that the chTBK1 ORF consists of 2190 base pairs (bp) encoding a deduced protein of 729 amino acid resi- dues. Multiple sequence alignment analysis demonstrated chTBK1 similarity to other birds and mammals, which indicates that it is evolutionarily conserved. Quantitative real-time PCR (qRT-PCR) results showed that chTBK1 was ubiquitously expressed in chicken tis- sues and expression was especially high in immune tissues. In addition, the expression of chTBK1 was significantly up-regulated by infection with avian leukosis virus subgroup J (ALV-J) both in vivo and in chicken embryo fibroblasts (CEFs) challenged with ALV-J or stimulated with poly I:C in vitro. Consistent with the activation of chTBK1, the interferon reg- ulatory factor 3 (IRF3) and IFNβ gene in CEFs were also up-regulated after challenge with ALV-J or polyI:C. In contrast, the expression of IRF3 and IFNβ in CEFs was significantly reduced by siRNA targeting the chTBK1 gene compared with a negative control (NC) during ALV-J infection or polyI:C transfection. In conclusion, our results demonstrated that chTBK1 may be an important immunoregulator for IRF3 and IFNβ induction in response to viral stim- ulation in chicken. OPEN ACCESS Citation: Wang Y, Yin Y, Lan X, Ye F, Tian K, Zhao X, et al. (2017) Molecular characterization, expression of chicken TBK1 gene and its effect on IRF3 signaling pathway. PLoS ONE 12(5): e0177608. https://doi.org/10.1371/journal. pone.0177608 Citation: Wang Y, Yin Y, Lan X, Ye F, Tian K, Zhao X, et al. (2017) Molecular characterization, expression of chicken TBK1 gene and its effect on IRF3 signaling pathway. PLoS ONE 12(5): e0177608. https://doi.org/10.1371/journal. pone.0177608 Editor: Dong-Yan Jin, University of Hong Kong, HONG KONG Molecular characterization and function of chicken TBK1 gene which belongs to the inhibitor of κB kinase (IKK) family [3]. As a non-canonical IKK family member, TBK1 possesses 61% sequence identity with IKKε (another non-canonical IKK member) and shares 27% primary sequence identity with IKKα/β (the canonical IKK mem- bers) [4]. Due to the sequence similarity, TBK1 is structurally identical to the other IKKs pro- teins, which present a trimodular architecture with an N-terminal kinase domain, an ubiquitin-like domain (absent in IKKα) and an α-helical scaffold/dimerization domain [5]. The kinase domain of TBK1, which contains the catalytic activity, is highly conserved [6]. The ubiquitin-like domain, which is adjacent to the kinase domain, is a regulatory component of the TBK1 kinase that positively controls TBK1 kinase activation and signal transduction [7]. The scaffold/dimerization domain, which extensively interacts with the kinase domain and the ubiquitin-like domain, contributes to the dimerization and substrate recognition of TBK1 [5]. In innate immunity, TBK1 was proposed to be a hub protein, which has been implicated in various pattern recognition receptors (PRRs). For instance, in the endosomal membrane, Toll- like receptor 4 (TLR4) and TLR3 recognized bacterial endotoxin or lipopolysaccharide (LPS) and virus or double-stranded RNA (dsRNA), respectively. Both TLR4 and TLR3 activated TBK1 via the recruitment of the adaptor protein called Toll/IL-1 receptor domain-containing adaptor-inducing IFNβ (TRIF) and tumor necrosis factor receptor-associated factor 3 (TRAF3) [8]. In the cytoplasm, the presence of virus particles or dsRNA initiated immune responses via retinoic acid-inducible gene I (RIG-1)-like receptors (RLRs), such as RIG-1 and melanoma differentiation-associated gene 5 (MDA-5), which assemble another adaptor mole- cule termed mitochondrion-associated adapter IFNβ promoter stimulator 1 (MAVS) as well as TRAF3 to evoke TBK1 [9,10]. Recently, an intracellular adaptor protein stimulator of inter- feron genes called STING, which was activated by cytoplasmic DNA receptors, also funneled into TBK1-specific activation [11, 12]. Initial studies showed that TBK1 possesses the ability to induce IκB degradation and NF-κB activity via TANK and IKKβ [13,14]. However, the role of TBK1 in NF-κB signaling pathways is controversial. TBK1 null fibroblasts exhibited normal NF-κB activation in response to tumor necrosis factor α (TNFα), interleukin-1 and virus infection [15]. Interestingly, following those discoveries, TBK1 and IKKε, a close homolog of TBK1, were confirmed as important kinases for IFN signaling induction [16]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0177608 May 11, 2017 Introduction TBK1, also termed NF-κB activating kinase or TRAF2-associated kinase, was identified as an important adaptor molecule linking upstream receptor signals to downstream gene activation in apoptotic, inflammatory and immune responses [1,2]. TBK1 is a serine-threonine kinase, Competing interests: The authors declare no conflicts of interest. PLOS ONE \| https://doi.org/10.1371/journal.pone.0177608 May 11, 2017 1 / 17 Cloning and sequencing of chicken TBK1 Based on the published reference sequence of TBK1 from Gallus gallus (GenBank accession number: NM_001199558.1), a pairs of gene specific primers (Table 1) for the amplification of the complete coding region were designed using Primer Premier 5 software (Premier BioSoft, Palo Alto, CA, USA). Spleen complementary DNA was used as the template for amplification. Polymerase chain reaction (PCR) was carried out with the initial denaturation at 94˚C for 5 min and 34 cycles of 94˚C for 45 sec, 60˚C for 30 sec and 72˚C for 1 min. A final elongation step was conducted at 72˚C for 10 min. The PCR fragments were purified by the Gel Extrac- tion Mini Kit (Qiagen, Germany) and were cloned into the pMD-19T vector (Takara, Japan). The plasmid DNA was isolated using a Plasmid Mini Kit (Qiagen, Germany) and was then sequenced by BGI Biotechnology Company (Beijing, China). Ethics statement The animal studies and sample collection were conducted according to the guidelines approved by the Sichuan Agricultural University Institutional Animal Care and Use Commit- tee in College of Animal Science and Technology, Sichuan Agricultural University, Sichuan, China (DKY-B201000805). Although both of the kinases have been found to involve IRF3/7 and they both share the common upstream adaptors, TBK1 and IKKε have largely distinct functions. TBK1 plays an indispensable role in IFN induction because the acti- vation of IRF3 and IFNβ is impaired in TBK1-defective (not IKKε-defective) murine fibro- blasts after virus infection [17]. Later, two important observations showed that TBK1 mainly functioned on ubiquitously expressed IRF3 to induce type I interferon activation, whereas IKKε preferred IRF7 and is required for the activation of IFN-stimulated genes by STAT1 [18,19]. While TBK1 was found to be constitutively expressed in many cells, IKKε was found to be primarily expressed in the cells of lymphoid tissues [18]. All of these results indicated that TBK1 functions as a key node protein to induce an early phase of IFN responses after chal- lenge by viruses and bacteria. TBK1 has been well-characterized in mammals, but few studies have investigated the sequence, tissues distribution and its possible function in chickens. To investigate these ques- tions, we cloned and characterized the full-length coding sequence of chTBK1 and examined the tissue-specific expression of chTBK1 mRNA. Moreover, we detected chTBK1 expression differences both in vivo and in vitro in chickens infected with ALV-J and in CEFs transfected with polyI:C, respectively. In addition, we measured the relative expression of IRF3 and IFNβ in CEFs stimulated with ALV-J or polyI:C. Furthermore, we examined the role of chTBK1 in TBK1-impaired CEFs challenged with ALV-J and polyI:C. 2 / 17 PLOS ONE \| https://doi.org/10.1371/journal.pone.0177608 May 11, 2017 Molecular characterization and function of chicken TBK1 gene F = forward primer, R = for reverse primer; AT-Annealing temperature (˚C) F = forward primer, R = for reverse primer; AT-Annealing temperature (˚C) F = forward primer, R = for reverse primer; AT-Annealing temperature (˚C) https://doi.org/10.1371/journal.pone.0177608.t001 ALV-J challenge and sample collection On the hatch day, 120 birds were randomly divided into two groups: one group was intraperi- toneally inoculated with a total ALV-J virus dose of 106 TCID50 in 0.4 ml, and the other group was treated with an equal volume of phosphate buffered saline only (0.4 ml) as the mock con- trol. Five chickens of each group were euthanized at 3, 5, 7, 9, 14, 24 and 42 days post-infection (dpi), and the immune tissues, including spleen, bursa of Fabricius and thymus, were collected and preserved at -80˚C. RNA extraction and reverse transcription Total RNA was extracted from tissue samples or CEFs using the TRIzol reagent (Invitrogen, USA) according to the manufacturer’s instructions. The RNA was eluted in 30 μl DNase/ RNase-Free water (Takaka, Japan) and was stored at -80˚C for reverse transcription. Total RNA was extracted from tissue samples or CEFs using the TRIzol reagent (Invitrogen, USA) according to the manufacturer’s instructions. The RNA was eluted in 30 μl DNase/ RNase-Free water (Takaka, Japan) and was stored at -80˚C for reverse transcription. Before reverse transcription, the total RNA for integrity and quality using electrophoresis (1% agarose gels with ethidium bromide) and a Nanodrop spectrophotometer (Nanodrop 2000C, Thermo Scientific, USA), respectively. Reverse transcription was carried out using the PrimeScript RT reagent Kit with gDNA Eraser (Takaka, Japan) according to the manufactur- er’s instructions Before reverse transcription, the total RNA for integrity and quality using electrophoresis (1% agarose gels with ethidium bromide) and a Nanodrop spectrophotometer (Nanodrop 2000C, Thermo Scientific, USA), respectively. Reverse transcription was carried out using the PrimeScript RT reagent Kit with gDNA Eraser (Takaka, Japan) according to the manufactur- er’s instructions. Molecular characterization and function of chicken TBK1 gene small intestine, brain, pectoral muscle and leg muscle of each chicken were harvested and immediately snap frozen in liquid nitrogen for total RNA extraction. Cell culture and treatment, RNA interference experiments CEFs were harvested from the 9-day-old embryonated SPF chicken eggs and were cultured in Dulbecco’s minimal essential medium containing 10% fetal bovine serum (Gibco, USA), 100 U/ml penicillin, and 100 μg/ml streptomycin. The cells were seeded in 12-well plates at 10×104 cells/ml and were maintained at 37˚C in a 5% CO2/air environment. At 80%-90% confluency, the cells were washed in serum-free medium and stimulated with ALV-J or polyI: C (Invitro- gen, USA). For virus challenge, the cells were incubated for 1 hour with 200 μl ALV-J virus (multiplicity of infection of 1) per well. PolyI:C was transfected into the cells using Lipofecta- mine 2000 (Invitrogen, USA) according to the manufacturer’s instructions. At 2, 6, 12 and 20 hours post-infection (hpi), the cells were collected in TRIzol reagent for further analysis. A small interfering RNA (siRNA) targeting the chTBK1 gene was designed and synthesized by a commercial company (Invitrogen, USA). Two siRNA sequences targeting 59–79 (siTBK1.1) and 983–1083 (siTBK1.2) of the chTBK1 ORF were selected with the corresponding siRNA sequences of 5’-GCTGTTGTCTGACATTCTAGG-3’ (siTBK1.1) and 5’-GCAGATGACCTTG CACAAAGT-3’(siTBK1.2), respectively. The negative control (NC) oligonucleotides were pur- chased from Invitrogen. SiRNA duplexes were transfected into cells using Lipofectamine 2000 according the manufacturer’s instructions. After 4 hours, the mix was removed and was replaced with DMEM with 5% FBS. At 22, 28 and 36 hours post-transfection, the cells were collected in TRIzol reagent to verify the knockdown of chTBK1 (to detect the interference efficiency). In addition, at 24 hours post-transfection, the cells were challenged with virus and polyI:C to evalu- ate the influence of chTBK1 knockdown on the expression of endogenous genes. PLOS ONE \| https://doi.org/10.1371/journal.pone.0177608 May 11, 2017 Viruses, chicken and tissue collection The ALV-J virus used in this study was obtained from the Veterinary Medicine of Sichuan Agricultural University (Ya’an, China) and was stored at -80˚C until inoculation. All of the experiments using infectious virus were conducted in biosafety level 2 (BSL-2) facilities at the Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Agri- cultural University (Ya’an, China). Specific pathogen free (SPF) chicken eggs (White Leghorn) were purchased from Merial Company (Beijing, China). The eggs hatched in the Sichuan Agricultural University (Ya’an, China) poultry farm and were reared in filtered-air, positive-pressure isolators with free access to feed and water (Sichuan, China). To detect the tissue distribution of chTBK1, three healthy chickens were euthanized at the age of 3 weeks and 11 tissue samples, including the heart, liver, spleen, lung, kidney, thymus, Table 1. Gene-specific primers used for clone and RT-PCR analyses in this study. Purpose Primer Sequence (5’-3’) Product size/bp AT Clone chTBK1-F CTCGAGATGCAGAGCACCTCGAATT 2200 60 chTBK1-R GGATCCGAGATACAGTCCACATTCC RT-PCR TBK1-F GGTTTGCCAGAATCGGAGT 227 61 TBK1-R TGTAAATACTCCTCTGTGCCGT GAPDH-F AGGACCAGGTTGTCTCCTGT 153 57 GAPDH-R CCATCAAGTCCACAACACGG IRF3-F TACACTGAGGACTTGCTGGAGGT 170 62 IRF3-R AAGATGGTGGTCTCCTGATCC IFNβ-F CCTCAACCAGATCCAGCATTAC 167 59 IFNβ-R CCCAGGTACAAGCACTGTAGTT Env-F TGCGTGCGTGGTTATTATTTC 144 56.6 Env-R AATGGTGAGGTCGCTGACTGT F = forward primer R = for reverse primer; AT Annealing temperature (˚C) Table 1. Gene-specific primers used for clone and RT-PCR analyses in this study. Table 1. Gene-specific primers used for clone and RT-PCR analyses in this study. PLOS ONE \| https://doi.org/10.1371/journal.pone.0177608 May 11, 2017 3 / 17 Molecular characterization and function of chicken TBK1 gene specific primer, 5 μl Ssofast EvaGreen (Bio-Rad) and 3.4 μl ddH2O. The optimum thermal cycling conditions were as follows: 98˚C for 2 min, 39 cycles of 98˚C for 10 s, 30 s at optimum temperatures, 72˚C for 10 s, and a final extension for 5 min with a temperature increment of 0.5˚C/s from 65˚C to 95˚C. The specificity of the qRT-PCR products were confirmed via melt- ing curve analysis and 1.5% agarose gel analysis. The ALV-J genome load in the ALV-J- infected chicken tissues was quantified using primers specific for the ALV-J Env gene. The rel- ative expression levels were calculated by the comparative Ct (2-ΔΔCt) method using glyceralde- hyde-3-phosphate-dehydrogenase (GAPDH) gene as an endogenous reference gene. The gene-specific primers used for qRT-PCR of ALV-J Env, GAPDH, chTBK1, IRF3 and IFNβ were listed in Table 1. Bioinformatics and statistical analysis The obtained nucleotide sequences were analyzed at NCBI (http://blast.ncbi.nlm.nih.gov/) and were compared against the sequence database using the BLAST server (http://www.ncbi. nlm.nih.gov/blast). The conserved domain prediction was analyzed by the SMART program (http://smart.emblheidelberg.de/). Multiple amino acid sequences alignment was performed using ClustalX 1.83. The secondary structure was predicted by the SOPMA software (http:// npsa-pbil.ibcp.fr/). The phylogenetic tree was constructed using the MEGA 5.05 software. The GenBank accession numbers of TBK1 from different species for the phylogenetic analysis were listed in Table 2. The relative gene expression levels were calculated relative to the expression of the GAPDH gene, which served as an endogenous reference gene. The statistical analysis was performed using SAS 9.0 through one-way ANOVA. The means ± SEM results were plotted to the figures using GraphPad Prism 5 software (GraphPad Software, La Jolla, CA, USA). The comparisons were considered significant at P<0.05 and extremely significant at P<0.01. PLOS ONE \| https://doi.org/10.1371/journal.pone.0177608 May 11, 2017 https://doi.org/10.1371/journal.pone.0177608.t002 Quantitative real-time PCR The expression of target genes was analyzed by quantitative Real-time PCR (qRT-PCR). qRT-PCR was performed on a CFX-96 qPCR thermal cycle instrument (Bio-Rad) and was car- ried out in a total volume of 10 μl with 1.0 μl complementary DNA preparations, 0.3 μl of each The expression of target genes was analyzed by quantitative Real-time PCR (qRT-PCR). qRT-PCR was performed on a CFX-96 qPCR thermal cycle instrument (Bio-Rad) and was car- ried out in a total volume of 10 μl with 1.0 μl complementary DNA preparations, 0.3 μl of each PLOS ONE \| https://doi.org/10.1371/journal.pone.0177608 May 11, 2017 4 / 17 Bioinformatic analysis of the chicken TBK1 gene We successfully cloned and sequenced the full-length coding sequence of chTBK1 based on the predicted TBK1 sequence of Gallus gallus (GenBank accession number: NM_001199558.1). Table 2. GenBank accession numbers of TBK1 homologs used in this study. Table 2. GenBank accession numbers of TBK1 homologs used in this study. Name of species Accession number Gallus gallus NP_001186487.1 Meleagris gallopavo XP_003202091.1 Taeniopygia guttata XP_002188051.2 Columba livia XP_005499866.1 Melopaittaous undulatus XP_005148126.1 Oryctolagus cunioulus XP_008254959.1 Homo sapiens NP_037386.1 Ovis aries XP_004006546.1 Capra hircus XP_005680278.1 Bos taurus NP_001179684.1 Sus scrofa NP_001098762.1 Mus musoulus NP_062760.3 Ornithorhynchus anatinus XP_007666451.1 Danio rerio NP_001038213.2 https://doi org/10 1371/journal pone 0177608 t002 5 / 17 PLOS ONE \| https://doi.org/10.1371/journal.pone.0177608 May 11, 2017 Molecular characterization and function of chicken TBK1 gene The full-length coding sequence of chTBK1 was 2190 bp encoding a putative protein of 729 amino acids residues (Fig 1A). Based on NCBI CD-Search and SMART software analysis, two conserved domains including kinase domain and ubiquitin-like domain were predicted (Fig 1B). The secondary structure analysis using SOPMA software indicated that chicken TBK1 was comprised of 47.19% α-helixes, 26.47% random coils connected by 19.07% extended strands and 7.27% β-turns (Fig 1C). The multiple alignments revealed that chTBK1 shares the highest identity (99.5%) with Gallus gallus followed by Meleagris gallopavo (97.4%), Teaniopy- gia gttata (95.9%), Columba livia (94.8%), Melopaittaous undulatus (93.4%), Oryctolagus cunioulus (86.7%), Homo sapiens (86.3%), Ovis aries (85.7%), Capre hirous (85.9%), Bos taurus (86.0%), Sus scrofa (86.0%), Mus musoulus (85.6%), Ornithorhynous anatinus (86.1%) and dis- played relatively low identity with Danio rerio (70.1%) (Fig 2). As observed in Fig 2, the ATP- binding site and S172 residue among the kinase domain of TBK1 were identical. To elucidate the genetic relationships of the chicken TBK1 protein with other species, we constructed a phylogenetic tree using MEGA 5.0 software with the neighbor joining (NJ) method and 1000 replicates (Fig 3). The TBK1 amino acid sequence from mammals, birds and fish segregated into 3 separate clusters, and the chicken TBK1 was grouped into the birds cluster containing Meleagris gallopavo, Teaniopygia gttata, Columba livia and Melopaittaous undulatus but was phylogenetically separated from that of other mammalian species. The phylogenetic tree analy- sis suggested that chTBK1 had a closer genetic relationship with that of other birds in compari- son to other vertebrates. These results were consistent with previous studies. Tissue distribution of chTBK1 mRNA in healthy chicken To assess the mRNA expression profile of the chTBK1 gene, 11 tissues were collected, and the transcript levels of target gene were compared using the GAPDH gene as the internal reference gene for normalization. As shown in Fig 4, the chTBK1 gene was ubiquitously expressed in all of the examined tissues. The most abundant chTBK1 expression levels were found in spleen, lung, thymus, bursa of Fabricius, which was followed by intestine, heart, kidney and brain. Low expression levels were found in the other tissues, including liver, pectoral muscle and leg mus- cle. The statistically analysis revealed that the transcripts level of chTBK1 in spleen, lung, thy- mus, bursa of Fabricius were significantly (P<0.01) higher than those in the other 7 tissue types. Response of chTBK1 in response to ALV-J infection in vivo To determine if chTBK1 is involved in chicken immune responses, the expression level of chTBK1 and the ALV-J Env gene at different time points following infection with ALV-J in spleen, bursa of Fabricius and thymus were determined by qRT-PCR. As shown in Fig 5A, the expression of chTBK1 was up-regulated after ALV-J infection when compared to the non- infected group. In the spleen, chTBK1 was activated at 5 (P<0.01), 7 (P<0.05) and 14 (P<0.01) dpi. In the bursa of Fabricius, a similar variation trend was found, but chTBK1 was increased at 5(P<0.05), 7 (P<0.05) and 14 (P<0.01) dpi. In the thymus, chTBK1 was up-regulated only at 7 (P<0.05) and 14 (P<0.01) dpi. As shown in Fig 5B, the ALV-J Env gene was quantifiable as early as 5 dpi in the spleen, and was detected later in the bursa of Fabricius. In the spleen, the expression of the ALV-J Env gene increased quickly, peaked at 14 dpi and maintained a higher expression level at 28 and 42 dpi. In the bursa of Fabricius and thymus, the expression of the ALV-J Env gene peaked at 14 dpi but phased down immediately. https://doi.org/10.1371/journal.pone.0177608.g001 Induction of chTBK1, IRF3 and IFNβ after in vitro challenge with ALV-J To further characterize the role of the chTBK1, we next investigated the potential role of chTBK1 in cellular antiviral responses. As only TBK1 (not IKKε) was found in mouse 6 / 17 PLOS ONE \| https://doi.org/10.1371/journal.pone.0177608 May 11, 2017 Molecular characterization and function of chicken TBK1 gene Fig 1. The chTBK1 ORF sequence, deduced amino acid sequence and the second structure of chTBK1. A. The translation start (ATG) codon is in bold and the stop (TGA) codon is markerd by asterisis (), the protein kinase domain is underlined and the ubiquitin-like domain is boxed. B. Schematic conserved domain structure of TBK1 protein, showing the protein kinase domain (KD) and ubiquitin-like domain (ULD). C. The longest lines, second longest lines, third longest lines and shortest lines stand for Alpha helixs (Hh), extended strands (Ee), beta turns (Tt), and random coils (Cc), respectively. Fig 1. The chTBK1 ORF sequence, deduced amino acid sequence and the second structure of chTBK1. A. The translation start (ATG) codon is in bold and the stop (TGA) codon is markerd by asterisis (), the protein kinase domain is underlined and the ubiquitin-like domain is boxed. B. Schematic conserved domain structure of TBK1 protein, showing the protein kinase domain (KD) and ubiquitin-like domain (ULD). C. The longest lines, second longest lines, third longest lines and shortest lines stand for Alpha helixs (Hh), extended strands (Ee), beta turns (Tt), and random coils (Cc), respectively. Fig 1. The chTBK1 ORF sequence, deduced amino acid sequence and the second structure of chTBK1. A. The translation start (ATG) codon is in bold and the stop (TGA) codon is markerd by asterisis (), the protein kinase domain is underlined and the ubiquitin-like domain is boxed. B. Schematic conserved domain structure of TBK1 protein, showing the protein kinase domain (KD) and ubiquitin-like domain (ULD). C. The longest lines, second longest lines, third longest lines and shortest lines stand for Alpha helixs (Hh), extended strands (Ee), beta turns (Tt), and random coils (Cc), respectively. https://doi.org/10.1371/journal.pone.0177608.g001 PLOS ONE \| https://doi.org/10.1371/journal.pone.0177608 May 11, 2017 7 / 17 Molecular characterization and function of chicken TBK1 gene Fig 2. Multiple sequence alignment of TBK1 protein sequence of chicken and other 14 species by the ClustalW program. Residues shaded in black are completely conserved across all species aligned, and residues shaded in grey refer to 80–90% identity. The ATP binding site is in green, the S172 is in blue. Induction of chTBK1, IRF3 and IFNβ after in vitro challenge with polyI:C The dsRNA generated during the virus replication could rapidly trigger host innate immune responses. To explore whether chTBK1 was implicated in chicken cell responses to dsRNA stimulation, the CEFs were transfected with polyI:C (a synthetic dsRNA) and were harvested at different time points. As shown in Fig 7, following transfection with 1.0 μg/ml polyI:C the transcription level of chTBK1, IRF3 and IFNβ in CEFs gradually increased from 2 hours and peaked at 6 hours. Induction of chTBK1, IRF3 and IFNβ after in vitro challenge with ALV-J Fig 2. Multiple sequence alignment of TBK1 protein sequence of chicken and other 14 species by the ClustalW program. Residues shaded in black are completely conserved across all species aligned, and residues shaded in grey refer to 80–90% identity. The ATP binding site is in green, the S172 is in blue. 8 / 17 PLOS ONE \| https://doi.org/10.1371/journal.pone.0177608 May 11, 2017 Molecular characterization and function of chicken TBK1 gene Fig 3. Phylogenetic trees analysis of chTBK1 proteins from different species. The phylogram was constructed with the MEGA 5.05 software using the neighbor-joining method based on an amino acid alignment (Clustal W) of the full-length protein. The scale bar indicates the branch length, and the bootstrap confidence values were shown at the nodes of the tree. https://doi.org/10.1371/journal.pone.0177608.g003 Fig 3. Phylogenetic trees analysis of chTBK1 proteins from different species. The phylogram was constructed with the MEGA 5.05 software using the neighbor-joining method based on an amino acid alignment (Clustal W) of the full-length protein. The scale bar indicates the branch length, and the bootstrap confidence values were shown at the nodes of the tree. https://doi.org/10.1371/journal.pone.0177608.g003 https://doi.org/10.1371/journal.pone.0177608.g003 embryonic fibroblasts by Northern blotting analysis, we choose CEFs as the cell model. After challenge with ALV-J, the chTBK1 gene sharply increased from 2 hpi and peaked at 6 and 12 hpi (Fig 6). Concurrently, the expression levels of IRF3 and IFNβ peaked at 6 hpi and then gradually decreased. Meanwhile, the ALV-J Env gene increased exponentially at 2, 6, 12 and 20 hpi. Induction of chTBK1, IRF3 and IFNβ after in vitro challenge with polyI:C ChTBK1 knockdown blocks IRF3 as well as IFNβ activation As expected, the expression of chTBK1 sharply decreased in CEFs transfected with siTBK1.1 and siTBK1.2 compared to the NC, regardless of whether the CEFs were not challenged or were challenged with ALV-J or polyI:C (Fig 8B). Interestingly, the transcription level of IRF3 and IFNβ were significantly reduced in chTBK1 knock-down CEFs challenged with ALV-J or polyI:C, as shown in Fig 8C and 8D, respectively. ChTBK1 knockdown blocks IRF3 as well as IFNβ activation To further understand the function of chTBK1, chTBK1 was knocked down in the CEFs. Two small interfering RNAs (siRNA) targeting different portions of chTBK1 (siTBK1.1, siTBK1.2) and a negative control (NC) siRNA were prepared. Before chTBK1 knock-down in CEFs chal- lenged with ALV-J or polyI:C, a pilot experiment evaluated the effectiveness of siTBK1.1 and siTBK1.2. As shown in Fig 8A, both chTBK1 siRNAs led to a 40–50% reduction of chTBK1 PLOS ONE \| https://doi.org/10.1371/journal.pone.0177608 May 11, 2017 9 / 17 Molecular characterization and function of chicken TBK1 gene Fig 4. Relative mRNA expression of chTBK1 gene in different tissues, including heart, liver, spleen, lung, kidney, thymus, brain, bursa of Fabricius, pectoral muscle, leg muscle, intestine. Data above were presented as mean ± SEM (n = 3). Error bars show the SEM of triplicate. Columns sharing different letters show extremely significant difference (P<0.01). https://doi.org/10.1371/journal.pone.0177608.g004 Fig 4. Relative mRNA expression of chTBK1 gene in different tissues, including heart, liver, spleen, lung, kidney, thymus, brain, bursa of Fabricius, pectoral muscle, leg muscle, intestine. Data above were presented as mean ± SEM (n = 3). Error bars show the SEM of triplicate. Columns sharing different letters show extremely significant difference (P<0.01). transcription level in CEFs at 22–36 hours post-transfection compared to the parallel control. We the challenged CEFs with ALV-J and poly I:C 24 hours after transfection with siTBK1, and 4 hours later all the cells were collected. As shown in Fig 8, the expression of chTBK1, IRF3 and IFNβ was determined by qRT-PCR. As expected, the expression of chTBK1 sharply decreased in CEFs transfected with siTBK1.1 and siTBK1.2 compared to the NC, regardless of whether the CEFs were not challenged or were challenged with ALV-J or polyI:C (Fig 8B). Interestingly, the transcription level of IRF3 and IFNβ were significantly reduced in chTBK1 knock-down CEFs challenged with ALV-J or polyI:C, as shown in Fig 8C and 8D, respectively. transcription level in CEFs at 22–36 hours post-transfection compared to the parallel control. We the challenged CEFs with ALV-J and poly I:C 24 hours after transfection with siTBK1, and 4 hours later all the cells were collected. As shown in Fig 8, the expression of chTBK1, IRF3 and IFNβ was determined by qRT-PCR. Discussion TBK1 is a critical adaptor molecule that is involved in the TLRs, RLRs and STING signaling pathways that induce type I IFN production after virus infection in mammals. To date, TBK1 has been well-characterized in a number of mammalian species and teleosts [20], but few stud- ies have been conducted in avian species. In this study, we cloned and characterized the PLOS ONE \| https://doi.org/10.1371/journal.pone.0177608 May 11, 2017 10 / 17 Molecular characterization and function of chicken TBK1 gene Fig 5. Relative mRNA expression of ALV-J Env and chTBK1 gene in vitro challenge with ALV-J in different immune tissues. A Relative mRNA expression of chTBK1 gene in the spleen, bursa, and thymus after challenge with ALV-J. Data above were presented as mean ± SEM (n = 3). Error bars show the SEM of triplicate. () and (*) represent significant (P < 0.05) and extremely significant (P <0.01) difference, respectively. B Relative mRNA expression of ALV-J Env gene in the spleen, bursa, and thymus of ALV-J infected chickens. https://doi org/10 1371/journal pone 0177608 g005 Fig 5. Relative mRNA expression of ALV-J Env and chTBK1 gene in vitro challenge with ALV-J in different immune tissues. A Relative mRNA expression of chTBK1 gene in the spleen, bursa, and thymus after challenge with ALV-J. Data above were presented as mean ± SEM (n = 3). Error bars show the SEM of triplicate. () and (*) represent significant (P < 0.05) and extremely significant (P <0.01) difference, respectively. B Relative mRNA expression of ALV-J Env gene in the spleen, bursa, and thymus of ALV-J infected chickens. https://doi org/10 1371/journal pone 0177608 g005 Fig 5. Relative mRNA expression of ALV-J Env and chTBK1 gene in vitro challenge with ALV-J in different immune tissues. A Relative mRNA expression of chTBK1 gene in the spleen, bursa, and thymus after challenge with ALV-J. Data above were presented as mean ± SEM (n = 3). Error bars show the SEM of triplicate. () and (*) represent significant (P < 0.05) and extremely significant (P <0.01) difference, respectively. B Relative mRNA expression of ALV-J Env gene in the spleen, bursa, and thymus of ALV-J infected chickens. Fig 5. Relative mRNA expression of ALV-J Env and chTBK1 gene in vitro challenge with ALV-J in different immune tissues. A Relative mRNA expression of chTBK1 gene in the spleen, bursa, and thymus after challenge with ALV-J. PLOS ONE \| https://doi.org/10.1371/journal.pone.0177608 May 11, 2017 Discussion Data above were presented as mean ± SEM (n = 3). Error bars show the SEM of triplicate. () and (*) represent significant (P < 0.05) and extremely significant (P <0.01) difference, respectively. B Relative mRNA expression of ALV-J Env gene in the spleen, bursa, and thymus of ALV-J infected chickens. https://doi.org/10.1371/journal.pone.0177608.g005 https://doi.org/10.1371/journal.pone.0177608.g005 chTBK1 gene. The full-length coding sequence chTBK1 consisted of 2190bp encoding for 729 amino acid residues. While chTBK1 clusters in the avian group, multiple sequence alignment analysis showed that chTBK1 has high homology with TBK1 of mammals, ranging from 85.6% to 97.4%. Conserved domains were identified in the N-terminus of the chTBK1 protein, including the protein kinase domain and the ubiquitin-like domain, which is identical with murine and human TBK1 [5,6]. Of note, the ATP-binding site (LGQGATANV) and Ser172 residue, which are located in the protein kinase domain, were completely conserved in all of the TBK1 kinases and is consistent with those found in other species [20]. The mammalian TBK1 crystal structure showed that the kinase domain of TBK1, which harbors an ATP-bind- ing site and a phosphorylation site (Ser172), is a structurally conserved protein domain con- taining the catalytic function of this protein kinase [6]. The ubiquitin-like domain functions as a protein-protein interaction domain, which is implicated in regulating kinase activity, sub- strate presentation and downstream signaling pathways [7]. Our results showed that chTBK1 is very similar to the human and murine orthologs in terms of the sequence and structure. The similarity is especially high in the conserved domain, which suggests that chTBK1 may have a similar function to the human and murine TBK1. chTBK1 gene. The full-length coding sequence chTBK1 consisted of 2190bp encoding for 729 amino acid residues. While chTBK1 clusters in the avian group, multiple sequence alignment analysis showed that chTBK1 has high homology with TBK1 of mammals, ranging from 85.6% to 97.4%. Conserved domains were identified in the N-terminus of the chTBK1 protein, including the protein kinase domain and the ubiquitin-like domain, which is identical with murine and human TBK1 [5,6]. Of note, the ATP-binding site (LGQGATANV) and Ser172 residue, which are located in the protein kinase domain, were completely conserved in all of the TBK1 kinases and is consistent with those found in other species [20]. Discussion The mammalian TBK1 crystal structure showed that the kinase domain of TBK1, which harbors an ATP-bind- ing site and a phosphorylation site (Ser172), is a structurally conserved protein domain con- taining the catalytic function of this protein kinase [6]. The ubiquitin-like domain functions as a protein-protein interaction domain, which is implicated in regulating kinase activity, sub- strate presentation and downstream signaling pathways [7]. Our results showed that chTBK1 is very similar to the human and murine orthologs in terms of the sequence and structure. The similarity is especially high in the conserved domain, which suggests that chTBK1 may have a similar function to the human and murine TBK1. The expression of TBK1 has been investigated in many vertebrate species. In contrast to its closest ortholog IKKε, TBK1 is ubiquitously expressed in most tissues and cell types [21]. In agreement with those observations, our results demonstrated that chTBK1 was differentially expressed in all of the examined tissues. Of note, it was highly expressed in the spleen, thymus, and lung, which are all immune-related tissues, suggesting that chTBK1 may be an important factor in the immune system of chickens. PLOS ONE \| https://doi.org/10.1371/journal.pone.0177608 May 11, 2017 11 / 17 The expression of TBK1 has been investigated in many vertebrate species. In contrast to its closest ortholog IKKε, TBK1 is ubiquitously expressed in most tissues and cell types [21]. In agreement with those observations, our results demonstrated that chTBK1 was differentially expressed in all of the examined tissues. Of note, it was highly expressed in the spleen, thymus, and lung, which are all immune-related tissues, suggesting that chTBK1 may be an important factor in the immune system of chickens. PLOS ONE \| https://doi.org/10.1371/journal.pone.0177608 May 11, 2017 11 / 17 Molecular characterization and function of chicken TBK1 gene Fig 6. Relative mRNA expression of chTBK1, IRF3, IFNβ and ALV-J Env gene in CEFs challenge with ALV-J, showing in A, B, C and D, respectively. Data above were presented as mean ± SEM (n = 3). Error bars show the SEM of triplicate. Columns sharing different letters show extremely significant difference (P<0.01). https://doi.org/10.1371/journal.pone.0177608.g006 Fig 6. Relative mRNA expression of chTBK1, IRF3, IFNβ and ALV-J Env gene in CEFs challenge with ALV-J, showing in A, B, C and D, respectively. Data above were presented as mean ± SEM (n = 3). Error bars show the SEM of triplicate. Discussion Columns sharing different letters show extremely significant difference (P<0.01). https://doi.org/10.1371/journal.pone.0177608.g006 Fig 7. Relative mRNA expression of chTBK1, IRF3 and IFNβ gene in CEFs transfection with polyI:C, showing in A, B and C, respectively. Data above were presented as mean ± SEM (n = 3). Error bars show the SEM of triplicate. Columns sharing different letters show extremely significant difference (P<0.01). https://doi.org/10.1371/journal.pone.0177608.g007 Fig 6. Relative mRNA expression of chTBK1, IRF3, IFNβ and ALV-J Env gene in CEFs challenge with ALV-J, showing in A, B, C and D, respectively. Data above were presented as mean ± SEM (n = 3). Error bars show the SEM of triplicate. Columns sharing different letters show extremely significant difference (P<0.01). https://doi.org/10.1371/journal.pone.0177608.g006 Fig 6. Relative mRNA expression of chTBK1, IRF3, IFNβ and ALV-J Env gene in CEFs challenge with ALV-J, showing in A, B, C and D, respectively. Data above were presented as mean ± SEM (n = 3). Error bars show the SEM of triplicate. Columns sharing different letters show extremely significant difference (P<0.01). https://doi.org/10.1371/journal.pone.0177608.g006 Fig 7. Relative mRNA expression of chTBK1, IRF3 and IFNβ gene in CEFs transfection with polyI:C, showing in A, B and C, respectively. Data above were presented as mean ± SEM (n = 3). Error bars show the SEM of triplicate. Columns sharing different letters show extremely significant difference (P<0.01). https://doi.org/10.1371/journal.pone.0177608.g007 Fig 7. Relative mRNA expression of chTBK1, IRF3 and IFNβ gene in CEFs transfection with polyI:C, showing in A, B and C, respectively. Data above were presented as mean ± SEM (n = 3). Error bars show the SEM of triplicate. Columns sharing different letters show extremely significant difference (P<0.01). https://doi.org/10.1371/journal.pone.0177608.g007 PLOS ONE \| https://doi.org/10.1371/journal.pone.0177608 May 11, 2017 12 / 17 Molecular characterization and function of chicken TBK1 gene Fig 8. Knockdown of chTBK1 reduced IRF3 and IFNβ gene expression in CEFs. A Effects of siTBK1 on the expression of endogenous chTBK1 in CEFs at different time. B, C, D Relative mRNA expression of chTBK1, IRF3 and IFNβ gene in chTBK1 knockdown CEFs challenge with ALV-J as well as polyI:C, compared to negative control (NC), respectively. Data above were presented as mean ± SEM (n = 3). Error bars show the SEM of triplicate. () and (*) represent significant (P < 0.05) and extremely significant (P <0.01) difference, respectively. https://doi.org/10.1371/journal.pone.0177608.g008 Fig 8. Knockdown of chTBK1 reduced IRF3 and IFNβ gene expression in CEFs. PLOS ONE \| https://doi.org/10.1371/journal.pone.0177608 May 11, 2017 Molecular characterization and function of chicken TBK1 gene tissues at 14 dpi when a large amount of ALV-J is distributed in the three organs, which indi- cated there was a strong correlation between ALV-J copy number and innate immune responses. These findings are consistent with observations of Newcastle disease virus and avian influenza virus infection [31]. In summary, increased chTBK1 expression was observed in chickens after infection with ALV-J in the three main immune organs compared to the con- trol, which suggests a potential antiviral role for chTBK1 in chickens. Because TBK1 was constitutively expression in almost all cell types, whereas IKKε was mainly found in immune cells [17], embryo fibroblasts are considered to be a good cell model to investigate TBK1. Previous studies reported that embryo fibroblasts primarily detect virus infection through cytoplasmic receptors called RLRs [32]. In chickens, MDA-5 has been inves- tigated and is involved in sensing dsRNA and influenza A virus [27,33,34]. As it is known that TBK1 is essential for the IFN responses against viruses or dsRNA recognized by both RIG-I and MDA5 [35], we speculated that chTBK1 would be up-regulate in CEFs after ALV-J or polyI:C challenge. As expected, the expression of chTBK1 was significantly increased at 2 hpi and peaked at 6 and 12 hpi in CEFs after ALV-J infection, suggesting that ALV-J could initiate the innate immune response of host cells as was found in the in vivo study. In addition, increases in the expression levels of the chTBK1 gene was also observed at 2 hours and peaked at 6 hours in CEFs transfected with polyI:C, suggesting the involvement of TBK1 in immune responses triggered by dsRNA in chicken cells. These results corroborated the in vivo data and were the first evidence to suggest that chTBK1 is involved in anti-virus immune responses both in vivo and in vitro. As mentioned above, chTBK1 is essential for anti-virus immune responses in chickens. In mammals, TBK1 was identified as a main kinase that mediates type I interferon expression by phosphorylation of IRF3/IRF7, especially in fibroblasts [17]. Surprisingly, in chickens, only IRF3 was found [36–38]. Moreover, chicken IRF3, which is closely related to mammalian IRF7 in structure, may induce IFNβ expression after virus or dsRNA stimulation [37,39]. This infor- mation suggestes that the chTBK1 gene may induce chIRF3 and IFNβ expression. PLOS ONE \| https://doi.org/10.1371/journal.pone.0177608 May 11, 2017 Discussion A Effects of siTBK1 on the expression of endogenous chTBK1 in CEFs at different time. B, C, D Relative mRNA expression of chTBK1, IRF3 and IFNβ gene in chTBK1 knockdown CEFs challenge with ALV-J as well as polyI:C, compared to negative control (NC), respectively. Data above were presented as mean ± SEM (n = 3). Error bars show the SEM of triplicate. () and (**) represent significant (P < 0.05) and extremely significant (P <0.01) difference, respectively. https://doi.org/10.1371/journal.pone.0177608.g008 ALV-J is a single-strand RNA virus belonging to the retroviridae. Similarly to human immunodeficiency virus, ALV-J replication cycle involves in provirus integration and the syn- thesis of viral proteins. During this cycle, the genomic information is carried in different forms, including single-strand RNA, RNA:DNA hybrid, single-strand DNA, dsDNA and dsRNA [22,23]. Previous studies in other retroviruses demonstrated that single-strand RNAs were recognized by the endosomes of specialized innate immune cells using TLR7, while reverse-transcribed DNA is believed to activate STING signaling pathways by cytoplasmic DNA sensors, such as cyclic GMP-AMP synthase and IFI16 [11,24,25]. Additionally, accord- ing to Liu et al, ALV-J is mainly recognized by the RIG-I-like signaling pathways in chicken [26]. Although RIG-I is absent in chickens, MDA5 (the closest ortholog of RIG-I), TLR7 and STING have been identified. Moreover, previous studies indicated that chMDA5 can function- ally compensate for the absence of RIG-I [27]. TBK1 is thought to be a common adaptor molecular in TLR, RLR and STING signaling pathways. Recently, a number of studies revealed that TBK1 is activated by various viruses, such as adenovirus [28], hepatitis C virus [29] as well as retrovirus like human immunodeficiency virus [25]. In this study, the expression of chTBK1 in the spleen was significantly up-regulated (P<0.01) as early as 5 dpi when the ALV-J Env gene was first detected. These results further confirmed that the spleen is a lymphoid organ that is reached early by the virus and is involved in the early innate host responses [30]. More- over, the transcription of chTBK1 was significantly up-regulated in all of the detected immune PLOS ONE \| https://doi.org/10.1371/journal.pone.0177608 May 11, 2017 13 / 17 Acknowledgments The authors would like to thank the Prof. Yong Huang provide us the ALV-J virus and give us the guidance in the experiment. We also would like to express our gratitude to the editor from AJE for revising the language patiently. Molecular characterization and function of chicken TBK1 gene In conclusion, we have conducted comprehensive structural characterizations and immune function analysis of chTBK1. The ORF of chTBK1 consists of 2190 bp encoding 729 amino acid residues. The putative chTBK1 protein is structurally and phylogenetically closely related to human and mouse TBK. ChTBK1 is highly expressed in immune or other immune-related tissues, including spleen, thymus, bursa of Fabricius and lungs. Furthermore, chTBK1 was sig- nificantly increase in vivo or in vitro challenge with ALV-J and polyI:C, respectively. Our results suggested that chTBK1 has an important role in virus infection or dsRNA stimulation in innate immunity. In addition, augmentation of chTBK1 resulted in IRF3 and IFNβ up-regu- lation in vitro after ALV-J infection or polyI:C transfection. Conversely, IRF3 and IFNβ induc- tion with reduced in TBK1-defective CEFs. Collectively, these results demonstrate that chTBK1 is required for type I interferon induction against viral infection and dsRNA stimula- tion. Importantly, IRF3 likely functions as a main adaptor molecular of chTBK1 signaling, which is similar to the role of mammalian IRF3. Project administration: YW QZ. Resources: XZ HY DL HX. Resources: XZ HY DL HX. Supervision: QZ YL. Supervision: QZ YL. Validation: FY HX. Validation: FY HX. Writing – original draft: YW YY. Writing – review & editing: HX YL QZ. PLOS ONE \| https://doi.org/10.1371/journal.pone.0177608 May 11, 2017 Author Contributions Conceptualization: YW YY XL. Conceptualization: YW YY XL. Conceptualization: YW YY XL. Formal analysis: YW YY XL. Funding acquisition: YW QZ. Investigation: YY XL FY KT. Project administration: YW QZ. As expected, significant increases in the expression levels of chicken IRF3 and IFNβ gene were observed in CEFs challenge with ALV-J and polyI:C, which is in agreement with the findings of previous studies [27,37]. We also observed that the chicken IRF3 and IFNβ genes have similar expres- sion level changes to the chTBK1 gene, which implied that the expression level of chicken IRF3 and IFNβ correlates with the activation of chTBK1. To address this potential correlation, we studied the effects of chicken TBK1 knockdown after challenge with ALV-J and polyI:C. After transfection with siTBK1.1 and siTBK1.2, the expression of chTBK1 was significantly reduced (40%-50%). Simultaneously, both IRF3 and IFNβ were significantly impaired after ALV-J and polyI:C treatment in chTBK1-deficient CEFs compared to the NC, which is consistent with the analysis of IRF3 and IFNβ expression in both TBK1- deficient cells and mice [15, 16]. These data further confirmed that chTBK1 induces IRF3 and IFNβ transcription in chicken cells. Previous studies have shown that upon activation TBK1 directly phosphorylates the C-terminal domain of IRF3 [16]. Phosphorylation ultimately results in IRF3 dimerization and translocation into the nucleus where the IRF3 homodimers cooperate with CREB-binding protein (CBP)/P300 to induce the expression of the type I interferon gene [40,41]. IFNβ is the main type I interferon group member. Thus, these previous results and our present observations suggest that chTBK1 likely functions as an adaptor molecule to resist a virus-induced antiviral signaling pathway in chicken cells. 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https://openalex.org/W4294958322	https://reunir.unir.net/bitstream/123456789/14419/1/learning_self_regulation_questionnaire.pdf	English	null	Learning Self-Regulation Questionnaire (SRQ-L): Psychometric and Measurement Invariance Evidence in Peruvian Undergraduate Students	Sustainability	2,022	cc-by	11,639	y * Correspondence: ﬁliberto.toledano.phd@gmail.com; Tel.: +52-5580094677 Citation: Merino-Soto, C.; Chávez-Ventura, G.; López- Fernández, V.; Chans, G.M.; Toledano-Toledano, F. Learning Self-Regulation Questionnaire (SRQ-L): Psychometric and Measurement Invariance Evidence in Peruvian Undergraduate Students. Sustainability 2022, 14, 11239. https://doi.org/10.3390/ su141811239 Abstract: Given the theoretical and applied importance of self-regulation in learning, our study aimed to report the internal structure of the psychometric properties of the Learning Self-Regulation Questionnaire. Five hundred and ninety-six Peruvian university students participated in their ﬁrst to tenth semesters on campuses in Lima, Trujillo, and Cajamarca. Nonparametric scalability, dimensionality, reliability (score and item levels), and latent invariance were analyzed. The results showed that reducing the number of response options was necessary. Reducing the number of items also produced better scaling. Two slightly related dimensions were strong internal validity and acceptable item reliability; furthermore, reliability was adequate. Age and gender had trivial correlations in item variability. Finally, differences between the semesters were obtained in the means, variances, and latent correlations. In conclusion, we propose a better deﬁnition of the constructs of autonomy and control measured by the SRQ-L. This article also discusses the limitations and implications of the study. Academic Editors: Africa Borges and Leire Aperribai Keywords: social cognition; self-regulation; validity; invariance; learning; higher education; educational innovation; professional education Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. sustainability sustainability sustainability Article Learning Self-Regulation Questionnaire (SRQ-L): Psychometric and Measurement Invariance Evidence in Peruvian Undergraduate Students César Merino-Soto 1 , Gina Chávez-Ventura 2 , Verónica López-Fernández 3, Guillermo M. Chans 4 and Filiberto Toledano-Toledano 5,6,7,* o-Soto 1 , Gina Chávez-Ventura 2 , Verónica López-Fernández 3, Guillermo M. Chans 4 T l d T l d 5 6 7 * 1 Facultad de Ciencias de la Salud, Escuela Profesional de Medicina, Universidad César Vallejo, Av. Larco 1770, Trujillo 13009, Peru j 2 Instituto de Investigación en Ciencia y Tecnología, Universidad César Vallejo, Av. Larco 1770, Urb. Las Flores, Distrito Víctor Larco Herrera, Trujillo 13009, Peru j 2 Instituto de Investigación en Ciencia y Tecnología, Universidad César Vallejo, Av. Larco 1770, Urb. Las Flores, Distrito Víctor Larco Herrera, Trujillo 13009, Peru 2 Instituto de Investigación en Ciencia y Tecnología, Universidad César Vallejo, Av. Larco 1770, Urb. Las Flores, Distrito Víctor Larco Herrera, Trujillo 13009, Peru 3 Department of Education, Universidad Internacional de La Rioja (UNIR), Avenida de la Paz 101, 26006 Logroño, Spain 4 Tecnologico de Monterrey, School of Engineering and Sciences, Mexico City 01389, Mexico 5 Unidad de Investigación en Medicina Basada en Evidencias, Hospital Infantil de México Federico Gómez, National Institute of Health, Márquez 162, Doctores, Cuauhtémoc, Mexico City 06720, Mexico 6 Unidad de Investigación Sociomédica, Instituto Nacional de Rehabilitación Luis Guillermo Ibarra Ibarra, Calzada México-Xochimilco 289, Arenal de Guadalupe, Tlalpan, Mexico City 14389, Mexico 7 Dirección de Investigación y Diseminación del Conocimiento, Instituto Nacional de Ciencias e Innovación para la Formación de Comunidad Cientíﬁca, INDEHUS, Periférico Sur 4860, Arenal de Guadalupe, Tlalpan, Mexico City 14389, Mexico * Correspondence: ﬁliberto.toledano.phd@gmail.com; Tel.: +52-5580094677 1. Introduction According to the reference website, these versions are slightly different and malleable for speciﬁc situations, suggesting transferability to other learning contents. The adaptations were developed for studies with medical students in training interviews [24] and organic chemistry [28]. In Peru, structural validity has been tested twice [27,29]. In these two studies, which had samples of 237 and 369 university students, respectively, the structure of the dimensions (autonomy and control) was empirically differentiated with satisfactory adjustment indices; the parameters of the items were also adequate to represent their dimensions validly. The relationship between control and autonomy was equal to zero [29], suggesting that they do not operate linearly to achieve learning. This situation would be in line with the study by Matos [27], which found no correlation between the two (0.08; p > 0.05). Other problems replicated in these studies were the factorial complexity of two items, 5 and 13, and their low factor loadings. Although these items were removed during their analysis, other types of psychometric problems may need to be explored to verify the lack of ﬁt of these items (the distribution of the items, the correlated errors, or the appropriate ordering of the response options). The answer to these problems still requires investigation because the theoretical or methodological explanations were not previously addressed in sufﬁcient detail, which may limit the interpretation of the scores. Exploring potential problems also involves the appropriate deﬁnition of the conceptual content of the factors to achieve valid interpretations of the scores. The practical implication of this psychometric clarity of the instrument (regarding its accuracy and content) is the correct identiﬁcation of the attribute in the students, given that control and autonomy are sources of inﬂuence on the student’s adaptive behavior. Other relevant aspects of the properties of the SRQ-L need to be addressed to build a complete knowledge of it. One of these is measurement invariance, a necessary condition to make valid comparisons between groups [30], for example, between men and women or completed academic years. The differences between these groups concerning self- regulation are not uniform, but there is a clear tendency not to corroborate the invariance of the parameters of the items. On the one hand, studies have shown that women self- regulate their learning more intensely than men [24], but it has also been found that there are no differences between the sexes [31]. 1. Introduction Learning is omnipresent throughout life, and self-regulation is particularly relevant in learning and performance in various age groups, for example, adults [1], university students [2,3], adolescents in secondary education [4,5], and children in elementary edu- cation [6,7]. The relevance is because self-regulation modulates cognitive, affective, and behavioral facets to achieve the desired success level [8]. Therefore, self-regulation is one of the relevant variables in understanding the student’s academic performance and adap- tation. The people who self-regulate their learning have a greater capacity to select and structure the content they must learn [2]. They can adapt teaching strategies to perform better [9–11], reﬂect, participate with initiative, engagement [12,13], perseverance, and Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/sustainability Sustainability 2022, 14, 11239. https://doi.org/10.3390/su141811239 Sustainability 2022, 14, 11239 2 of 17 proactivity in their learning [11,14,15] have ﬁrmer self-efﬁcacy beliefs [11,16,17], become goal-oriented in a particular domain [18], adjust more easily to university life [14], and persist in their careers [19–21], especially during the COVID-19 pandemic’s conﬁnement and health restrictions [22]. This study is based on the cognitive perspective of self-regulation of learning from self-determination theory [23,24]. According to this, the regulation of behavior depends on the extent to which the individual perceives themselves as controlled (that is, pressured by external demands or situations) or autonomous (internal control and internalization of self-regulation processes). This perception affects intrinsic motivation, the expression of positive effects, ﬂexibility, and the capacity for autonomous choices [11–14]. Its relevance and transversal quality [25] make it an essential competency in all academic semesters [26]. In the university context, particularly in Peru, it is necessary to have instruments with adequate evidence of validity and reliability. One instrument that measures learning self-regulation is the Learning Self-Regulation Questionnaire (SRQ-L) [24]. It is an adaptation to the ﬁeld of learning from the clinical area. The Hispanic version of the instrument came from a Peruvian study, which was rigorously translated from English to Spanish spoken in Peru [27]. However, it could be recognized that the phrasing is generalizable in different Hispanic countries. The obtained scale integrates the content of two versions, which is also available on the author’s website (http://selfdeterminationtheory.org/self-regulation-questionnaires/ (accessed on 10 September 2021)). 1. Introduction In another case, it was observed that women have greater autonomy than men [24], while in another, no differences were observed in control [2]. The differential performance of the instrument may also inﬂuence these differences. Therefore, interpreting the variability between these groups may contain Sustainability 2022, 14, 11239 3 of 17 3 of 17 undetected bias or parameters that violate the measurement invariance of the SRQ-L. Since this property of the internal structure has not been veriﬁed before, it needs to be resolved. p p y Another aspect is the reliability of the differences between scales and the identiﬁcation of abnormal differences, both psychometric parameters that help describe the discrepancies between the scores while controlling the measurement error [32,33]. This type of informa- tion is practical because it is exposed in the metric of the observed score [32] and directly inﬂuences the interpretation of the score to describe the student’s position in the measured attribute and the study of the differences between both measured constructs (control and autonomy). Compared to the estimation of reliability coefﬁcients, the information on the error variability in the observed score metric has practical and nonacademic use. y p Regarding reliability, studies usually report internal consistency coefﬁcients, specif- ically the alpha coefﬁcient; its magnitude varies, typically from 0.60 but less than 0.90 on the two subscales [19,24,27,28,34–37]. More appropriate internal consistency estimates may be required because the alpha coefﬁcient requires several assumptions to be met, such as the absence of correlated errors and the tau-equivalence relationship between the items and their construct [38]. The fulﬁllment of these conditions avoids overestimating or underestimating the reliability through the alpha coefﬁcient. The objective of the present study is to contribute to a more rigorous evaluation of the construct validity through a validated Self-Regulation of Learning Questionnaire (SRQ-L) by considering two aspects: the internal structure and the measurement invariance, overcoming the gaps from previous studies [29], and thus obtain higher metric quality during its application in university learning contexts. This metric quality can be deﬁned by the validity of the items, the dimensional structure, the invariability of its parameters, and the functioning of the current response scaling. These properties were not previously investigated or were performed with outdated analysis strategies, so this situation suggests a source of potential inconsistency with future studies from an updated view of the internal structure of the SRQ-L. 1. Introduction Due to the importance of adapting and using tests in education, this instrument in its Spanish version (SRQ-L) demonstrated solid psychometric properties, with the methodological strength necessary to interpret the results in the desired way. It is a functional, valid, reliable, and culturally relevant scale for higher education students, replicating these ﬁndings in other contexts and populations. 2.3. Procedure Design. The study was cross-sectional and instrumental [40,41] and employed a quantitative methodology. Data Collection. Authorization was obtained from the directors of each university, and we solicited the support of the Psychological Orientation area to evaluate the students by psychologists and psychology interns trained for this purpose in an estimated 15 min. The test was applied in the middle of the academic semester, coordinating with the teachers to facilitate classroom access. The instruments were applied in groups of approximately 35 students, to whom the evaluation objectives were explained, and participation was voluntary, following the ethical principles of psychologists [42,43]. These instructions also communicated the anonymity of the response, the support in case of questions about the survey content, the absence of participation incentives, and the freedom not to continue ﬁlling out the survey. The applied material consisted of informed consent and the study instrument. Analysis. The quantitative analysis focused on carefully examining (a) the univariate characteristics of the items, (b) the internal structure, (c) and the reliability. The differences according to variables that could explain the variability in self-regulation were identiﬁed using the latent variables within the methodological framework of the analysis of structural invariance. The analysis’ general strategy was to apply several approaches to reduce the dependence of the conclusions on a single analytical procedure [44]. Item analysis. As the content of the items referred to two control attributes as in- dividual expressions [45], their involvement could generate possible response patterns associated with the functional distribution of the response categories [45,46]. To examine it, we estimated thresholds for the items, which are points of intersection between adjacent response categories associated with the probability of choosing between one or another conditional category at the level of the latent attribute (or construct). The ordering (or dis- ordering) of these thresholds would indicate the good (or bad) performance of the response categories [46]. It was estimated within the partial credit model [47,48], derived from the Rasch modeling for polytomous items [49]. For this estimation, the eRm program [50] was used within the IANA graphical interface [51] in the R program [52]. Additionally, the correlation between sex and age was examined as a partial expression of the content validity of the items [53,54]. Nonparametric analysis. 2.1. Participants Five hundred and ninety-six evaluated university students from the cities of Trujillo (320, 54%), Lima (29, 29%), and Cajamarca (101, 17%), Peru, were selected through non- probabilistic convenience sampling [39], related to the researchers’ access to professional career directors and the facilities provided by teachers for access to classrooms. Participants excluded from the study were the ones who had studied a previous professional career, were not present at the time of the survey administration, did not sign the informed consent, and did not complete the survey due to personal choice. 51% of the participants were male, 88% were single, and 72% did not work. The ages ﬂuctuated between 16 and 40 years (M = 19.85; SD = 2.82). Their socioeconomic level was medium to medium-high. Of the participants, 77% were between the ﬁrst- and third-semester studies, 22% between the fourth and sixth semesters, and 1% between the seventh and tenth semesters. They were enrolled in the professional careers of Engineering (39%), Administration (24%), Accounting (20%), Law (14%), Psychology (2%), and Com- munications (1%). To compare the participants per their study semester, we categorized three groups: from the 1st to the 2nd semester (141, 24%), the 3rd semester (320, 54%), and the 4th to the 10th (134, 22%). All students in the study signed the informed consent as a condition for completing the survey. Sustainability 2022, 14, 11239 4 of 17 2.2. Instrument Self-Regulatory Learning Questionnaire (SRQ-L) [24]. It is a self-report test consisting of 14 ordinal, 5-point items (from 1 = Not true at all to 5 = Completely true for me). It was translated and adapted to the Peruvian context by Matos [27], the version used in this study. Its structure comprised two factors: Autonomy (6 items) and Control (8 items). The ﬁrst refers to the importance assigned to learning due to internal regulation and is based on intrinsic motivation; the basic psychological needs of competency, autonomy, and relationship were satisﬁed without searching for external stimuli. The second was oriented toward seeking rewards, external approval, or punishment avoidance [23,24]. 2.3. Procedure A nonparametric approach [55] was applied to the ordinal items of the SRQ-L [56] to verify several fundamental and precursor properties [57] of the instrument scores but with independence from the strong assumptions of latent variable models. Three essential characteristics [58] were explored for the completion of the mono- tone homogeneity model (MHM): scalability (using the coefﬁcient H), local independence (responses to the items are not mutually inﬂuenced; examined through three conditional association indices, W(1), W(2), and W(3) [59]), and monotonicity (incremental function between the item and the latent attribute; evaluated by comparing the number of current and expected violations to the monotonic model [55]). The procedure was performed using the Mokken program [52,60]. SEM analysis. The evaluation of dimensionality was complemented by the conﬁr- matory factor analysis for categorical data, using the weighted least square mean and Sustainability 2022, 14, 11239 5 of 17 5 of 17 variance adjusted estimator (WLSMV) [61]; ﬁt was assessed using approximate ﬁt indices: CFI (≥0.95), TLI (≥0.95), RMSEA (≤0.05), Gamma-hat (G-h) [62], and SRMR (≤0.05). Re- garding the measurement invariance, the appropriate sequence of steps was implemented for categorical variables [63], starting with successfully implementing restrictions on the parameters of the items. We started with conﬁgurational invariance, then introduced the cumulative constraint of equality of thresholds, factor loadings, intercepts, and, ﬁnally, residuals. Since identifying the adjustment in the measurement invariance is still a matter of debate [64], together with the CFI, the centrality index (Mc) [65] was also used, given its statistical robustness [64]. After corroborating the measurement invariance, the latent differences of the means were evaluated and estimated using d [66]; also, we assessed the differences of the variances through the standardized variance heterogeneity index, SVH [67], and the correlations using the standardized index q [66]. All SEM analyses were performed with the lavaan program [52,68]. Reliability. Reliability was estimated at the item and score level for each subscale. Regarding the items, it was calculated using the coefﬁcient corrected by attenuation [69], given its lower bias and computational ease [70]; the minimum acceptable value is around 0.30 [71]. At the scoring level: (a) consistent with the nonparametric model, the MS coefﬁcient was estimated [56], and (b) consistent with linear SEM modeling, the coefﬁcient ω was estimated [72]. For comparison purposes, the coefﬁcient α was obtained. 2.3. Procedure p p p Practical indices of the measurement error of both scores were also estimated, using the standard error of measurement (SEM) and the standard error of measurement of the difference (SEMD) [33]. For the latter, the formula q SEM2 F1 + SEM2 F2 was used, where SEMF1 and SEMF2 correspond to the SEM of each compared score. To obtain the critical values, the SEMD was multiplied by the z values derived from the standardized normal curve (1.43, 1.64, 1.96, and 2.57) and consistent with the 0.15, 0.10, 0.05, and 0.01 levels, two-tailed (respectively). Finally, to estimate the statistical abnormality of the difference between the F1 and F2 scores obtained by a subject from a clinicometric approach, the standard deviation of the difference was calculated [73]: SDD = SDp2 −2rxy, where SD is the standard deviation in the standardized score metric (for this study, t-score, that is, M = 50, SD = 10), and rXY is the correlation between both compared scores (usually Pearson’s linear correlation). 3.1. Item Analysis Results are presented in Table 1. The means of the F1 (Autonomy) items were generally higher than those of F2 (Control), indicating a degree of independence of the behaviors in both factors. In contrast, an apparent more signiﬁcant variability of behaviors was found in F2 (observed from the standard deviation). On the other hand, the distributional non-normality was moderately variable (skewness and kurtosis). The correlations between the items of the scale F1 with F2 tended to be lower than the inter-item correlations within the same scale, suggesting clear divergent and convergent relationships (respectively). For the correlations of the items with sex and age, the Type I error was controlled by adjusting the nominal alpha by the Bonferroni method (in F1: 0.05/6 items = 0.008; in F2: 0.05/8 items = 0.006), ﬁnding that none of the correlations with the sex and age of the participants was statistically signiﬁcant. Although no cut-off points were established to indicate the magnitude of the item–criterion correlations, the magnitude of the correlations obtained could be considered between the trivial and low levels. The examination of the ordering of the response options (see Figure 1) showed a pattern of disorder, strongly concentrated in options 1 and 2, and 4 and 5; a tendency to less differentiation between thresholds 3 and 4 was also observed. Because these observations (disordering and less differentiation) decrease the appropriate interpretation of the scale scores [46], category 1 was merged with 2, and category 3 merged with 4 and 5. The new scaling with three response options effectively maintained the ordering of the thresholds, except in Items 7 Sustainability 2022, 14, 11239 6 of 17 hree re- It 7 6 of 17 hree re- It 7 and 14 of the F2 scale (Control). Without making other modiﬁcations, these items were kept for the following analyses to evaluate their effect on parameterization with factor analysis. kept for the following analyses to evaluate their effect on parameterization with factor analysis. and 14 of the F2 scale (Control). Without making other modiﬁcations, these items were kept for the following analyses to evaluate their effect on parameterization with factor analysis. kept for the following analyses to evaluate their effect on parameterization with factor analysis. 3.1. Item Analysis for the following analyses to evaluate their effect on parameterization with factor analys F1 Autonomy F2 Control First analysis Second analysis (After the collapse of answer options) Second analysis (After the collapse of answer options) Figure 1. Distribution of the thresholds of the items in their dimensions. Figure 1. Distribution of the thresholds of the items in their dimensions. F2 Control F1 Autonomy F1 Autonomy First analysis F2 Control Second analysis First analysis Second analysis (After the collapse of answer options) Second analysis Second analysis y (After the collapse of answer options) y (After the collapse of answer options) Figure 1. Distribution of the thresholds of the items in their dimensions Figure 1. Distribution of the thresholds of the items in their dimensions. 3.2. Nonparametric Dimensional Analysis 3.2. Nonparametric Dimensional Analysis Scalability. The result of the Mokken modeling appears in Table 2. The scalability coefﬁcients (H) were obtained in the total and three-semester samples. The decision to remove the items was based on (a) the pattern of comparatively low H coefﬁcients (<0.30) [74], (b) their comparatively high standard errors, and (c) the moderate invariance of the two previous patterns in the compared groups (three levels of academic semesters). Therefore, Items 12 and 11 of F1 (Autonomy) and Items 4, 5, and 13 of F2 (Control) showed the lowest scalability, considering that the lower limit of their conﬁdence interval (in 95%) was below 0.30. The ﬁnal evaluation result of the items appears in the last column of the scalability analysis in Table 2. 7 of 17 Sustainability 2022, 14, 11239 Table 1. Descriptive and correlational characteristics of the SRQ-L items (n = 596). Descriptive Information Correlations M SD Skew. Kurt. au1 au3 au6 au9 au11 au12 au2 au4 au7 au8 au10 au14 au5 au13 Gender Age Autonomy (F1) au1 3.790 0.993 −0.138 −0.806 1.000 −0.130 0.029 au3 3.948 1.062 −0.664 −0.199 0.514 1.000 −0.024 −0.043 au6 4.310 0.898 −1.14 0.774 0.228 0.257 1.000 −0.006 −0.070 au9 4.227 0.942 −0.973 0.223 0.209 0.316 0.459 1.000 0.070 −0.139 au11 4.413 0.900 −1.57 2.186 0.238 0.260 0.330 0.315 1.000 0.050 0.090 au12 4.153 0.981 −0.898 0.078 0.176 0.161 0.198 0.239 0.280 1.000 −0.015 0.056 Control (F2) au2 1.621 0.977 1.447 1.314 0.022 0.017 0.031 0.055 −0.053 0.081 1.000 −0.109 −0.071 au4 2.894 1.371 0.034 −1.05 0.144 0.284 0.152 0.168 0.103 0.169 0.266 1.000 −0.008 −0.056 au7 2.148 1.296 0.779 −0.551 0.020 −0.001 0.125 0.063 0.002 0.081 0.453 0.299 1.000 −0.044 −0.098 au8 2.384 1.300 0.541 −0.691 −0.075 −0.073 0.170 0.152 0.011 0.149 0.362 0.139 0.479 1.000 −0.069 −0.131 au10 2.122 1.227 0.751 −0.436 −0.041 0.044 0.173 0.156 −0.079 0.099 0.409 0.386 0.477 0.414 1.000 −0.026 −0.080 au14 2.297 1.406 0.670 −0.854 0.060 0.056 0.136 0.086 0.040 0.131 0.377 0.269 0.632 0.361 0.462 1.000 −0.075 −0.075 au5 3.930 1.085 −0.682 −0.216 0.117 0.128 0.512 0.403 0.252 0.177 0.146 0.185 0.208 0.246 0.232 0.159 1.000 0.011 −0.128 au13 3.968 1.096 −0.852 0.054 0.148 0.140 0.229 0.276 0.322 0.633 0.105 0.136 0.112 0.219 0.154 0.181 0.200 1.000 0.013 −0.002 Note. Skew.: Skewness coefﬁcient. Kurt.: Kurtosis coefﬁcient. Correlations are not statistically signiﬁcant (see text). au1 . . . aun: SRQ-L items. Correlations: Pearson correlation coefﬁcient. 3.2. Nonparametric Dimensional Analysis Finally, the local independence of the reduced version, evaluated with the indices W(1), W(2), and W(3) [59], indicated that the items of F1 (Autonomy) do not contain associations between signiﬁcant items in magnitude (W(1) between 0.111 and 0.937; W(2) between 2.426 and 3.605; W(3) between 0.015 and 1.893). In F2 (Control), there was substantial inconsistency between the indices (concerning Item 2): W(1) between 0.009 and 0.618; W(2) between 3.322 and 6.714; W(3) between 0.004 and 2.355. Therefore, the ﬁnal detection of local independence was evaluated by linear SEM modeling as a conﬁrmatory option [76]. 3.3. Structural Equations Modeling (SEM)-Based Parametric Analysis Dimensionality. Dimensionality modeling with all items showed poor results: WLSMV- χ2 = 1926.135 (df = 76, p < 0.001), CFI = 0.775, TLI = 0.730, RMSEA = 0.202 (90% CI = 195, 0.210), SRMR = 0.168. However, the factor loadings were acceptable; for example, in F1 they were between 0.456 (h2 = 0.208; Item 1) and 0.784 (h2 = 0.615; Item 12); in F2, between 0.474 (h2 = 0.225; Item 4) and 0.770 (h2 = 0.593; Item 7); and the covariation between the factors was high (r = 0.621, cov = 0.165, p < 0.01). This result was considered questionable, and it was a conclusion strengthened by the observation of the modiﬁcation indices (MI), in which several potential and strong factor-item respeciﬁcations were detected, not deﬁned by the original structure (MI > 200.0, ZEPC = 0.500), and a strong covariation of residuals existed between several items (e.g., MI > 100.0, ZEPC > 0.80). These possible respeciﬁcations were consistently corroborated by a method based on the statistical power and size of the Table 2. Results of the Mokken analysis (scalability and monotonicity) of the SRQ-L. Scalability (H Coefﬁcient) Monotonicity (n = 596) Initial Analysis Final Analysis Total Sample (n = 596) Level 1 Semester (n = 136) Level 2 Semester (n = 309) Level 3 Semester (n = 131) Total Sample (n = 596) #vi #zsig CRIT H s.e. H s.e. H s.e. H s.e. H s.e. 3.2. Nonparametric Dimensional Analysis Autonomy (F1) au1 0.339 0.036 0.320 0.068 0.365 0.055 0.320 0.066 0.413 0.039 0 0 0 au3 0.314 0.034 0.339 0.072 0.325 0.049 0.273 0.065 0.399 0.037 0 0 0 au6 0.345 0.035 0.281 0.078 0.370 0.051 0.332 0.055 0.395 0.041 1 0 19 au9 0.315 0.033 0.332 0.064 0.345 0.044 0.239 0.069 0.363 0.040 0 0 0 au11 0.288 0.043 0.235 0.084 0.300 0.067 0.336 0.061 – – – – – au12 0.187 0.037 0.105 0.069 0.205 0.053 0.187 0.073 – – – – – Total 0.296 0.029 0.269 0.055 0.316 0.044 0.279 0.048 0.392 0.033 Control (F2) au2 0.393 0.035 0.322 0.084 0.486 0.042 0.262 0.077 0.414 0.040 2 0 17 au4 0.258 0.032 0.183 0.067 0.254 0.046 0.354 0.057 – – au7 0.451 0.026 0.413 0.056 0.478 0.037 0.421 0.053 0.522 0.027 0 0 0 au8 0.374 0.027 0.359 0.054 0.407 0.038 0.315 0.059 0.445 0.032 1 0 9 au10 0.439 0.025 0.389 0.057 0.474 0.033 0.407 0.058 0.465 0.030 0 0 0 au14 0.424 0.026 0.340 0.063 0.474 0.034 0.400 0.053 0.490 0.028 1 0 5 au5 0.290 0.036 0.323 0.059 0.327 0.048 0.154 0.089 – – – – – au13 0.257 0.039 0.271 0.067 0.280 0.055 0.200 0.084 – – – – – Total 0.367 0.022 0.326 0.046 0.401 0.029 0.329 0.047 0.472 0.026 Note. s.e.: standard error of H. #vi: number of monotonicity violations. #zsig: number of statistically signiﬁcant violations. CRIT: Combined and weighted count of #vi and #zsig. Table 2. Results of the Mokken analysis (scalability and monotonicity) of the SRQ-L. Note. s.e.: standard error of H. #vi: number of monotonicity violations. #zsig: number of statistically signiﬁcant violations. CRIT: Combined and weighted count of #vi and #zsig. Monotonicity. With the reduced version of the SRQ-L, no violation (#vi) was found in the monotonicity ratio greater than two between the items or that was statistically signiﬁcant (#zsig), and the CRIT statistic for each item was less than 40 [75]. Overall, the results indicate that the monotonic homogeneity model is satisfactory. Local independence. Finally, the local independence of the reduced version, evaluated with the indices W(1), W(2), and W(3) [59], indicated that the items of F1 (Autonomy) do not contain associations between signiﬁcant items in magnitude (W(1) between 0.111 and 0.937; W(2) between 2.426 and 3.605; W(3) between 0.015 and 1.893). 3.2. Nonparametric Dimensional Analysis In F2 (Control), there was substantial inconsistency between the indices (concerning Item 2): W(1) between 0.009 and 0.618; W(2) between 3.322 and 6.714; W(3) between 0.004 and 2.355. Therefore, the ﬁnal detection of local independence was evaluated by linear SEM modeling as a conﬁrmatory option [76]. 3.2. Nonparametric Dimensional Analysis Association with gender and age: biserial-point, and Pearson coefﬁcients correlation, respectively. Table 1. Descriptive and correlational characteristics of the SRQ-L items (n = 596). Sustainability 2022, 14, 11239 8 of 17 Table 2. Results of the Mokken analysis (scalability and monotonicity) of the SRQ-L. Scalability (H Coefﬁcient) Monotonicity (n = 596) Initial Analysis Final Analysis Total Sample (n = 596) Level 1 Semester (n = 136) Level 2 Semester (n = 309) Level 3 Semester (n = 131) Total Sample (n = 596) #vi #zsig CRIT H s.e. H s.e. H s.e. H s.e. H s.e. Autonomy (F1) au1 0.339 0.036 0.320 0.068 0.365 0.055 0.320 0.066 0.413 0.039 0 0 0 au3 0.314 0.034 0.339 0.072 0.325 0.049 0.273 0.065 0.399 0.037 0 0 0 au6 0.345 0.035 0.281 0.078 0.370 0.051 0.332 0.055 0.395 0.041 1 0 19 au9 0.315 0.033 0.332 0.064 0.345 0.044 0.239 0.069 0.363 0.040 0 0 0 au11 0.288 0.043 0.235 0.084 0.300 0.067 0.336 0.061 – – – – – au12 0.187 0.037 0.105 0.069 0.205 0.053 0.187 0.073 – – – – – Total 0.296 0.029 0.269 0.055 0.316 0.044 0.279 0.048 0.392 0.033 Control (F2) au2 0.393 0.035 0.322 0.084 0.486 0.042 0.262 0.077 0.414 0.040 2 0 17 au4 0.258 0.032 0.183 0.067 0.254 0.046 0.354 0.057 – – au7 0.451 0.026 0.413 0.056 0.478 0.037 0.421 0.053 0.522 0.027 0 0 0 au8 0.374 0.027 0.359 0.054 0.407 0.038 0.315 0.059 0.445 0.032 1 0 9 au10 0.439 0.025 0.389 0.057 0.474 0.033 0.407 0.058 0.465 0.030 0 0 0 au14 0.424 0.026 0.340 0.063 0.474 0.034 0.400 0.053 0.490 0.028 1 0 5 au5 0.290 0.036 0.323 0.059 0.327 0.048 0.154 0.089 – – – – – au13 0.257 0.039 0.271 0.067 0.280 0.055 0.200 0.084 – – – – – Total 0.367 0.022 0.326 0.046 0.401 0.029 0.329 0.047 0.472 0.026 Note. s.e.: standard error of H. #vi: number of monotonicity violations. #zsig: number of statistically signiﬁcant violations. CRIT: Combined and weighted count of #vi and #zsig. Monotonicity. With the reduced version of the SRQ-L, no violation (#vi) was found in the monotonicity ratio greater than two between the items or that was statistically signiﬁcant (#zsig), and the CRIT statistic for each item was less than 40 [75]. Overall, the results indicate that the monotonic homogeneity model is satisfactory. Local independence. 3.3. Structural Equations Modeling (SEM)-Based Parametric Analysis Dimensionality. Dimensionality modeling with all items showed poor results: WLSMV- χ2 = 1926.135 (df = 76, p < 0.001), CFI = 0.775, TLI = 0.730, RMSEA = 0.202 (90% CI = 195, 0.210), SRMR = 0.168. However, the factor loadings were acceptable; for example, in F1 they were between 0.456 (h2 = 0.208; Item 1) and 0.784 (h2 = 0.615; Item 12); in F2, between 0.474 (h2 = 0.225; Item 4) and 0.770 (h2 = 0.593; Item 7); and the covariation between the factors was high (r = 0.621, cov = 0.165, p < 0.01). This result was considered questionable, and it was a conclusion strengthened by the observation of the modiﬁcation indices (MI), in which several potential and strong factor-item respeciﬁcations were detected, not deﬁned by the original structure (MI > 200.0, ZEPC = 0.500), and a strong covariation of residuals existed between several items (e.g., MI > 100.0, ZEPC > 0.80). These possible respeciﬁcations were consistently corroborated by a method based on the statistical power and size of the Sustainability 2022, 14, 11239 9 of 17 MI [77]. These problems occurred more frequently in the items detected as problematic by the Mokken nonparametric analysis (Items 11, 12, 5, and 13). y Taking into account the items selected in the Mokken scaling, the adjusted model showed higher ﬁt indicators: WLSMV-χ2 = 118.391 (df = 26, p < 0.001), CFI = 966, TLI = 0.953, RMSEA = 0.077 (IC 90% = 0.063, 0.092), SRMR = 0.090. When reviewing the MIs, trivial factor-item coefﬁcients were found towards a nonhypothesized item; however, strongly correlated residuals were found with Items 1 and 3 (ZEPC = 0.563) and Items 6 and 9 (ZEPC = 0.498). The procedure of Saris et al. [77] detected an inconsistency in the criteria based on power and the magnitude variation of the respeciﬁcation between Items 1 and 3; therefore, these were discarded. There was consistency in the association of Items 6 and 9, but the content analysis did not reveal an explicit conceptual link, so it was also discarded. Therefore, the ﬁnal model appears in Table 3, where it is shown that the items maintained strong factor loadings with their individual factors and were statistically signiﬁcant (F1, λ > 0.65, z > 9.0; in F2, λ > 0.62, z > 11.0). 3.3. Structural Equations Modeling (SEM)-Based Parametric Analysis The linear covariation between the factors was 0.09, 95% CI = 0.01, 0.169 (cov = 0.040, p > 0.10), showing orthogonal association. The correlation at the direct score level was 0.108 (95% CI = 0.028, 0.187). Table 3. Conﬁrmatory factor analysis and item reliability of the ﬁnal version of the SRQ-L. λF1 λF2 h2 ritc ritem au1 0.674 0.454 0.436 0.300 au3 0.689 0.474 0.503 0.400 au6 0.739 0.546 0.412 0.268 au9 0.704 0.496 0.431 0.294 au2 0.635 0.403 0.514 0.341 au7 0.872 0.760 0.696 0.626 au8 0.666 0.444 0.519 0.348 au10 0.723 0.522 0.576 0.429 au14 0.819 0.671 0.609 0.479 Descriptive statistics M 16.275 10.572 SD 2.754 4.642 Skew. −0.583 0.765 Kurt. 0.212 −0.118 Clinicometric indicators SEMF1 SEMF2 SEMD DED Value 1.470 2.060 6.943 13 Z 85% 2.116 2.966 10 19 90% 2.418 3.389 11 22 95% 2.882 4.038 14 26 99% 3.787 5.307 18 34 Note. λ: factor loadings. h2: commonality. ritem: reliability of the item. Skew: Skewness coefﬁcient. Kurt.: Kurtosis coefﬁcient. ritc: corrected item–test correlation. F1: Autonomy. F2: Control. SEM: standard error of measurement of F1 and F2. SEMD: standard error measurement of difference (reliable difference). DED: abnormal deviation (abnormal difference). Table 3. Conﬁrmatory factor analysis and item reliability of the ﬁnal version of the SRQ-L. Note. λ: factor loadings. h2: commonality. ritem: reliability of the item. Skew: Skewness coefﬁcient. Kurt.: Kurtosis coefﬁcient. ritc: corrected item–test correlation. F1: Autonomy. F2: Control. SEM: standard error of measurement of F1 and F2. SEMD: standard error measurement of difference (reliable difference). DED: abnormal deviation (abnormal difference). Tau-equivalent model. To verify if the items were similar in their validity with their construct, the tau-equivalent model (equal factor loads) was tested to ﬁt, ﬁnding a poor ﬁt, WLSMV-χ2 = 1445.526 (df = 36, p < 0.001), CFI = 0.892, TLI = 0.882, RMSEA = 0.088 (90% CI = 0.063, 0.092), SRMR = 0.090. The scaled difference [78] between this model and the previously accepted congeneric was statistically signiﬁcant, ∆χ2 = 48.65 (df = 7). 3.5. Invariance Measurement invariance. After verifying the internally most acceptable model, we evaluated the measurement invariance. Table 4 presents the estimates and adjustment differences. The groups chosen for this objective were study semesters carried out by the participants, grouped into three categories: Group 1 or initial (the ﬁrst two semesters), Group 2 or intermediate (3rd semester), and Group 3 or advanced (the 4th to the 10th semesters). Age and gender were not included in this analysis because they showed correla- tions around zero with the SQR-L items. The ﬁrst stage of the invariance was verifying the factorial structure (conﬁgurational invariance), where the results were acceptable, although with some slight variation in some indices (Mc and RMSEA). Although the standardized factor loadings of the model in each group were not reported, the magnitudes of the differences [79] were in the trivial (≤0.10) or small (≥0.1) range, and none in moderate magnitudes (≥0.2) or large (≥0.3). The invariance of the thresholds was also satisfactory, and its difference with the conﬁgurational model was slight. The magnitude of minor dif- ferences was maintained in comparing the rest of the evaluated models, even reaching the invariance of the residuals. At this level of strict invariance, the ﬁt indices were uniformly satisfactory, except for Mc. Table 4. Measurement invariance parameters (semester groups: initial, intermediate, and advanced). Invariance Models Fit Estimates Fit Differences WLSMV-χ2 (df) CFI TLI G-h Mc RMSEA (90% CI) ∆WLSMV-χ2 (df) ∆CFI ∆TLI ∆G-h ∆Mc ∆RMSEA Conﬁguration 159.314 (78) 0.973 0.963 0.970 0.933 0.073 (0.056, 0.089) – – – – – – Thresholds 171.101 (92) 0.974 0.969 0.971 0.935 0.066 (0.050, 0.081) 17.79 (14) 0.001 0.006 0.001 0.002 −0.007 Loads, thresholds 183.521 (106) 0.975 0.974 0.971 0.936 0.061 (0.046, 0.075) 13.21 (14) 0.001 0.005 0.000 0.001 −0.005 Loads, thresholds, intercepts 206.634 (110) 0.968 0.969 0.965 0.922 0.067 (0.053, 0.081) 10.07 * (14) −0.007 −0.005 −0.006 −0.014 0.006 Residuals 200.111 (124) 0.975 0.978 0.971 0.938 0.056 (0.041, 0.070) 1.40 (14) 0.007 0.009 0.006 0.016 −0.011 Note. G-h: Gamma-hat index. Mc: McDonald index. CFI: Comparative Fit Index. TLI: Tucker-Lewis Index. RMSEA: Root Mean Square Error of Approximation. ∆: difference. * p < 0.05. Table 4. Measurement invariance parameters (semester groups: initial, intermediate, and advanced). Structural invariance. After ensuring the invariance of the measurement, Table 5 shows the results of the structural comparisons between groups of the latent parameters (means, variances, and correlations). For the comparison of g1 vs. 3.4. Reliability The MS coefﬁcient was 0.684 and 0.772 for F1 (Autonomy) and F2 (Control), respec- tively. The reliability estimated by the ω coefﬁcient for categorical variables [72] was 0.715 (s.e. = 0.031, 95% CI = 0.621, 0.776, bca bootstrap) for F1, and 0.803 (s.e. = 0.015, 95% CI = 0.767, 0.830, bca bootstrap) for F2. Compared with the alpha coefﬁcient, it was 10 of 17 Sustainability 2022, 14, 11239 10 of 17 substantially lower in F1 (α = 0.634, s.e. = 0.029, 95% CI = 0.572, 0.688, bca bootstrap) and F2 (α = 0.774, s.e. = 0.016, 95% CI = 0.740, 0.804, bca bootstrap). At the item level, reliability in F1 was acceptable but slightly low (close to 0.30) [58,59], while in F2, it was satisfactory (>0.33) but moderately variable. The clinicometric application parameters appear in Table 3. 3.5. Invariance * p < 0.05. Table 5. Structural invariance (means, variances, and latent covariances). Variances and Latent Correlations Note. ∆no-Z: nonstandardized difference; ∆Z: standardized difference. g1, g2, g3: semester groups compared (initial, intermediate, and advanced, respectively). F1: Autonomy factor. F2: Control factor. SVH: standardized variance heterogeneity. Ng1 = 136. Ng2 = 309. Ng3 = 131. * p < 0.05. Note. ∆no-Z: nonstandardized difference; ∆Z: standardized difference. g1, g2, g3: semester groups compared (initial, intermediate, and advanced, respectively). F1: Autonomy factor. F2: Control factor. SVH: standardized variance heterogeneity. Ng1 = 136. Ng2 = 309. Ng3 = 131. * p < 0.05. 3.5. Invariance g2 and g3 groups, the reference group was g1; for the comparison between g2 and g3, the reference group was g3. Regarding the latent means, the standardized differences detected that in the Autonomy construct (F1), the differences between groups were close to zero and statistically insigniﬁcant, except that the students of the g3 group (fourth to tenth semesters) scored slightly higher (between 0.30 and 0.50) [66] than students in the third semester (group g2). On the other hand, in the Control construct (F2), the group from the third semester (g2) showed lower scores compared to the students from the ﬁrst two semesters (g1), but the size of the difference was negligible. The group of advanced semesters (g3) scored more than Group g2 (3rd semester), of small magnitude. Regarding the degree of individual differences (latent variances) in the compared semesters, students from more advanced semesters (g3, 4th to 10th semester) in Autonomy (F1), and students from 3rd semester (g2) in Control (F2), showed more variability. However, the comparisons with the SVH index were small (<0.56) [67], comparing groups within Sustainability 2022, 14, 11239 11 of 17 11 of 17 each factor and between factors. Finally, in the evaluation of the heterogeneity of the correlations [80], it was found that the relationships between the Autonomy and Control factors are different between the three groups (Q = 6.880, gl = 2, p < 0.05); the magnitude of this difference, I2 [81], can be considered strong (I2 = 90.92%). As shown in Table 5, the covariation was higher in Group g3 than in the rest. Table 5. Structural invariance (means, variances, and latent covariances). Comparison of Latent Means Variances and Latent Correlations F1 (Autonomy) F2 (Control) g1 g2 g3 g1 g2 g3 g1 g2 g3 SVH Variances g1 (1st to 2nd semester) – – F1 0.746 0.779 1.062 0.116 g2 (3rd semester) F2 0.401 1.041 0.462 0.321 ∆no-Z −0.075 – −0.323 * – – SVH 0.300 0.143 0.393 ∆Z −0.085 – −0.317 * – – Correlations g3 (4th to 10th semester) g1 g2 g3 ∆no-Z 0.029 0.116 – −0.011 0.322 * – r(F1,F2) −0.075 0.097 0.244 ∆Z 0.028 0.322 – −0.017 0.459 * – Note. ∆no-Z: nonstandardized difference; ∆Z: standardized difference. g1, g2, g3: semester groups compared (initial, intermediate, and advanced, respectively). F1: Autonomy factor. F2: Control factor. SVH: standardized variance heterogeneity. Ng1 = 136. Ng2 = 309. Ng3 = 131. 4. Discussion However, the statistical Sustainability 2022, 14, 11239 12 of 17 12 of 17 ﬁt of this full version using nonparametric scaling was not satisfactory; this is usually related to multidimensionality or excess variance irrelevant to the measured construct. The correlated errors between the items detected by both procedures (nonparametric and SEM) conﬁrmed that the structure of the original SRQ-L contained inter-item relationships irrelevant to the construct measured and even that some items could be better related to the other factor. Furthermore, these problems strongly inﬂuenced the correlation between the constructs because the high correlation (>0.50) initially estimated between them was drastically reduced in the shortened version (zero). Regarding reliability, the estimates showed acceptable magnitudes to describe the con- struct in a group of participants. However, they are possibly less precise if the abbreviated SRQ-L describes an intervention’s individual effects. It is accepted that higher levels of reliability are required, such as >0.89. Interestingly, the brevity does not appear to have seriously affected the reliability of the scores, and the small number of items did not bias toward high reliability. This information may suggest that the content is not redundant or repetitive and still displays moderately different behaviors linked to its constructs. On the other hand, the clinical value indicators reported in Table 3 are helpful for research but especially for professional practice [32,33]. Multiplied with the standardized values of the normal curve, the statistically infrequent (abnormal) differences show an extreme range, especially at the 95% and 99% levels. Applied use may require choosing the 90% level to reasonably determine abnormal differences or the 85% level to reduce Type II error. y yp Regarding the study’s implications, the reduced version can be easily incorporated into screening evaluations during some study semester periods, allowing space for other instruments. On the other hand, it can also be very efﬁcient in measuring the change in self-regulatory skills since it is sensitive to interventions in the educational context. Indeed, some experimental studies have shown that self-regulation skills can be strengthened [4,82]. The development of a reduced version has known advantages for evaluation practice. For example, it reduces examinee fatigue when a set of measures is applied at a ﬁxed time or in a longitudinal design. It reduces the number of estimable parameters when a short measure is used in structural equation modeling; sampled behaviors maintain stronger statistical covariations, and replicability is more guaranteed. 4. Discussion The purpose of the study was to evaluate the internal structure of the psychometric properties of the SRQ-L [24] in a group of Peruvian university students. The motivation was to explore its psychometric functioning in greater depth, given that the trend of previous studies in moderately similar groups [29] did not seem to identify some of its properties and failed to evaluate other important ones, speciﬁcally, measurement invariance, response scaling performance, divergent item–factor relationships, and correlated errors. The analyses related to dimensionality showed results that seem to optimize the valid- ity of the short version, given that the content relationships of the SRQ-L were maximized by selecting those items with better scaling and discriminative power; that is, they bet- ter differentiated the subject’s position on the score continuum. The selected items, as a whole, helped to deﬁne better the score derived from them, through more discriminative, representative items and with better psychometric evidence. In the reduced version, the Autonomy construct now includes behaviors of following suggestions to optimize learning and actively participating in class to understand and improve skills. In Control, this is represented by behaviors to avoid the disapproval of others, actively participating in class, and following suggestions to achieve a good grade and have a good image in the eyes of others. Two of the items in this smaller version (Items 7 and 14 of the Control scale) showed a signiﬁcantly different scaling compared to the rest of the items because the distance between their thresholds was minimal, and one of them still showed threshold disorder. However, these items maintained sufﬁciently high inter-item correlations, high scalability, and a pattern of statistical indicators indistinguishable from the rest of the items. Therefore, they were chosen for the ﬁnal version of the SQR-L. Because previous studies did not examine this, we are unsure if this result comes from a sampling variation, representing a consistent feature of these two items. At this point, one implication is that further examination is required on a new sample to characterize this particular result. q p p The instrument’s content derived from our analyses reduced the behaviors sampled from the original version (six items in the Autonomy factor and eight in the Control factor), which may limit the validity of the initially designed construct. 4. Discussion Although the original version is not excessively long, a parsimonious measure with good psychometric properties is possibly better accepted. A ﬁnal implication of the study is that since the instrument is used in various regions worldwide, our results provide working hypotheses on internal structure and group differences. The reduced version also achieved invariance of its main psychometric parameters be- cause the compared groups of students belonging to different academic semesters showed homogeneity in autonomy and control for learning, with a slightly greater tendency in students from the more advanced academic semesters. This ﬁnding would indicate that autonomy in learning supports, during professional training, learning achievements, well- being, and adjustment in the classroom [14]. In other contexts, promoting self-regulated learning in students during their ﬁrst year at university is necessary to enable them to stay longer and avoid dropping out [83]. The difference could be explained by the period in which the data collection was carried out (the middle of the academic semester). The results might have been different if the measurement had been done at the beginning. One of the indices that showed inconsistency with others (CFI, TLI, G-h) was the Mc [65], a fact possibly associated with the different validity of the items with their construct (factor loads) or the imbalance of the groups compared in a three-group context [84], as occurred in the present study. However, since the rest of the adjustment indices and comparisons between them were satisfactory, the validity of the invariance evaluations can also be considered satisfactory. It can be concluded that the construct is measured invariantly among the three semesters compared. The minor differences in structural invariance (e.g., means, variances, and latent correlations) are not tests of measurement invariance because the differences, even slight, are related to the latent attribute once it has been controlled for differences in the measurement (i.e., measurement invariance). 13 of 17 13 of 17 Sustainability 2022, 14, 11239 Regarding the study limitation, we did not evaluate the replicability of the results since a similar-sized sample was not available in this study phase. Although the replicability of the results can be partially concluded from the invariance study [85], a complete study with larger sample size is required. Another limitation is the possible lack of representativeness of the participants because it does not ensure the generalization of the results. 5. Conclusions The Hispanic version of the SRQ-L applied to Peruvian students is a measure with satisfactory evidence of validity in its internal structure. The results showed that the two constructs were maintained but without two items from the Autonomy scale and two from the Control scale. Because disorder and trivial distinction of some response thresholds were detected, the scaling of the items needed to be modiﬁed from an original scaling of ﬁve to one of three response options. p p The items of this new version show moderate scalability (measured from Mokken Scal- ing Analysis) and a satisfactory ﬁt to the monotonic homogeneity model. From structural equation modeling, the items showed to be moderately or strongly associated with their constructs. The reliability of the scores was appropriate for use in interpreting the construct in groups, and the reliability of the items was acceptable. The measurement invariance was maintained in the groups of the chosen semesters. The structural invariance indicated that this characteristic was not fulﬁlled concerning the mean and latent variance between some semesters. This result was explained as possible natural differences among the groups. The size of these differences was generally small. Finally, the association between the two constructs measured by SRQ-L (autonomy and control) varied between the compared semester groups, indicating a possible evolution of these constructs aligned with their dependency. Although these results point to a more complete and optimal psychometric characterization of SRQ-L, the possible intercultural variability should be evaluated and avoided inducing its validity from one or more studies [86–88]. Author Contributions: Conceptualization, C.M.-S. and G.C.-V.; methodology, C.M.-S.; software, C.M.-S.; validation, C.M.-S., G.C.-V. and V.L.-F.; formal analysis, C.M.-S.; investigation, C.M.-S., G.C.-V. and V.L.-F.; resources, F.T.-T. and G.M.C.; data curation, C.M.-S.; writing—original draft preparation, C.M.-S., G.C.-V. and V.L.-F.; writing—review and editing, F.T.-T., G.C.-V., V.L.-F. and G.M.C.; visualization, G.C.-V., V.L.-F., F.T.-T. and G.M.C.; supervision, F.T.-T. and G.M.C.; project administration, F.T.-T. and G.M.C.; funding acquisition, F.T.-T. All authors have read and agreed to the published version of the manuscript. Funding: This work presents some results of the HIM/2015/017/SSA.1207 research project “Effects of mindfulness training on psychological distress and quality of life of the family caregiver”, Main researcher: Filiberto Toledano-Toledano. The present research was funded with federal funds for health research. It was approved by the Commissions of Research, Ethics and Biosafety (Comisiones de Investigación, Ética y Bioseguridad), Hospital Infantil de México Federico Gómez, National Institute of Health. 4. Discussion The study did not prove the population representativeness of the participating sample, and the degree of representativeness is inconclusive. Finally, it is unknown how the scores obtained from this new version are associated with behaviors and constructs relevant to practical academic life, such as academic performance, student well-being, and other adaptive behaviors. These limitations suggest that, although this version of the SRQ-L can achieve a better psychometric deﬁnition in the sample studied, additional studies are required to verify its results. References 1. Hardy, J.H.; Day, E.A.; Steele, L.M. Interrelationships Among Self-Regulated Learning Processes: Toward a Dynamic Process- Based Model of Self-Regulated Learning. J. Manag. 2019, 45, 3146–3177. [CrossRef] 2. 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Conclusions The funding agency had no control over the study’s design, the collection, analysis, and interpretation of the data, or the writing of the manuscript. Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Commissions of Research, Ethics and Biosafety (Comisiones de Investigación, Ética y Bioseguridad), Hospital Infantil de México Federico Gómez National Institute of Health. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. 14 of 17 Sustainability 2022, 14, 11239 14 of 17 Data Availability Statement: The datasets used during the current study are available from the corresponding author upon reasonable request. Data Availability Statement: The datasets used during the current study are available from the corresponding author upon reasonable request. Acknowledgments: The authors acknowledge the ﬁnancial and technical support of the Writing Lab, Institute for the Future of Education, Tecnologico de Monterrey, Mexico, in producing this work. Acknowledgments: The authors acknowledge the ﬁnancial and technical support of the Writing Lab, Institute for the Future of Education, Tecnologico de Monterrey, Mexico, in producing this work. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. References 2017, 107, 1–12. [CrossRef] 38. Elosua, P.; Zumbo, B. Coeﬁcientes de ﬁabilidad para escalas de respuesta ordenada. Psicothema 2008, 20, 896–901. 39. Aquiahuatl Torres, E.C. Metodología de la Investigación Interdisciplinaria. Tomo I Investigación Monodisc Mexico City, Mexico, 2015. y 40. Ato, M.; López-García, J.J.; Benavente, A. Un sistema de clasiﬁcación de los diseños de investigación en psicología. 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https://openalex.org/W4391836797	https://link.springer.com/content/pdf/10.1007/s13304-023-01739-6.pdf	English	null	Clinical predictors of postoperative complications in the context of enhanced recovery (ERAS) in patients with esophageal and gastric cancer	Updates in surgery	2,024	cc-by	6,251	Abstract The overall frequency of postoperative complications in patients with esophageal and gastric cancer diverges between studies. We evaluated the frequency and assessed the relationship between complications and demographic and clinical features. For this observational study, data were extracted from the ERAS Registry managed by the University of Verona, Italy. Patients were evaluated and compared for postoperative complications according to the consensus-based classification and the Clavien–Dindo scale. The study population was 877 patients: 346 (39.5%) with esophageal and 531 (60.5%) with gastric cancer; 492 (56.2%) reported one or more postoperative complications, 213 (61.6%) of those with esophageal and 279 (52.5%) of those with gastric cancer. When stratified by consensus-based classification, patients with esophageal cancer reported general postoperative complications more frequently (p < 0.001) than those with gastric cancer, but there was no difference in postoperative surgical complications between the two groups. Multiple logistic regression models revealed an association between postoperative complications and the Charlson Comorbidity Index (adjusted odds ratio [OR] 1.22; 95% confidence interval [CI] 1.08–1.36), operation time (adjusted OR, 1.08; 95% CI 1.00–1.15), and days to solid diet intake (adjusted OR, 1.39; 95% CI 1.20–1.59). Complications in patients with esophageal and gastric cancer are frequent, even in those treated according to ERAS principles, and are often associated with comorbidities, longer operative time, and longer time to solid diet intake. Keywords Gastric and esophageal cancer · Enhanced recovery after surgery · Postoperative complications · Surgery · Prehabilitation Updates in Surgery (2024) 76:1855–1864 https://doi.org/10.1007/s13304-023-01739-6 Updates in Surgery (2024) 76:1855–1864 https://doi.org/10.1007/s13304-023-01739-6 ORIGINAL ARTICLE Christian Geroin1 · Jacopo Weindelmayer1 · Serena Camozzi1 · Barbara Leone1 · Cecilia Turolo1 · Selma Hetoja1 · Maria Bencivenga1 · Michele Sacco1 · Carlo Alberto De Pasqual1 · Eugenia Mattioni1 · Giovanni de Manzoni1 · Simone Giacopuzzi1 Christian Geroin1 · Jacopo Weindelmayer1 · Serena Camozzi1 · Barbara Leone1 · Cecilia Turolo1 · Selma Hetoja1 · Maria Bencivenga1 · Michele Sacco1 · Carlo Alberto De Pasqual1 · Eugenia Mattioni1 · Giovanni de Manzoni1 · Simone Giacopuzzi1 Received: 2 June 2023 / Accepted: 19 December 2023 / Published online: 15 February 2024 © The Author(s) 2024 1 General and Upper G.I. Surgery Division, University of Verona, Borgo Trento, Verona, Italy Clinical predictors of postoperative complications in the context of enhanced recovery (ERAS) in patients with esophageal and gastric cancer Christian Geroin1 · Jacopo Weindelmayer1 · Serena Camozzi1 · Barbara Leone1 · Cecilia Turolo1 · Selma Hetoja1 · Maria Bencivenga1 · Michele Sacco1 · Carlo Alberto De Pasqual1 · Eugenia Mattioni1 · Giovanni de Manzoni1 · Simone Giacopuzzi1 * Christian Geroin christian.geroin@univr.it Introduction complications [5–8], and from 10 to 40% in patients with gastric cancer [9, 10]. Previous studies investigating post- operative complications reported a wide range of incidence rates due to the use of inconsistent definitions and report- ing systems (major, minor or overall complications), as well as various classifications (e.g., Clavien-Dindo, complica- tions list) [2, 3, 6, 7, 11]. Recently, two international expert consensus classifications for complications have helped to improve consistency in their reporting in esophagectomy [5] and gastrectomy (intraoperative, postoperative general, postoperative surgical) [2], thereby facilitating comparison of outcomes. Postoperative complications in patients with esophagogastric cancer are associated with increased mortality, longer hospi- tal length of stay (LOS), and higher healthcare costs [1–5]. The overall 30 days frequency in esophagogastric cancer varies, with morbidity rates from 24 to 59% in patients with esophageal cancer, with predominant pulmonary and cardiac Several studies have focused on reducing complications by improving surgical techniques [12]. Currently, periopera- tive care is being standardized through the implementation of the Enhanced Recovery After Surgery (ERAS) protocol 1 General and Upper G.I. Surgery Division, University of Verona, Borgo Trento, Verona, Italy (0123 3456789) 3456789) 1856 Updates in Surgery (2024) 76:1855–1864 [13, 14]. The application of ERAS protocols in esophago- gastric cancer surgery [13, 14] has been associated with better outcomes and lower hospital costs as compared with conventional care [15–17]. Systematic reviews and meta- analyses in esophagectomy have reported fewer pulmonary complications and shorter hospital length of stay (LOS), without increasing readmissions [18–20]. Similarly, the implementation of ERAS in gastrectomy has demonstrated a reduction in intraabdominal drain and nasogastric tube usage, facilitated a faster return to solid diet, and resulted in shorter LOS, without increasing complications [12]. Despite improvements in postoperative care and surgical techniques, surgery is still associated with high morbidity rates ranging from 26 to 64% in esophageal cancer [18, 19] and from 5 to 33.9% in gastric cancer [15, 21, 22]. While this raises ques- tions about the benefit of ERAS for reducing postoperative morbidity [23], the studies may not have adequately captured the true incidence rate of complications for several reasons. First, they may have underestimated the rate by considering only “major” complications. Second, they did not classify complications as proposed by the international consensus [18, 19], which limits comparison of incidence rates among study canters. Moreover, these cohorts did not consistently report adherence to the ERAS program, except for one study [15]. Subjects The study population was patients with a diagnosis of esophageal, esophagogastric junction, and gastric cancer who underwent surgery with radical intent in the con- text of an ERAS program [15] at our Institution between November 2013 and December 2022. Exclusion criteria were resection performed without curative intent, multio- rgan resection, and urgent procedures. We collected the following clinical, oncological, surgi- cal data, and postoperative complications [4]: age, sex, body-mass index (BMI, weight in kg divided by height in meters squared), smoking history, serum albumin level, preoperative comorbidities, the Charlson Comorbidities Index (CCI) calculated without including the esophageal and gastric cancer score, history of major surgery, Ameri- can Society of Anesthesiologists (ASA) score, tumor his- tology, location, and TNM pathological stage. We also recorded neoadjuvant therapy, type of surgery, intra- operatory infusion, operative time, days to ward transfer, days to sitting/standing upright, days to independent gait without external aid, nutrition intake, and days to hospital discharge. Occurrence of one or more postoperative com- plications was recorded up to 90 days after the operation [15] according to a consensus-based classification (postop- erative general, postoperative surgical complications) [2] and the Clavien–Dindo scale (CD). Compliance with the protocol was evaluated according to the 15 ERAS items [15, 17]. To overcome these possible limitations, we conducted a prospective study using the ERAS Registry of our Institution and applied an international classification of complications [2]. We reported the level of adherence to ERAS [15] and provided a detailed description of several clinical features. The aim was to assess and compare the complications rates in a large patient cohort with esophageal and gastric can- cer and to examine the relationship with demographic and clinical variables. Our findings may be useful for identify- ing at-risk patients and complications-associated comor- bidities that could benefit from earlier therapeutic strategies (prehabilitation). Introduction Because adherence to the entire ERAS pathway is cru- cial for obtaining the greatest benefits, any lack of adherence could increase the complications rate. Finally, many of these cohorts were retrospective studies [18, 21, 22], which may have introduced potential referral bias. They also failed to compare between the clinical and demographic character- istics of patients with and without complications and those with esophageal and gastric cancer. natural history, risk factors, and comorbidity in patients with esophageal or gastric cancer [15]. Statistical analysis Data are expressed as mean ± standard deviation (SD) and range for continuous variables, counts, and percentages for categorical variables. We compared the groups using the Mann–Whitney U test for continuous variables and the chi-squared test or Fisher’s exact test (if ≤ 5 expected frequencies) for categorical variables. Logistic regression models were created to estimate unadjusted and adjusted odds ratio (OR; 95% confidence interval [CI]) for one or more complications (dependent variable) in relation to sociodemographic and clinical characteristics (independ- ent variables). Independent variables were chosen accord- ing to exploratory analysis results and clinical relevance. All tests were statistically significant at p < 0.05. Statistical analysis was performed using SPSS statistical software version 20 (IBM-SPSS, Armonk, NY, USA). Demographic, surgical, and post‑operative data of total sample The study population was 877 patients, 346 (39.5%) treated for esophageal and 531 (60.5%) for gastric cancer; 615 (70.1%) were male and 262 (29.9%) were female, with a mean age of 68.7 ± 12.6 years. A total of 648 (73.8%) patients presented one or more preoperative comorbidi- ties: cardiovascular (n = 486, 55.4%), respiratory (n = 133, 15.2%), metabolic (n = 226, 25.8%), and kidney (n = 59, 6.7%) disease, with an average of CCI of 1.2 ± 1.5. Previ- ous major surgery was reported in 154 (17.6%) patients. The ASA score was II in 537 (61.5%). Adenocarcinoma was the most frequent type of tumor (overall, n = 750, 85.8%) with pathological stage T grade 3 in 234 (27.4%), stage N grade 0 in 406 (47.8%), and stage M grade 0 in 782 (91.7%). The majority of patients (n = 530, 60.6%) under- went neoadjuvant therapy. Different types of surgery were employed: Ivor-Lewis was the most frequent technique (n = 251, 64.5%) in patients with esophageal cancer, while total gastrectomy was the most frequent (n = 290, 59.9%) in patients with gastric cancer. The total average amount of intraoperative infusion was 3129.4 ± 1578.3 mL, the operative time was 6.6 ± 2.6 h, the time to ward trans- fer was 0.5 ± 1.4 days, the time to position the patient in sitting/standing position by the physiotherapist was 1.4 ± 1.7 days, and the time to independent unaided gait was 3.1 ± 3.2 days. The carbohydrate load was delivered in 771 (88.2%) patients. The time to return to solid diet was 4.8 ± 4.7 days, and hospital discharge occurred at 10.2 ± 8.8 days on average (Table 1). Adherence to ERAS after its implementation was appropriate for many items, with a compliance rate of 70% or higher for 9 and 14 out of 15 items for esophageal and gastric cancer, respectively (Fig. 1, panel A, B). Patients with esophageal cancer reported grade IIIa and IVa complications more frequently but fewer grade I compli- cations than those with gastric cancer (CD score, Table 2). In addition, patients with postoperative complications were older, had preoperative comorbidities more often (e.g., car- diovascular, respiratory, metabolic), a higher CCI score, ASA score grade III, greater amount of intraoperative liquid infusion, and a more frequent squamous tumor histology located predominately in the esophagus than patients with- out postoperative complications. Demographic, surgical, and post‑operative data of total sample Patients with postoperative complications and esophageal tumor underwent mini-inva- sive intervention and Ivor-Lewis surgery more frequently. They were also noted to have longer operative time and more days to ward transfer, stand/sit upright and walk unaided, return to liquid/solid diet intake, and hospital discharge than patients without complications (Table 1). The univariate logistic regression model yielded an asso- ciation between postoperative complications and many of clinical and demographic features (Table 1). After adjusting for all variables in the model, multivariate logistic regres- sion analysis confirmed associations with the variables: CCI (adjusted OR, 1.22; 95% CI 1.08–1.36), operative time (adjusted OR, 1.08; 95% CI 1.00–1.15), and number of days to return to solid diet intake (adjusted OR, 1.39; 95% CI 1.20–1.59) (Table 3). Methods For this observational study, data were extracted from the ERAS Registry managed by the General and Upper G.I. Surgery Division, University of Verona, Verona, Italy. The ERAS Registry prospectively collects data on symptoms, Updates in Surgery (2024) 76:1855–1864 1857 complication was recorded in 133 (71.1%) patients with esophageal cancer, while a combined form (more than one general complication) was recorded in 54 (28.9%). Only one isolated surgical complication was recorded in 73 (83%) patients, while a combined form (more than one surgical complication was recorded in 15 (17%). Only one isolated general complication was record in 158 (79.7%) patients with gastric cancer, while a combined form (more than one general complication was record in 40 (20.3%). Only one isolated surgical complication was recorded in 92 (73.6%), while a combined form (more than one surgi- cal complication) was recorded in 33 (26.4%). A complete list of complications is presented in Fig. 2, panel B. Discussion In this observational study of the ERAS Registry, we found that one or more postoperative complications were recorded for 56.2% of patients with esophageal and gastric cancer. When stratified by a consensus based-classification [2], postoperative general complications were more frequent and severe in patients with esophageal cancer. We noted several associations between postsurgical complications and clini- cal and demographic features; however, few remained after we adjust for all variables in the model, such as higher CCI score, longer operative time, and more days to return to solid diet. We found that 492 (56.2%) patients reported one or more postoperative complications: 213 (61.6%) of those treated for esophageal and 279 (52.5%) of those treated for gas- tric cancer. When stratified by the consensus-based clas- sification, the patients with esophageal cancer reported postoperative general complications more frequently (54%, p < 0.001) than those with gastric cancer (37%). No dif- ference in postoperative surgical complications was noted between those with esophageal (25.5%) and gastric can- cer (23.8%) (Fig. 2, panel A). Only one isolated general Updates in Surgery (2024) 76:1855–1864 1858 Table 1 Demographic and clinical features of patients with esophageal or gastric cancer with or without postoperative complications Variable Total sample Without complications With complications Without vs. with complications (p value) Patients, no, % 877 385 (43.8) 492 (56.2) Sex, no. (%) Male 615 (70.1) 259 (67.3) 356 (72.4) 0.103b Female 262 (29.9) 126 (32.7) 136 (27.6) Age, year, mean (SD) 68.7 (12.6) 66.9 (12.7) 70.1 (12.4) < 0.001d Body-mass index (kg/m2), mean (SD) 25.1 (4.3) 25 (4.2) 25.2 (4.4) 0.864d Smoking history, no. (%) 141 (16.1) 53 (13.8) 88 (17.9) 0.099b Serum albumin level, g/L, mean (SD) 38.02 (6.3) 38.2 (5.8) 37.8 (6.6) 0.329d Preoperative comorbidities, no. (%) Cardiovascular 486 (55.4) 188 (48.8) 298 (60.6) 0.001b Respiratory 133 (15.2) 43 (11.2) 90 (18.3) 0.004b Metabolic diseases 226 (25.8) 84 (21.8) 142 (28.9) 0.018b Kidney 59 (6.7) 21 (5.5) 38 (7.7) 0.183b Charlson comorbidity index, mean (SD) 1.2 (1.5) 0.8 (1.2) 1.4 (1.6) < 0.001d Previous major surgery, no. Discussion (%) 154 (17.6) 57 (14.8) 97 (19.7) 0.058b American Society of Anesthesiologists score (%) I 32 (3.7) 21 (5.5) 11 (2.2) 0.012b II 537 (61.5) 264 (68.8) 273 (55.8) < 0.001b III 279 (32) 92 (24) 187 (38.2) < 0.001b IV 25 (2.9) 7 (1.8) 18 (3.7) 0.102b Cancer characteristics Esophageal 346 (100) 133 (38.4) 213 (61.6) 0.009b Gastric 531 (100) 252 (47.5) 279 (52.5) Tumor histology, no. (%) Adenocarcinoma 750 (85.8) 350 (91.4) 400 (81.5) < 0.001b Squamous 99 (11.3) 23 (6) 76 (15.5) < 0.001b Mixed 4 (0.5) 0 4 (0.8) 0.136c NET/Others 21 (2.4) 10 (2.6) 11 (2.2) 0.723b Tumor location, no. (%) Esophagus 251 (28.9) 96 (25.2) 155 (31.8) 0.034b Esophagogastric junction 235 (27) 92 (24.1) 143 (29.3) 0.090b Stomach 383 (44.1) 193 (50.7) 190 (38.9) 0.001b Pathological stage (T) 0 121 (14.2) 56 (15) 65 (13.4) 0.541b I 156 (18.3) 71 (19) 85 (17.7) 0.619b II 112 (13.2) 51 (13.7) 61 (12.7) 0.679b III 234 (27.4) 97 (26) 137 (28.3) 0.449b IV 230 (27) 98 (26.3) 132 (27.9) 0.689b Pathological stage (N) 0 406 (47.6) 174 (46.6) 232 (48.3) 0.625b I 160 (18.8) 71 (19) 89 (18.5) 0.855b II 109 (12.8) 45 (12.4) 64 (13.3) 0.582b III 173 (20.8) 82 (22) 91 (19.9) 0.276b Pathological stage (M) 0 782 (91.7) 348 (93.3) 434 (90.4) 0.131b I 71 (8.3) 25 (6.7) 46 (9.6) 0.131b Neoadjuvant therapy, no. (%) 530 (60.6) 228 (59.4) 302 (61.6) 0.498b Type of surgery, no. (%) Updates in Surgery (2024) 76:1855–1864 1859 Table 1 (continued) Variable Total sample Without complications With complications Without vs. with complications (p value) Esophagus Hybrid or mini-invasive intervention 166 (48.1) 49 (37.1) 117 (54.9) 0.001b Open 179 (51.9) 83 (62.9) 96 (45.1) Stomacha Mini-invasive intervention 62 (12.8) 35 (14.9) 27 (10.8) 0.183b Open 422 (87.2) 200 (85.1) 222 (89.2) Type of surgery, no. Discussion (%) Esophagus Ivor-Lewis 251 (64.5) 109 (73.7) 142 (58.9) 0.005b McKeown 84 (21.6) 20 (13.5) 64 (26.6) Esophagogastrectomyb 54 (13.9) 19 (12.8) 35 (14.5) Stomach Total gastrectomy 290 (59.9) 143 (60.9) 147 (59) 0.684c Subtotal gastrectomy 194 (40.1) 92 (39.1) 102 (41) Intra-operatory infusion, mL 3129.4 (1578.3) 2798.20(1049.9) 3381.93 (1845.8) < 0.001d Operation time, hours, mean (SD) 6.6 (2.6) 6.1 (2.3) 7.02 (2.7) < 0.001M Ward transfer, days, mean (SD) 0.5 (1.4) 0.3 (0.6) 0.7 (1.8) < 0.001d Physiotherapy Verticalization, days, mean (SD) 1.4 (1.7) 1.2 (0.6) 1.6 (2.3) < 0.001d Independent gait, days, mean (SD) 3.1 (3.2) 2.4 (1.2) 3.6 (4.1) < 0.001d Nutrition Carbohydrate load, no. (%) 771 (88.2) 339 (88.1) 432 (88.3) 0.894b Liquid diet, days mean (SD) 2.05 (3.4) 1.4 (1.2) 2.5 (4.4) < 0.001d Solid diet, days mean (SD) 4.8 (4.7) 3.6 (1.2) 5.8 (6.1) < 0.001d Hospital discharge, days mean (SD) 10.2 (8.8)d 6.7 (1.5) 12.9 (11.1) < 0.001d a Stomach does not include n = 46 patients who underwent esophagectomy; Mini-invasive for both esophagus and stomach denotes laparoscopic surgery b Chi-square test c Fisher’s exact test d Mann–Whitney U Test; in bold: statistically significant values with p < 0.05. Missing: serum albumin level n = 20; Charlson Comorbidity Index n = 23; American Society of Anesthesiologists score n = 4; tumor histology n = 3; tumor location n = 8; clinical stage (t) n = 24; clinical stage (n) n = 29; clinical stage (m) n = 24; neoadjuvant therapy n = 3; Type of surgery, esophagus, hybrid or mini-invasive vs open n = 1; stomach, hybrid or mini-invasive vs open n=47 c Fisher’s exact test d Mann–Whitney U Test; in bold: statistically significant values with p < 0.05. Abbreviations: SD standard deviation, NET neuroendocrine tumor; Other interventions: Ivor-Lewis, McKeown, esophagogastrectomy for esoph- ageal surgery; other interventions total gastrectomy and subtotal gastrectomy for stomach cancer Discussion Missing: serum albumin level n = 20; Charlson Comorbidity Index n = 23; American Society of Anesthesiologists score n = 4; tumor histology n = 3; tumor location n = 8; clinical stage (t) n = 24; clinical stage (n) n = 29; clinical stage (m) n = 24; neoadjuvant therapy n = 3; Type of surgery, esophagus, hybrid or mini-invasive vs open n = 1; stomach, hybrid or mini-invasive vs open n = 47 e Esophagectomy: includes n = 46 patients with gastric cancer, plus 3 missing values; Ivor-Lewis n = 1; McKeown n = 1; esophagogastrectomy n = 1; minimally invasive esophagectomy n = 1; total gastrectomy n = 47; subtotal gastrectomy n = 47; minimally invasive gastrectomy n = 47; intra-operatory infusion n = 105; operative time n = 4; ward transfer n = 4; verticalization n = 14; independent gait n = 23; carbohydrate load n = 3; liquid diet = 11; solid diet n = 19; hospital discharge n = 23 Despite improvement in postoperative care and surgi- cal techniques achieved in ERAS program, surgery is still associated with high morbidity rates in both esophageal [18, 19] and gastric cancer [15, 21, 22]. It may be, possi- ble, however, that the studies underestimated complications rates because they focused on “major” complications. In our cohort, the overall incidence of postoperative complications was slightly higher for the patients treated for gastric cancer, as we included all types of complications, including those with CD < 3. These data extend previous findings and sug- gest that complications are frequent in patients with esopha- geal and gastric cancer also in those treated according to ERAS principles. For example, esophagectomy was associ- ated with a higher morbidity rate than gastrectomy, despite major advances in surgery, anesthesia, and perioperative care, including minimally invasive procedures. We noted that patients with complications were more likely to have multiple comorbidities. Indeed, Updates in Surgery (2024) 76:1855–1864 1860 cardiovascular, respiratory, and metabolic comorbidi- ties (e.g., diabetes mellitus) are frequent in patients with h l d i [6 24] P i di and anastomotic leakage [24]. Vascular disease, particu- larly arterial calcification, is an independent predictor for li i i ll i l k [24] Fig. 1 Compliance with ERAS items (n = 15) for esophageal (panel A) and gastric (panel B) cancer; POD denotes postoperative days Fig. 1 Compliance with ERAS items (n = 15) for esophageal (panel A) and gastric (panel B) cancer; POD denotes postoperative days Fig. 1 Compliance with ERAS items (n = 15) for esophageal (panel A) and gastric (panel B) cancer; POD denotes postoperative days and anastomotic leakage [24]. Vascular disease, particu- larly arterial calcification, is an independent predictor for severe complications, especially anastomotic leakage [24]. In patients undergoing laparoscopic total gastrectomy, the impact of comorbidities is considerable. For instance, pul- monary disease is associated with a higher risk of postop- erative complications [25]. These data may also provide cardiovascular, respiratory, and metabolic comorbidi- ties (e.g., diabetes mellitus) are frequent in patients with esophageal and gastric cancer [6, 24]. Previous studies suggested that patients with comorbidities and those using polypharmacy (≥ 5 medications) are more susceptible to developing complications [24]. Heart failure, hyperten- sion, and renal insufficiency are reported to be independ- ent clinical predictors associated with major complications 1861 Updates in Surgery (2024) 76:1855–1864 Fig. 2 Panel A Comparison of complications in patients treated for esophageal or gastric cancer according to a consensus-based classification; panel B percent- age of postoperative general and surgical complications. The category “minor/other” includes complications that cannot be classified within the consensus- based classification. Abbrevia- tions: CPR cardiopulmonary resuscitation, CCU coronary care unit, ICU intensive care unit, EF ejection fraction, CVVH continuous veno-venous hemofiltration, NG nasogastric Fig. 2 Panel A Comparison of complications in patients treated for esophageal or gastric cancer according to a consensus-based classification; panel B percent- age of postoperative general and surgical complications. The category “minor/other” includes complications that cannot be classified within the consensus- based classification. Abbrevia- tions: CPR cardiopulmonary resuscitation, CCU coronary care unit, ICU intensive care unit, EF ejection fraction, CVVH continuous veno-venous hemofiltration, NG nasogastric Updates in Surgery (2024) 76:1855–1864 1862 Table 2 Clavien-Dindo classification of surgical complications in patients with esophageal or gastric cancer In bold: statistically significant values a Chi-square test b Fisher’s exact test; Missing: n = 14 (n = 9 esophageal cancer, n = 5 stomach cancer) Variable Total sample Esophageal cancer Gastric cancer p value Patients, no. (%) 478 204 274 I 71 (8.1) 19 (5.5) 52 (9.8) 0.026a II 212 (24.2) 77 (22.3) 135 (25.4) 0.340a III 2 (0.2) 0 2 (0.4) 0.523b IIIa 120 (13.7) 65 (18.8) 55 (10.4) < 0.001a IIIb 27 (3.1) 13 (3.8) 14 (2.6) 0.327a IV 1 (0.1) 0 1 (0.2) 1000b IVa 36 (4.1) 26 (7.5) 10 (1.9) < 0.001a IVb 2 (0.2) 1 (0.3) 1 (0.2) 1000b V 7 (0.8) 3 (0.9) 4 (0.8) 1000b Table 3 Clinical and demographic variables associated with postop- erative complications in patients with esophageal or gastric cancer patients with esophageal and gastric cancer may be at high risk of anastomosis leakage [26]. However, ERAS consensus guidelines recommend offering patients drink and food at will from day 1 after total gastrectomy [14]. These findings suggest the need to implement prehabilitation of nutrition support and physiotherapy, which could benefit patient well- being during perioperative periods [27]. Proper postopera- tive care for optimizing pulmonary function may be required for patients with pulmonary disease [25]. Table 3 Clinical and demographic variables associated with postop- erative complications in patients with esophageal or gastric cancer Significant associations at p < 0.05; Bold indicates statistically sig- nificant values. OR odds ratio, CI confidence interval, ASA American Society of Anesthesiologists score; in bold: statistically significant values. Missing: Charlson Comorbidity Index (n = 23); American Society of Anesthesiologists score (n = 4); operative time (n = 4); ward transfer (n = 4); verticalization(n = 14); independent gait (n = 23; solid diet (n = 19) Adjusted Independent variable OR 95% CI p value Age, year 1.01 0.99–1.02 0.084 Charlson comorbidity index, score 1.22 1.08–1.36 0.001 ASA III, score 1.39 0.97–1.98 0.066 Operative time, h 1.08 1.00–1.15 0.034 Ward transfer, days 0.89 0.68–1.17 0.441 Verticalization, days 1.13 0.81–1.56 0.468 Independent gait, days 1.05 0.89–1.23 0.561 Solid diet, days 1.39 1.20–1.59 < 0.001 Adherence to the entire ERAS pathway is crucial for achieving the greatest benefits, and any lack of adherence may increase the complications rate. Except for one study [15], other previous studies did not consistently report adher- ence to the ERAS program In our study, all our patients were treated according to ERAS principles [13, 14], with a 70% adherence rate or higher for many items. Nonetheless, we observed no decrease in complications rates when com- pared with outcomes in a non-ERAS program. This obser- vation is shared by previous studies that suggested that the ERAS program does not increase the complications rate as compared with conventional care but rather leads to better outcomes, reducing LOS [12] and hospital costs [15–17]. Despite the high adherence, there are plausible reasons for the lack of reduction of morbidity rate: heterogeneity/scant standardization in the number and the definition of ERAS components, resulting in low adherence by clinicians [23]; the unclear contribution of each item to prevent complica- tions from arising; and finally, the difference in associated comorbidities, not all of which eligible for adherence to the ERAS program [28]. an explanation for the observed association between com- plications and longer operative time in our cohort. The impact of operative time is likely to be experienced mostly by patients with poor physical and nutritional status [6]. Longer operative time is also a component of complex surgical intervention. The benefits of minimally invasive surgery notwithstanding (e.g., reduced pain), many stud- ies have found that the procedure involves a steep learning curve, which increases operative time and may also lead to differences in outcomes between trainees and experienced surgeons. The present study has several limitations. First, our sam- ple was made up of a mixed population. Conflict of interest The authors declare no conflict of interest. Conflict of interest The authors declare no conflict of interest. Ethical approval All procedures performed in this study involving human participants were under the ethical standards of the institutional and/or national research committee and with the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. The appropriate ethics committee/institutional review board (IRB) of our institute (Comitato Etico per la Sperimentazione dell’Azienda Ospe- daliera Istituti Ospitalieri di Verona) approved data collection (code 2175CESC). The need for informed consent was waived by the Ethics Committee for the Experimentation of the Hospital, Hospitaller Insti- tutes of Verona. This article does not contain any studies with animal subjects performed by any authors. 6. Liu XL, Wang RC, Liu YY, Chen H, Qi C, Hu LW, Yi J, Wang W (2021) Risk prediction nomogram for major morbidity related to primary resection for esophageal squamous cancer. Medicine (Baltimore) 100(31):e26189 7. Jezerskyte E, van Berge Henegouwen MI, van Laarhoven HWM, van Kleef JJ, Eshuis WJ, Heisterkamp J, Hartgrink HH, Rosman C, van Hillegersberg R, Hulshof M, Sprangers MAG, Gisbertz SS, Dutch Upper GICG (2021) Postoperative complications and long- term quality of life after multimodality treatment for esophageal cancer: an analysis of the prospective observational cohort study of esophageal-gastric cancer patients (POCOP). Ann Surg Oncol 28(12):7259–7276 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/. 8. Liang Z, Luo K, Wang Y, Zeng Q, Ling X, Wang S, Dragomir MP, Li Q, Yang H, Xi M, Chen B (2023) Clinical and dosimetric pre- dictors for postoperative cardiopulmonary complications in esoph- ageal squamous cell carcinoma patients receiving neoadjuvant chemoradiotherapy and surgery. Ann Surg Oncol 30(1):529–538 9. Esophageal can- cer requires more complex invasive surgical procedures than gastric cancer, predisposing patients to more severe complications. Stratification based on type of cancer and severity (CD < 3B vs. CD ≥ 3B for esophageal and CD < 3A We noted that patients with complications need more time before returning to a solid diet. One explanation for the delay in resuming solid food intake is that surgeons and nurses may be reluctant to initiate early oral feeding since Updates in Surgery (2024) 76:1855–1864 1863 vs. CD ≥ 3A for gastric cancer) might have revealed fur- ther clinical predictors specific for each disease. Second, we stratified our sample by a consensus-based classification conceptualized for gastric [2] but not for esophageal cancer.i a complications list after gastrectomy for cancer. Gastric Cancer 22(1):172–189 vs. CD ≥ 3A for gastric cancer) might have revealed fur- ther clinical predictors specific for each disease. Second, we stratified our sample by a consensus-based classification conceptualized for gastric [2] but not for esophageal cancer.i ( ) 3. Baiocchi GL, Giacopuzzi S, Vittimberga G, De Pascale S, Pas- torelli E, Gelmini R, Vigano J, Graziosi L, Vagliasindi A, Rosa F, Steccanella F, Demartini P, Reddavid R, Berselli M, Elmore U, Romario UF, Degiuli M, Morgagni P, Marrelli D, D’Ugo D, Rosati R, De Manzoni G (2022) Clinical outcomes of patients with complicated post-operative course after gastrectomy for can- cer: a GIRCG study using the GASTRODATA registry. Updates Surg. https://doi.org/10.1007/s13304-022-01318-1 These limitations notwithstanding, our study findings indicate that postoperative complications are frequent in patients with esophageal and gastric cancer, and that they are often associated with comorbidities, longer operative time, and longer time to solid diet intake. Such patients may benefit from prehabilitation and from nutrition support and physiotherapy in particular. y g g Surg. https://doi.org/10.1007/s13304-022-01318-1 4. Baiocchi GL, Giacopuzzi S, Reim D, Piessen G, Costa PMD, Reynolds JV, Meyer HJ, Morgagni P, Gockel I, Santos LL, Jensen LS, Murphy T, D’Ugo D, Rosati R, Fumagalli Romario U, Degiuli M, Kielan W, Monig S, Kolodziejczyk P, Polkowski W, Pera M, Schneider PM, Wijnhoven B, de Steur WO, Gisbertz SS, Hart- grink H, van Sandick JW, Botticini M, Holscher AH, Allum W, De Manzoni G (2020) Incidence and Grading of Complications After Gastrectomy for Cancer Using the GASTRODATA Reg- istry: A European Retrospective Observational Study. Ann Surg 272(5):807–813 Funding Open access funding provided by Università degli Studi di Verona within the CRUI-CARE Agreement. No funds were received to conduct this study. Conflict of interest The authors declare no conflict of interest. Strong VE, Song KY, Park CH, Jacks LM, Gonen M, Shah M, Coit DG, Brennan MF (2010) Comparison of gastric cancer sur- vival following R0 resection in the United States and Korea using an internationally validated nomogram. Ann Surg 251(4):640–646 10. Brenkman HJF, Gisbertz SS, Slaman AE, Goense L, Ruurda JP, van Berge Henegouwen MI, van Hillegersberg R (2017) Dutch upper gastrointestinal cancer audit, postoperative outcomes of minimally invasive gastrectomy versus open gastrectomy dur- ing the early introduction of minimally invasive gastrectomy in the netherlands: a population-based cohort study. Ann Surg 266(5):831–838 Declarations 5. Low DE, Kuppusamy MK, Alderson D, Cecconello I, Chang AC, Darling G, Davies A, D’Journo XB, Gisbertz SS, Griffin SM, Hardwick R, Hoelscher A, Hofstetter W, Jobe B, Kitagawa Y, Law S, Mariette C, Maynard N, Morse CR, Nafteux P, Pera M, Pramesh CS, Puig S, Reynolds JV, Schroeder W, Smithers M, Wijnhoven BPL (2019) Benchmarking complications associated with esophagectomy. Ann Surg 269(2):291–298 References Tang Z, Lu M, Qu C, Zhang Y, Li L, Li S, Qi L, Cheng C, Tian H (2022) Enhanced recovery after surgery improves short-term out- comes in patients undergoing esophagectomy. Ann Thorac Surg 114(4):1197–1204 24. van Kooten RT, Bahadoer RR, Peeters K, Hoeksema JHL, Steyer- berg EW, Hartgrink HH, van de Velde CJH, Wouters M, Tollenaar R (2021) Preoperative risk factors for major postoperative compli- cations after complex gastrointestinal cancer surgery: a systematic review. Eur J Surg Oncol 47(12):3049–3058 15. Weindelmayer J, Mengardo V, Gasparini A, Sacco M, Torroni L, Carlini M, Verlato G, de Manzoni G (2021) Enhanced recovery after surgery can improve patient outcomes and reduce hospital cost of gastrectomy for cancer in the west: a propensity-score- based analysis. Ann Surg Oncol 28(12):7087–7094 25. Jeong O, Jung MR, Ryu SY (2018) Impact of various types of comorbidities on the outcomes of laparoscopic total gas- trectomy in patients with gastric carcinoma. J Gastric Cancer 18(3):253–263 16 Weindelmayer J, Verlato G, Alberti L, Poli R, Priolo S, Bovo C, de Manzoni G (2019) Enhanced recovery protocol in esophagectomy, is it really worth it? A cost analysis related to team experience and protocol compliance. Dis Esophagus. https://doi.org/10.1093/dote/ doy114 26. He H, Ma Y, Zheng Z, Deng X, Zhu J, Wang Y (2022) Early versus delayed oral feeding after gastrectomy for gastric cancer: a systematic review and meta-analysis. Int J Nurs Stud 126:104120 17. Giacopuzzi S, Weindelmayer J, Treppiedi E, Bencivenga M, Ceola M, Priolo S, Carlini M, de Manzoni G (2017) Enhanced recovery after surgery protocol in patients undergoing esophagectomy for cancer: a single center experience. Dis Esophagus 30(4):1–6 27. Tukanova KH, Chidambaram S, Guidozzi N, Hanna GB, McGregor AH, Markar SR (2022) Physiotherapy regimens in esophagectomy and gastrectomy: a systematic review and meta- analysis. Ann Surg Oncol 29(5):3148–3167 g p p g 18. Findlay JM, Gillies RS, Millo J, Sgromo B, Marshall RE, Maynard ND (2014) Enhanced recovery for esophagectomy: a systematic review and evidence-based guidelines. Ann Surg 259(3):413–431 28. 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Mortensen K, Nilsson M, Slim K, Schafer M, Mariette C, Braga M, Carli F, Demartines N, Griffin SM, Lassen K (2014) Enhanced Recovery After Surgery, Consensus guidelines for enhanced recovery after gastrectomy: enhanced Recovery After Surgery (ERAS(R)) Society recommendations. Br J Surg 101(10):1209–29 23. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. References World J Surg 43(10):2490–2498 19. Markar SR, Karthikesalingam A, Low DE (2015) Enhanced recov- ery pathways lead to an improvement in postoperative outcomes following esophagectomy: systematic review and pooled analysis. Dis Esophagus 28(5):468–475 20 Puccetti F, Wijnhoven BPL, Kuppusamy M, Hubka M, Low DE (2022) Impact of standardized clinical pathways on esophagec- tomy: a systematic review and meta-analysis. 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https://openalex.org/W2589041862	http://repository.unair.ac.id/85928/1/Aggregatibacter%20actinomycetemcomitans.pdf	English	null	Antibacterial effects of 70% ethanol and water extract of cacao beans (Theobroma cacao L.) on Aggregatibacter actinomycetemcomitans	Dental Journal	2,017	cc-by-sa	6,290	104 104 Dental Journal (Majalah Kedokteran Gigi) 2016 June; 49(2): 104–109 Research Report Research Report abstract Background: Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) is a Gram negative bacteria that form a subgingival plaque causing periodontitis. Nowadays, many natural resources can be used as a basic ingredient of drugs. One of the resources used as an antibacterial material is cacao bean. It contains of polyphenol flavonoids, such as catechin, epicatechin, anthocyanin, and proanthocyanidin. Chemical compounds contained in ethanol extract and water extract are different in quantity from those in cocoa beans. Purpose: This research aimed to find out difference in antibacterial activity between the 70% ethanol and water extract of cacao beans (Theobroma cacao L.) on A. actinomycetemcomitans. Method: This research was an in vitro laboratory experiment. The serial dilutions was performed on the 70% ethanol and water extract of cacao beans a concentration of 100% to 3.125%. At each concentration, the 70% ethanol and water extract of cacao beans were added with grown bacterial suspension of A. actinomycetemcomitans. After they were incubated for 24 hours, the bacteria grown on Luria Berthani media were observed. Bacteria colonies then were measured in CFU/ml. Result: There were significant differences in bacterial colonies grown at the concentrations of 6.25% and 3.125% between the 70% ethanol extract of cacao beans and the water extract of cacao beans as p-value = 0.000 (p<0.05). Conclusion: 70% Ethanol beans and water extract of cacao beans have antibacterial activity against A. Actinomycetemcomitans. The concentrations of MIC and MBC extracts were 6.25% and 12.5% respectively. Keywords: Aggregatibacter actinomycetemcomitans; Cacao beans; antibacterial effect Correspondence: Hendrik Setia Budi, Department of Oral Biology, Faculty of Dental Medicine, Universitas Airlangga. Jln. Mayjend. Prof. Dr. Moestopo no. 47 Surabaya 60132, Indonesia. E-mail: hendriksetiabudi@gmail.com and Gram-negative bacteria. One of the Gram-negative anaerobic bacteria that plays a role in the formation of subgingival plaque causing periodontitis is Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans).1,4 Antibacterial effect of 70% ethanol and water extract of cacao beans (Theobroma cacao L.) on Aggregatibacter actinomycetemcomitans Ayu Rafania Atikah, Hendrik Setia Budi, and Tuti Kusumaningsih Departement of Oral Biology Faculty of Dental Medicine, Universitas Airlangga Surabaya - Indonesia Dental Journal (Majalah Kedokteran Gigi) p-ISSN: 1978-3728; e-ISSN: 2442-9740. Accredited No. 56/DIKTI/Kep./2012. Open access under CC-BY-SA license. Available at http://e-journal.unair.ac.id/index.php/MKG DOI: 10.20473/j.djmkg.v49.i2.p104-109 materials and method This study was an in vitro laboratory experimental research using randomized post test only control group design. This study was conducted at the Laboratory of Microbiology, Faculty of Dental Medicine, Universitas Airlangga from June to October 2015. Samples were A. actinomycetemcomitans bacteria taken from the microbiology laboratory, Faculty of Dental Medicine, Universitas Airlangga, and then cultured in Brain Heart Infusion Broth media (BHIB). Minimal sample size eligible to be analyzed is determined by Lemeshow formula, as many as five. Cocoa beans contain polyphenolic flavonoid of flavonol class, namely monomeric catechin and epicatechin flavonoids as well as proanthocyanidin polymerized flavonoids.10 Flavonols are classified into a flavonoid group composed of several molecules of phenols (polyphenols).11 Flavonols contained in cocoa stimulate peripheral blood mononuclear cells to secrete interleukin-5 (IL-5 ) and stimulate immunoglobulin A (IgA) production that protects the oral cavity from Streptococcus mutans.8 y Manufacture of 70% ethanol extract of cocoa beans and water extract of cocoa beans was conducted. The 70% ethanol extract of cocoa beans and the water extract of cocoa beans were obtained from Integrated Service Unit (UPT) Materia Medika Batu, East Java. Cocoa beans used were non-fermented cocoa beans and macerated using 70% ethanol and water solvent. Having obtained the 70% ethanol extract of cocoa beans and the water extract of cocoa beans, the preparation of A. actinomycetemcomitans bacteria then was performed by suspending the BHIB media until turbidity standard was equivalent to 0.5 Mc Farland (1.5 × 108 CFU/ ml). Manufacture of the 70% ethanol extract of cocoa beans and the water extract of cocoa beans at various concentrations of 100%, 50%, 25%, 12.5%, 6.25%, and 3.125% was performed using serial dilution method. Cocoa beans can also inhibit the growth of Streptococcus mutans at an effective concentration of 12.5%.12 The extract water of cocoa beans and the 70% ethanol extract of cocoa beans even have antibacterial activity against Escherichia coli and Bacillus subtilis. A research on cocoa beans extracted with the ethanol 70% shows a higher antimicrobial activity against Streptococcus mutans and C. albicans bacteria than with water.13 Cocoa, furthermore, will generate a different antibacterial activity when extracted with different polarities of its solvents.14 Extraction method varies depending on the samples to be tested. Several different solvents used include water, 70% ethanol, 70% methanol, acetonitrile, diethyl ether, and acetone. introduction Periodontal disease is an infectious disease of the oral cavity that can be suffered by teens and adults. Based on Health Research (RISKESDAS) in 2013, the prevalence of oral and dental problems, including periodontal disease reached 25.9%. The most common periodontal tissue diseases are gingivitis and periodontitis.1-3 A. actinomycetemcomitans are Gram-negative bacteria that have a small, non-motile, capnophilic, fermentative coccobacillus form. A. actinomycetemcomitans can be found in dental plaque, periodontal pockets, and buccal mucosa in 36% of patients in the normal population.1 A.actinomycetemcomitans release virulence factors, deliver adhesion on oral surfaces, inactivate the host immune response, as well as induce inflammation and tissue Periodontitis is an infectious disease resulting in inflammation of the tissues supporting teeth, gingival attachment loss progressively, and bone loss. The disease is caused by the induction of facultative anaerobic bacteria 105 105 Atikah, et al./Dent. J. (Majalah Kedokteran Gigi) 2016 June; 49(2): 104–109 damage. A. actinomycetemcomitans are also frequently detected in periodontal tissues of healthy people and then can be grouped into a part of the normal flora in oral cavity.5 Polyphenol level, furthermore, will be higher when extracted with a lower solvent polarity. Solvent water will tend to dissolve inorganic compounds and salts of acids or bases.14 The use of water compared to the 70% ethanol as cosolvent can generate higher residual polyphenols, so the solvent water is considered to be better because it can maintain a high quantity of beneficial compounds.17 Currently, some herbal ingredients have been developed as an alternative to medicine ingredients, one of which is cocoa beans. Indonesia ranks on the third position in the world cocoa producers. Cocoa production in Indonesia has the potential to be improved, but it depends on political and local economic factors.6 The size of cocoa growing areas in Indonesia in 2002 has reached 776, 900 hectares scattered in all provinces, except in the capital city of Jakarta (DKI).7 This study aimed to determine whether there were differences in antibacterial activity between the 70% ethanol extract of cocoa beans and the water extract of cocoa beans against A. actinomycetemcomitan bacteria. Cocoa is composed of stems, fruits and seeds, flowers, roots, bark seeds, and leaves. introduction Cocoa (Theobroma cacao L.) has polyphenolic compounds, such as catechin, epicatechin, anthocyanin, proanthocyanidin, phenolic acid, condensed tannins, flavonoids, and other small compounds.8 Cocoa polyphenols are useful as an antioxidant, anticarcinogenic, anti-inflammatory, and antimicrobial.9 Generally, the majority of people consume cocoa only on the seeds alone. Several researches also have shown that cocoa bean skin and cocoa beans can be used as antibacterial ingredients.7 Dental Journal (Majalah Kedokteran Gigi) p-ISSN: 1978-3728; e-ISSN: 2442-9740. Accredited No. 56/DIKTI/Kep./2012. Open access under CC-BY-SA license. Available at http://e-journal.unair.ac.id/index.php/MKG DOI: 10.20473/j.djmkg.v49.i2.p104-109 results 12,5% 12,5% dilution were planted in Luria Berthani agar media using streak technique as a cross-check to see the growth of bacterial colonies. It was necessary to do because of dark-colored extract material and turbidity occured at any concentration. This research used A. actinomycetemcomitan bacteria incubated for 1 x 24 hours at 37° C in the BHIB media, synchronized with the 0.5 McFarland standard. Materials used were non-fermented cocoa beans from Kota Batu, Malang. Next, cocoa extracts were made in UPT Materia Medika Batu in East Java by maceration method using 70% ethanol and water. Furthermore, based on the observation results on the number of bacterial colonies grown in the Luria Berthani media expressed in colony forming units (CFU), 70% ethanol extract of cocoa beans and water extract of cocoa beans could be able to inhibit and kill A. actinomycetemcomitan bacteria (Table 2). Based on the analysis results of chemical compounds contained in the 70% ethanol extract of cocoa beans as well as in the water extract of cocoa beans, there were different dosages of flavonoids, catechins, epicatechin, anthocyanin, and proanthocyanidin contained (Table 1). Catechin, epicatechin, and anthocyanin contained in the 70% ethanol extract of cocoa beans were higher than in the water extract of cocoa beans. Meanwhile, flavonoids and proanthocyanidin contained in the water extract of cocoa beans were higher than in the 70% ethanol extract of cocoa beans. Figure 2. a) Streak results of the 70% ethanol extract of cocoa b bacteria at any concentration as well as the controls grow results of the water extract of cocoa beans against A concentration as well as the controls grown in the Luria the 70% ethanol extract of cocoa beans against A. concentration as well as the controls grown in the Luria the water extract of cocoa beans against A. actinomycetem well as the controls grown in the Luria Berthani media. ure 2. a) Streak results of the 70% ethanol extract of cocoa bean bacteria at any concentration as well as the controls grown in results of the water extract of cocoa beans against A.act concentration as well as the controls grown in the Luria Ber the 70% ethanol extract of cocoa beans against A. acti concentration as well as the controls grown in the Luria Ber the water extract of cocoa beans against A. actinomycetemcom well as the controls grown in the Luria Berthani media. materials and method actinomycetemcomitan bacteria at a concentration o 50% in tube 2; a concentration of 25% in tube 3; and a c concentration of 6.25% in tube 5; and a concentration of 3.125 o. Chemical compounds 70% ethanol extract of cocoa beans Water ex b Flavonoids 1.32% 2 Catechins 2.01% 1 Epicatechin 1.36% 1 Anthocyanins 1.74% 1 Proanthocyanidin 1.93% 2 3,125% 12,5% 25% 50% 3,125% 6,25% 25% 50% 100% 100% 6,25% A B Figure 1. Serial dilution (A) the 70% ethanol extract of cocoa beans and (B) the water extract of cocoa beans against A. actinomycetemcomitan bacteria at a concentration of 100% in tube 1; a concentration of 50% in tube 2; a concentration of 25% in tube 3; and a concentration of 12.5% in tube 4; a concentration of 6.25% in tube 5; and a concentration of 3.125% in tube 6. Table 1. The analysis results of the chemical compounds contained in 70% ethanol extract of cocoa beans and the water extract of of cocoa beans Table 1 The analysis results of the chemical compounds contained ble 1 The analysis results of the chemical compounds contained in The reading of the results of antibacterial activity in the 70% ethanol extract of cocoa beans and the water extract of cocoa beans against the growth of A. actinomycetemcomitans bacteria was conducted by measuring the number of colonies grown on bacteria subculture as much as 0.1 ml of each tube as well as a positive control and a negative control on the Luria Berthani media. Planting on the Luria Berthani media was conducted using spreader, and incubated at 37 ° C for 24 hours. No. Chemical compounds 70% ethanol extract of cocoa beans Water extract of cocoa beans 1. Flavonoids 1.32% 2.18% 2. Catechins 2.01% 1.22% 3. Epicatechin 1.36% 1.08% 4. Anthocyanins 1.74% 1.67% 5. Proanthocyanidin 1.93% 2.51% and the water extract of of cocoa beans No. Chemical compounds 70% ethanol extract of cocoa beans Wate 1. Flavonoids 1.32% 2. Catechins 2.01% 3. Epicatechin 1.36% 4. Anthocyanins 1.74% 5. Proanthocyanidin 1.93% and the water extract of of cocoa beans o. Chemical compounds 70% ethanol extract of cocoa beans Water ex Flavonoids 1.32% 2 Catechins 2.01% 1 Epicatechin 1.36% 1 Anthocyanins 1.74% 1 Proanthocyanidin 1.93% 2 The results showed 90% of A. actinomycetemcomitans bacterial growth was inhibited compared with the positive control used as MIC. The results also indicated 99.9% of A. materials and method 70% ethanol (ethyl alcohol) is a solvent which has a low boiling point and is widely used by industries. 70% ethanol has a boiling point of 70° C so that extraction temperature is used to attract all the components in the basic materials.15 70% ethanol is widely used as a solvent of various chemical or natural materials devoted to drugs. In in vitro research, 70% ethanol can disrupt transport ions, namely Na +, K +, and ATP. The use of 70% ethanol in antibacterial material has a risk to human health, especially in liver because of the residual concentration and ethanol exposure.16 The determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) in the 70% ethanol extract of cocoa beans and the water extract of cocoa beans against A. actinomycetemcomitan bacteria was started from the preparation of the test tube as much as 9 tube. Six test tubes were filled with bacterial suspensions of A. actinomycetemcomitans as much as 0.05 ml that had been standardized to 0.5 Mc Farland and mixed with BHIB media as well as the extracts at the various concentrations (100%, 50%, 25%, 12.5%, 6.25 %, and 3.125). The test tube K + (as positive control) containing 0.05 ml bacterial suspension of A. actinomycetemcomitans were planted in the BHIB media, while the test tube K- (as negative control) contained only media BHIB and control extracts. Control is useful to make sure that no bacterial contamination in the media. 106 Atikah, et al./Dent. J. (Majalah Kedokteran Gigi) 2016 June; 49(2): 104–109 Table 1. The analysis results of the chemical compounds contained in 70% ethanol extract of cocoa beans and the water extract of of cocoa beans No. Chemical compounds 70% ethanol extract of cocoa beans Water extract of cocoa beans 1. Flavonoids 1.32% 2.18% 2. Catechins 2.01% 1.22% 3. Epicatechin 1.36% 1.08% 4. Anthocyanins 1.74% 1.67% 5. Proanthocyanidin 1.93% 2.51% Table 1. The analysis results of the chemical compounds contained and the water extract of of cocoa beans Figure 1. Serial dilution (a) the 70% ethanol extract of cocoa beans against A. actinomycetemcomitan bacteria at a concentrati 50% in tube 2; a concentration of 25% in tube 3; and concentration of 6.25% in tube 5; and a concentration of 3 No. Chemical compounds 70% ethanol extract of cocoa beans Wate 1. Flavonoids 1.32% 2. Catechins 2.01% 3. Epicatechin 1.36% 4. Anthocyanins 1.74% 5. Dental Journal (Majalah Kedokteran Gigi) p-ISSN: 1978-3728; e-ISSN: 2442-9740. Accredited No. 56/DIKTI/Kep./2012. Open access under CC-BY-SA license. Available at http://e-journal.unair.ac.id/index.php/MKG DOI: 10.20473/j.djmkg.v49.i2.p104-109 materials and method actinomycetemcomitans bacterial death compared with the positive control was used as MBC. MIC and MBC were determined by measuring the number of colonies grown on the Luria Berthani media manually and expressed as CFU/ ml. The calculation was repeated five times by three different observers, and then the mean values were measured. B A A Processing data was conducted using a statistical analysis test for normality with Kolmogorov-Smirnov test to see whether the data obtained were normally distributed or not. Next, homogeneity test using Levene test was performed. A statistical test for antibacterial power difference in the 70% ethanol extract of cocoa beans and the water extract of cocoa beans at every concentration then was carried out using Independent t-test. Figure 1. Serial dilution (a) the 70% ethanol extract of cocoa beans against A. actinomycetemcomitan bacteria at a concentrati 50% in tube 2; a concentration of 25% in tube 3; and concentration of 6.25% in tube 5; and a concentration of 3 3,125% 12,5% 25% 50% 3,125% 6,25% 25% 50% 100% 100% 6,25% ure 1. Serial dilution (a) the 70% ethanol extract of cocoa beans and against A. actinomycetemcomitan bacteria at a concentration 50% in tube 2; a concentration of 25% in tube 3; and a c concentration of 6.25% in tube 5; and a concentration of 3.12 3,125% 12,5% 25% 50% 3,125% 6,25% 25% 50% 100% 100% 6,25% Figure 1. Serial dilution (A) the 70% ethanol extract of cocoa beans and (B) the water extract of cocoa beans against A. actinomycetemcomitan bacteria at a concentration of 100% in tube 1; a concentration of 50% in tube 2; a concentration of 25% in tube 3; and a concentration of 12.5% in tube 4; a concentration of 6.25% in tube 5; and a concentration of 3.125% in tube 6. materials and method Proanthocyanidin 1.93% 3,125% 12,5% 25% 50% 3,125% 6,25% 25% 50% 100% 100% 6,25% able 1. The analysis results of the chemical compounds contained in and the water extract of of cocoa beans gure 1. Serial dilution (a) the 70% ethanol extract of cocoa beans and against A. actinomycetemcomitan bacteria at a concentration o 50% in tube 2; a concentration of 25% in tube 3; and a c concentration of 6.25% in tube 5; and a concentration of 3.125 o. Chemical compounds 70% ethanol extract of cocoa beans Water ex Flavonoids 1.32% 2 Catechins 2.01% 1 Epicatechin 1.36% 1 Anthocyanins 1.74% 1 Proanthocyanidin 1.93% 2 3,125% 12,5% 25% 50% 3,125% 6,25% 25% 50% 100% 100% 6,25% A B Figure 1. Serial dilution (A) the 70% ethanol extract of cocoa beans and (B) the water extract of cocoa beans against A. actinomycetemcomitan bacteria at a concentration of 100% in tube 1; a concentration of 50% in tube 2; a concentration of 25% in tube 3; and a concentration of 12.5% in tube 4; a concentration of 6.25% in tube 5; and a concentration of 3.125% in tube 6. Table 1. The analysis results of the chemical compounds contained in 70% ethanol extract of cocoa beans and the water extract of of cocoa beans No. Chemical compounds 70% ethanol extract of cocoa beans Water extract of cocoa beans 1. Flavonoids 1.32% 2.18% 2. Catechins 2.01% 1.22% 3. Epicatechin 1.36% 1.08% 4. Anthocyanins 1.74% 1.67% 5. Proanthocyanidin 1.93% 2.51% Table 1. The analysis results of the chemical compounds contained and the water extract of of cocoa beans Figure 1. Serial dilution (a) the 70% ethanol extract of cocoa beans against A. actinomycetemcomitan bacteria at a concentratio 50% in tube 2; a concentration of 25% in tube 3; and concentration of 6.25% in tube 5; and a concentration of 3 No. Chemical compounds 70% ethanol extract of cocoa beans Wate 1. Flavonoids 1.32% 2. Catechins 2.01% 3. Epicatechin 1.36% 4. Anthocyanins 1.74% 5. Proanthocyanidin 1.93% 3,125% 12,5% 25% 50% 3,125% 6,25% 25% 50% 100% 100% 6,25% able 1. The analysis results of the chemical compounds contained in and the water extract of of cocoa beans gure 1. Serial dilution (a) the 70% ethanol extract of cocoa beans and against A. results The comparative test on the MIC and MBC in the 70% ethanol extract cocoa beans and the water extract of cocoa beans was conducted using serial dilution method. The results showed that the extracts at the concentrations of 100%, 50%, 25%, and 12.5% contained no bacterial growth. Meanwhile, the mean bacterial growth in the 70% ethanol extract cocoa beans at the concentration of 6.25% was 11.4. On the other hand, the mean bacterial growth in the water extract of cocoa beans at the same concentration was 7.8 (Table 2). Serial dilution method is a method usually used in a comparative research of MIC and MBC in both the 70% ethanol extract of cocoa beans and the water extract of cocoa beans against A.actinomycetemcomitan bacteria (Figure 1). Before the antibacterial power difference analysis test on the 70% ethanol extract of cocoa beans and the water extract of cocoa beans against A. actinomycetemcomitan bacteria was performed, the normality test using Kolmogorov- Smirnov test as well as the homogeneity test using Levene test were conducted in each group. The results of the During the observation of MIC and MBC in the 70% ethanol extract of cocoa beans and the water extract of cocoa beans at every concentration, the results of serial 107 107 ration of ube 4; a ration of ube 4; a Atikah, et al./Dent. J. (Majalah Kedokteran Gigi) 2016 June; 49(2): 104–109 g y 50% in tube 2; a concentration of 25% in t concentration of 6 25% in tube 5; and a concen against A. actinomycetemcomitan bacteria at a 50% in tube 2; a concentration of 25% in t concentration of 6 25% in tube 5; and a concen Figure 2. a) Streak results of the 70% ethanol extract of cocoa beans against A. actinomycetemcomitan bacteria at any concentration as well as the controls grown in the Luria Berthani media; b) streak results of the water extract of cocoa beans against A.actinomycetemcomitan bacteria at any concentration as well as the controls grown in the Luria Berthani media; c) replanting results o the 70% ethanol extract of cocoa beans against A. actinomycetemcomitan bacteria at any concentration as well as the controls grown in the Luria Berthani media; d) replanting results o the water extract of cocoa beans against A. actinomycetemcomitan bacteria at any concentration a 3,125% 12,5% 25% 50% 3,125% 6,25% 12,5% 25% 50% 100% 100% 6,25% A Figure 2. results a) Streak results of the 70% ethanol extract of cocoa beans against A. actinomycetemcomitan bacteria at any concentration as well as the controls grown in the Luria Berthani media; b) streak results of the water extract of cocoa beans against A.actinomycetemcomitan bacteria at any concentration as well as the controls grown in the Luria Berthani media; c) replanting results o the 70% ethanol extract of cocoa beans against A. actinomycetemcomitan bacteria at any concentration as well as the controls grown in the Luria Berthani media; d) replanting results o the water extract of cocoa beans against A actinomycetemcomitan bacteria at any concentration a 3,125% 12,5% 25% 50% 3,125% 6,25% 12,5% 25% 50% 100% 100% 6,25% B C D Figure 2. a) Streak results of the 70% ethanol extract of cocoa beans against A. actinomycetemcomitan bacteria at any concentration as well as the controls grown in the Luria Berthani media; b) streak results of the water extract of cocoa beans against A.actinomycetemcomitan bacteria at any concentration as well as the controls grown in the Luria Berthani media; c) replanting results of the 70% ethanol extract of cocoa beans against A. actinomycetemcomitan bacteria at any concentration as well as the controls grown in the Luria Berthani media; d) replanting results of the water extract of cocoa beans against A. actinomycetemcomitan bacteria at any concentration as well as the controls grown in the Luria Berthani media. 1 1 B C D D Figure 2. a) Streak results of the 70% ethanol extract of cocoa beans against A. actinomycetemcomita bacteria at any concentration as well as the controls grown in the Luria Berthani media; b) strea results of the water extract of cocoa beans against A.actinomycetemcomitan bacteria at an concentration as well as the controls grown in the Luria Berthani media; c) replanting results o the 70% ethanol extract of cocoa beans against A. actinomycetemcomitan bacteria at an concentration as well as the controls grown in the Luria Berthani media; d) replanting results o th t t t f b i t A ti t it b t i t t ti Figure 2. a) Streak results of the 70% ethanol extract of cocoa beans against A. results actinomycetemcomita bacteria at any concentration as well as the controls grown in the Luria Berthani media; b) strea results of the water extract of cocoa beans against A.actinomycetemcomitan bacteria at an concentration as well as the controls grown in the Luria Berthani media; c) replanting results o the 70% ethanol extract of cocoa beans against A. actinomycetemcomitan bacteria at an concentration as well as the controls grown in the Luria Berthani media; d) replanting results o Figure 2. a) Streak results of the 70% ethanol extract of cocoa beans against A. actinomycetemcomitan bacteria at any concentration as well as the controls grown in the Luria Berthani media; b) streak results of the water extract of cocoa beans against A.actinomycetemcomitan bacteria at any concentration as well as the controls grown in the Luria Berthani media; c) replanting results of the 70% ethanol extract of cocoa beans against A. actinomycetemcomitan bacteria at any concentration as well as the controls grown in the Luria Berthani media; d) replanting results of the water extract of cocoa beans against A. actinomycetemcomitan bacteria at any concentration as well as the controls grown in the Luria Berthani media. 11 well as the controls grown in the Luria Berthani media. 11 well as the controls grown in the Luria Berthani media. Table 2. The number of A. actinomycetemcomitan bacterial colonies grown in the Luria Berthani media at various concentrations of 70% ethanol extract of cocoa beans and water extract of cocoa beans Treatment Group N The means number of the bacterial colonies (x108 CFU/ml) 70% Ethanol extract Water extract Positive control Negative control At the concentration of 100% 5 0 0 109 0 At the concentration of 50% 5 0 0 At the concentration of 25% 5 0 0 At the concentration of 12.5% 5 0 0 At the concentration of 6.25% 5 11.4 7.8 At the concentration of 3.125% 5 27.2 18 Note: n: number of replication; 0: no growth on the negative control; 109: the number of bacterial colonies grown as the positive control 1 g 1 well as the controls grown in the Luria Berthani media. Table 2. The number of A. actinomycetemcomitan bacterial colonies grown in the Luria Berthani media at various concentrations of 70% ethanol extract of cocoa beans and water extract of cocoa beans g well as the controls grown in the Luria Berthani media. The number of A. Dental Journal (Majalah Kedokteran Gigi) p-ISSN: 1978-3728; e-ISSN: 2442-9740. Accredited No. 56/DIKTI/Kep./2012. Open access under CC-BY-SA license. Available at http://e-journal.unair.ac.id/index.php/MKG DOI: 10.20473/j.djmkg.v49.i2.p104-109 discussion How to cultivate antibacterial materials, thus, is a thing that needs attention. Antibacterial material can be extracted by using various methods, one of which is by solvent extraction (maceration). Solvents widely used for polyphenol extraction method are water and 70% ethanol. Cocoa, on the other hand, will yield a different antibacterial activity when extracted with a different solvent polarity.14 This research was an in vitro research aimed to see the difference in antibacterial power beween the 70% ethanol extract of cocoa beans and the water extract of cocoa beans against A. actinomycetemcomitan bacteria. Antibacterial agents used in this research were non-fermented cocoa beans since non-fermented cocoa beans actually have higher antibacterial effects than fermented ones. Fermentation and drying processes, however, can reduce the active components of natural materials, such as polyphenol.18 This research also used a serial dilution method with the concentrations of 100%, 50%, 25%, 12.5%, 6.25%, and 3.125%. A. actinomycetemcomitan bacteria grown on the BHIB media were considered as the positive control (+), whlie the bacteria grown on the BHIB media and the control extracts were considered as as the negative control (-). Serial dilution method was used because it can give the significant calculation results of microorganism colonies at small concentrations. Thus, if the serial dilution method is not conducted, there will be a number of colonies stacked so that the calculation of the colonies will be not accurate.19 Serial dilution method has widely been used for many years.20 Ethanol is a polar solvent that dissolves polar compounds, while water is an inorganic solvent that dissolves inorganic materials. In addition, cacao bean is an inorganic material because it is derived from plants. To dissolve the cocoa beans using ethanol, therefore, will take a long process because of evaporation process. The process of evaporation can affect the polyphenol content in the extract. Water, on the other hand, is a solvent that can maintain a high quantity of essential compounds contained in natural materials.17 Water solvent will dissolve inorganic compounds and salts of acids and bases.14 Cocoa beans diluted with water is more soluble and requires a shorter time than cocoa diluted with ethanol. The analysis results of chemical compounds in the 70% ethanol extract of cocoa beans contain flavonoids (1.32%), catechin (2.01%), epicatechin (1.36%), anthocyanin (1.74%), and proanthocyanidin (1.93%). The water extract of cocoa beans contain flavonoids (2.18%), catechin (1.22%), epicatechin (1.08%), anthocyanin (1.67%), and proanthocyanidin (2.51%). Dental Journal (Majalah Kedokteran Gigi) p-ISSN: 1978-3728; e-ISSN: 2442-9740. Accredited No. 56/DIKTI/Kep./2012. Open access under CC-BY-SA license. Available at http://e-journal.unair.ac.id/index.php/MKG DOI: 10.20473/j.djmkg.v49.i2.p104-109 results actinomycetemcomitan bacterial colonies grown in the Luria Berthani media at various concentration of 70% ethanol extract of cocoa beans and water extract of cocoa beans Table 3. The mean and standard deviation of the number of bacterial colonies of A. actinomycetemcomitans Treatment control Mean ± SD Significance 70% Ethanol extract Water extract At the concentration of 6.25% 11.4 ± 0.54772 a 7.8 ± 0.83666 b P = 0.000 At the concentration of 3.125% 27.2 ± 1.64317 a 18.0 ± 1.22474 b P = 0.000 Note: a b: significant difference Table 3. The mean and standard deviation of the number of bacterial colonies of A. actinomycetemcomitans normality and homogeneity tests showed that data obtained in the water extracts of cocoa beans at the concentrations of 6.25% and 3.125% were normally distributed and homogeneous (p>0.05). Whereas, data obtained in the 70% ethanol extract of cocoa beans at the concentration of 6.25% were not normally distributed (p <0.05), and at the concentration of 3.125% was not homogeneous (p<0.05). Next, Kruskal-Wallis test was carried out to test significant differences in antibacterial activities between the 70% ethanol extract of cocoa beans and the water extract of cocoa beans since there were groups which were not normally distributed and not homogeneous. The results showed value of p was less than 0.05 (p = 0.000), indicating that there was a difference in antibacterial activity between the Atikah, et al./Dent. J. (Majalah Kedokteran Gigi) 2016 June; 49(2): 104–109 108 70% ethanol extract of cocoa beans and the water extract of cocoa beans at the concentrations of 6.25% and 3.125% (Table 3). bacterial enzyme system. Proanthocyanidin, furthermore, is condensed tannins that can inhibit the formation of cell walls and lead to termination of the crosslinking peptide that combines the glycan chains of peptidoglycan in another chain, causing cell membrane damage.28 references 16. Darmono. Farmasi forensik dan toksikologi. Jakarta: UI Press; 2009. p. 23. 1. Carranza P, Klokkevold F, Newman M, Takei H. Carranza’s clinical periodontology. St. Louis, MO: Saunders Elsevier; 2009. p. 187- 192 17. Boakye S. Levels of selected pesticide residues in cocoa beans From Ashanti and Brong Ahafo Regions of Ghana. Dissertation. Ghana: Faculty of Physicial Science College of Science; 2012. 2. Riset Kesehatan Dasar (Riskesdas) Nasional. Jakarta: Badan Penelitan dan Pengembangan Kesehatan, Departemen Kesehatan Republik Indonesia; 2013. p. 113. 18. Smullen J, Koutsou GA, Foster HA, Zumbe A,Storey DM. The antibacterial activity of plant extract containing polyphenols against Streptococcus mutans. Caries Res 2007; 41: 342-9. 3. Chauhan VS, Chauhan RS, Devkar N, Vibhute A, More S. Gingival and periodontitis diseases in children and adolescents. J Dent & Allie Sci 2012 ; 1(1): 26. 19. Mejos Jay Al, CaraignJ.W, De Pano J, Labador AV, Macapagal EM. Bacterial colony isolation using serial dilution techniques. Quezon City: Institute of Biology, College of Science, University of the Phillipines; 2010. p. 6. 4. Newman M, Takei H, Klokkevold P, Carranza F. Carranza’s clinical periodontology. St. Louis, MO: Saunders Elsevier; 2012. p. 185- 93. 20. Seeley HW, VanDemark PJ. Selected exercises from microbes in action a laboratory manual of microbiology. 3 rd ed. Sanfrancisco: Freeman and Company; 1981. p. 37-41. 5. Umeda EJ, Priscila LL, Maria RL, Marcia PAM. Differential transcription of virulence genes in Aggregatibacter actinomycetemcomitans serotypes. Brazil: Departement of microbiology, Institute of Biomedical Sciences, University of Sao Paulo; 2013. p. 4-6. 21. V.S.T. Saito, T.F. dos Santos, C.G. Vinderola, C. Romano, J.R. Nicoli, L.S. Araújo, M.M. Costa, J.L. Andrioli, A.P.T. Uetanabaro. Viability and Resistance of Lactobacilli Isolated from Cocoa Fermentation to Simulated Gastrointestinal Digestive Steps in Soy Yogurt. Journal of food and science 2014; 79(2): doi. 10.1111/1750-3841.12326. 6. Gu F, Tan L , Wu H, Fang Y , Xu F , Chu Z, Wang Q. Comparison of cocoa beans from China, Indonesia and Papua New Guinea. Foods 2013; 2(2): 183-97. of food and science 2014; 79(2): doi. 10.1111/1750-3841.12326. 22. Kenneth T. Structure and function of bacterial cells. Online Textbook of Bacteriology2009; 5. 7. Misnawi. Pemanfaatan Biji Kakao sebagai Sumber Antioksidan Alami. Jember: Pusat Penelitian Kopi dan Kakao Indonesia; 2005. p. 6. 23. Zainal H, Made A, Kasno, Anggraini AD. Uji aktivitas antibakteri propolis lebah madu Trigona spp. Prosiding Seminar Nasional Indonesia, 2006; p. 204-14. 8. references Arlorio M, Coisson JD, Travaglia F, Varsaldi F, Miglio G, Lombardi G, Martelli A. Antioxidant and biological activity of phenolic pigments from Theobroma cacao hulls extracted with supercritical CO2. Food Research International 2005; 38(8-9): 1009-14. 24. Ariza BTS, Mufida D, Fatima N. In vitro antibacterial activity of cocoa ethanolic extract against Escherichia coli. International Food Research Journal 2014; 21(3): 935-40. CO2. Food Research International 2005; 38(8-9): 1009-14. 9. Hii CL, Law CL, Suzannah S, Misnawi, Cloke M. Polyphenols in cocoa (Theobroma cacao L.). As J Food Ag-Ind 2009; 2(04): 702- 22.f 25. Misnawi, Wulandari P, Suswati E, Rianul A. Antibacterial effect of ethanol extract cocoa beans (Theobroma cacao) on growth in vitro By Shigella dysentriae. Jurnal Medika Planta 2012; 1(5): 73. 10. Sulistyowati, Misnawi. Effects of alkali concentration and conching temperature on antioxidant activity and physical properties of chocolate. International Food Research Journal 2008; 15(3): 297- 304. 26. Sabir A. Aktivitas antibakteri flavonoid propolis Trigona sp terhadap bakteri Streptococcus mutans (in vitro). Majalah Kedokteran Gigi (Dental Journal) 2005; 38(2): 135-41. 26. Sabir A. Aktivitas antibakteri flavonoid propolis Trigona sp te 27. Chinami Hirao, Eisaku Nishimura, Masanori Kamei, Tomoko Ohshima, Nobuko Maeda. Antibacterial effects of cocoa on periodontal pathogenic bacteria. Journal of Oral Biosciences 2010; 52(3): 283-291. 11. Lee SY, Yoo SS, Lee MJ, Kwon IB, dan Pyun YR. Optimization of Nib Roasting in Cocoa bean processing with lotte-better taste and color process. Food Sci Biotechnol 2001; 10: 286-93. 12. Purnamasari DA, Munadziroh E, Yogiartono RM. Konsentrasi ekstrak biji kakao sebagai material alam dalam menghambat pertumbuhan Streptococcus mutans. Jurnal PDGI 2010; 59(1): 14-8. 28. Duskova M, Karpiskova R. Antimicrobial resistance of Lactobacilli Isolated from food. Czech J Food Sci 2013; 31(1): 27-32. 29. Surbhi Leekha, Christine L. Terrell, and Randall S. Edson. General principles of antimicrobial therapy. Mayo Clin Proc 2011;86(2): 156–167. 13. Gianmaria F. Ferrazzano, Ivana Amato, Aniello Ingenito, Antonino De Natale, Antonino Pollio. Anti-cariogenic effects of polyphenols from plant stimulant beverages (cocoa, coffee, tea). Fitoterapia 2009; 80(5): 255–262 30. Akinyemi KO, Oluwa OK, Omomigbehin EO. Antimicrobial activity of crude extracts of three medicinal plants used in south-west Nigerian folk medicine on some food borne bacterial pathogens. African Journal of Traditional, Complementary and Alternative Medicines 2006; 3(4): 13-22. 14. John NA, Fang Z, Kebitsamang JM, Mohamed LB, Camel L. Quantification of Total Polyphenolic Content and Antimicrobial Activity of Cocoa (Theobroma cacao L.) Bean Shells. discussion (Majalah Kedokteran Gigi) 2016 June; 49(2): 104–109 109 109 15. Manuchair Ebadi. Pharmacodynamic basic of herbal medicine. 2nd ed. New york: Taylor & Francis; 2007.p. 647. discussion Proanthocyanidin contained in cocoa beans was 58%, catechin amounted to 37%, and anthocyanin amounted to 4%, thus indicating that the water extract of cocoa beans contain more useful chemical compounds than the ethanol extract of cocoa beans.10 In the antibacterial activity test, properties owned by the cell wall of bacteria may affect the ability of cocoa bean extracts to inhibit tested bacterial growth.21 A.actinomycetemcomitan bacteria are gram-negative bacteria that have a cell wall in the form of peptidoglycan surrounded by a membrane structure, called as outer membrane. Outer membrane of Gram-negative bacteria has lipopolysaccharide or endotoxin components that can prevent an antibacterial agent to penetrate into the cells.22,23 In the layers of lipopolysaccharide, Gram-negative bacteria have a system of selection against strange substances so that Gram negative bacteria are more resistant to strange substances than Gram positive bacteria.21 The presence of polyphenols, such as flavonoids in cocoa beans can penetrate the membrane of Gram-negative bacteria to mediate the cell response.24 In the 70% ethanol extract of cocoa beans and the water extract of cocoa beans at the concentration of 6.25%, moreover, there was still bacterial colony growth on the Luria Berthani media after incubated for 1x24 hours. It means that not all bacteria die, or there are barriers to the growth of colonies by 90%.28 Nevertheless, in the 70% ethanol extract of cocoa beans and the water extract of cocoa beans at the concentration of 12.5%, there was no colony growth. It indicates that the 70% ethanol extract of cocoa beans and the water extract of cocoa beans at this concentration could kill bacteria by 99.9% of the total average bacteria that managed to grow on the positive control and were considered as bactericide.29,30 Flavonoids have antibacterial activity through the barrier function of bacterial DNA gyrase so that replication capabilities and bacterial translation can be inhibited.25 Flavonoids may cause damage to the permeability of bacterial cell wall, microsomes, and lysosomes as a result of the interaction of flavonoids with bacterial DNA.26 Catechins, moreover, are natural polyphenolic compounds and secondary metabolites included in the constituent group of tannins.21 Catechins may damage cytoplasmic membrane causing the release of important metabolites that inactivate It can be concluded that water and 70% ethanol extract of cocoa beans have antibacterial activities against A. actinomycetemcomitans. Both the extracts also have the same MIC and MBC at concentrations of 6.25 and 12.5%. Atikah, et al./Dent. J. Dental Journal (Majalah Kedokteran Gigi) p-ISSN: 1978-3728; e-ISSN: 2442-9740. Accredited No. 56/DIKTI/Kep./2012. Open access under CC-BY-SA license. Available at http://e-journal.unair.ac.id/index.php/MKG DOI 10 20473/j dj k 49 i2 104 109 references Pakistan Journal of Nutrition 2012; 11(7): 574-579 Dental Journal (Majalah Kedokteran Gigi) p-ISSN: 1978-3728; e-ISSN: 2442-9740. Accredited No. 56/DIKTI/Kep./2012. Open access under CC-BY-SA license. Available at http://e-journal.unair.ac.id/index.php/MKG DOI: 10 20473/j djmkg v49 i2 p104 109
https://openalex.org/W2914297388	https://bmcsurg.biomedcentral.com/track/pdf/10.1186/s12893-019-0472-1	English	null	Technical considerations and outcomes for ileal ureter replacement: a retrospective study in China	BMC surgery	2,019	cc-by	4,608	Zhong et al. BMC Surgery (2019) 19:9 https://doi.org/10.1186/s12893-019-0472-1 Zhong et al. BMC Surgery (2019) 19:9 https://doi.org/10.1186/s12893-019-0472-1 Open Access © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background: Ileal ureter replacement is an alternative treatment for various length ureter defects. We present our experience and outcome of ileal ureter replacement in China. Methods: We retrospectively collected data of patients who underwent ileal ureter replacement between January 2010 and January 2015. We reviewed the medical history, indications for surgery, operative data, perioperative data, and outcomes. Besides, follow-up data included symptom, urine routine test, serum creatinine, serum electrolyte status, and radiographic test. Results: There were 23 patients who underwent ileal ureter replacement by the same surgeon. Twenty patients were performed unilateral ileal ureter replacement, two patients underwent a combination of ileal ureter replacement and Boari flap-psoas hitch, and one received bilateral ileal ureter replacement. Among these patients, the main cause leading to surgical treatment was iatrogenic injuries (n = 15), especially urinary surgery procedure (n = 11). The median follow-up time was 45 months. There were 6 early complications and 6 late complications after operation. Only one patient suffered from small bowel-related complication and was cured by conservative treatment. Only the patient who underwent bilateral ileal ureter replacement had metabolic acidosis. And 22 patients (95.7%) had a good renal function. Conclusions: Ileal ureter replacement is an efficacious and safe procedure for the therapy of long ureteral defects. With appropriate technical considerations, the complication rate may decrease. Keywords: Ileum, Ureter replacement, Reconstructive surgical procedures, Outcome bladder. Ileal ureter replacement is occasionally considered as the last resort for more extensive defects that are not amenable to reconstruction by other means. Technical considerations and outcomes for ileal ureter replacement: a retrospective study in China Wenlong Zhong1†, Peng Hong1†, Guangpu Ding1†, Kunlin Yang1, Xuesong Li1, Junsheng Bao2, Guochang Bao3, Liang Cui4, Changping Men5, Zhe Li6, Peng Zhang6, Ning Chu7 and Liqun Zhou1 Background Long segment ureteral defect, occasionally requiring a definitive reconstructive procedure, presents a complex challenge to urologists. Depending on the length and position of the ureteral injury, ureteroureterostomy, psoas hitch, Boari flap, and autotransplantation can be used as alternative techniques for treatment [1]. How- ever, all of these techniques have inherent limitations owing to the restricted availability of the ureter or Ileal ureter replacement was introduced by Shoemaker [2] in 1906 to treat long segment ureteral defect and was popularized by Goodwin et al. [3] in 1959. Studies were subsequently conducted to evaluate different alternative materials for the ureter; meanwhile, an increasing number of cases that underwent ileal ureteral substitution were reported in published studies [4–8]. Currently, the use of ileal segment for ureteral replacement has become a valu- able alternative in reconstructive urology. * Correspondence: pineneedle@sina.com; zhoulqmail@sina.com † †Wenlong Zhong and Peng Hong and Guangpu Ding contributed equally to this work. However, technical difficulties and surgical complications limit the extended use of the procedure. To our knowledge, published studies on the use of this procedure 1Department of Urology, Peking University First Hospital, Institute of Ur National Urological Cancer Center, Peking University, No.8 Xishiku St, Xi District, Beijing 100034, China Full list of author information is available at the end of the article 1Department of Urology, Peking University First Hospital, Institute of Urology, National Urological Cancer Center, Peking University, No.8 Xishiku St, Xicheng District, Beijing 100034, China 1Department of Urology, Peking University First Hospital, Institute of Uro National Urological Cancer Center, Peking University, No.8 Xishiku St, Xic District, Beijing 100034, China Full list of author information is available at the end of the article Methods A retrospective study of ileal ureter replacement in patients with long ureteral defects was conducted from January 2010 to January 2015. Preoperative radiographic examinations, such as antegrade and retrograde pyelog- raphy, nuclear renography, computed tomography urog- raphy (CTU), and magnetic resonance urography (MRU) were used to evaluate the defects. Data on patient characteristics, indications for surgery, intraoperative variables, surgical complications, and postoperative out- comes were collected. The present study was approved by Peking University First Hospital ethics committee. Written, informed consent was obtained from all indi- vidual participants in the study. Surgical technique The procedure for ileal ureter replacement was similar to that described in a previous study [9]; however, sev- eral minor surgical steps were used during our proced- ure. Ureteral exposure was achieved with mid-line incision. After the involved ureteral segment was dis- sected, the renal pelvis or ureter proximal to the injury site was widely spatulated for subsequent anastomosis. An appropriate ileal segment was divided 25–30 cm proximal from the ileocecal junction after measuring the length of the defect. The isolated ileal segment was used to bridge the defect in an isoperistaltic direction. Bowel continuity was restored with stapled side-to-side anasto- mosis using two linear staplers (Fig. 1, a and b) and Zhong et al. BMC Surgery (2019) 19:9 Zhong et al. BMC Surgery (2019) 19:9 Page 2 of 7 Zhong et al. BMC Surgery (2019) 19:9 Page 2 of 7 interrupted Lembert sutures were subsequently used to strengthen the anastomotic edge (Fig. 1, c and d). in the Asian population remain limited. In the present study, we describe our technical considerations in ileal ureter replacement and present a retrospective experience among patients with long ureteral injuries in China. g g g A 6–8 F ureter stent was inserted into the isolated ileal segment. The ureter stent was fixed to the proximal and distal ends of an ileal graft to prevent dislocation. Pyeloileal and ureteroileal anastomoses were performed in an end-to-end fashion. For the bilateral ureteral replacement, a reverse “7” shaped reconstruction was performed, with the 2 proximal anastomoses on the same ileal graft. In most cases, a distal anti-reflux nipple valve was created (Fig. 2). Ileocystostomy was performed in a two-layer fashion with a running mucosa-to-mucosa suture and interrupted seromuscular–detrusor muscle suture. For the patients with preoperative borderline renal function (defined as 1.5–2.0 mg/dl), a bladder flap measuring 4 cm wide at the apex and 6–8 cm wide at the base was created, and the vesico–psoas hitch technique was performed (Fig. 3). The distal ileum with an anti-reflux nipple was anastomosed to the bladder flap. Two suction drains were placed near the proximal and distal anastomoses, and a 20F indwelling Foley cath- eter was inserted into the bladder. Follow-up Patient follow-ups were conducted 1 and 6 months after surgery and at least once a year thereafter. The patients routinely received physical examination, blood test (including blood gas analysis, serum creatinine test, and electrolyte test), urine routine test, and radiographic examination (abdominal ultrasonog- raphy, computed tomography urography/magnetic resonance urography, and voiding cystogram) at each visit. Antegrade nephrostogram was performed post- operatively on the patient with a nephrostomy tube. The length of follow-up was defined as the interval from surgery to the last visit. Fig. 1 Our modified stapled side-to-side anastomosis. a Side-to-side anastomosis at the anti-mesenteric edge, completed using a linear stapler through the vertical incision, b. Open ends of the proximal and distal ilea, closed using another linear stapler load, c and d. The anastomotic edge was strengthened by interrupted Lembert sutures ed stapled side-to-side anastomosis. a Side-to-side anastomosis at the anti-mesenteric edge, completed using a linear staple cal incision, b. Open ends of the proximal and distal ilea, closed using another linear stapler load, c and d. The anastomotic hened by interrupted Lembert sutures Fig. 1 Our modified stapled side-to-side anastomosis. a Side-to-side anastomosis at the anti-mesenteric edge, completed using a linear stapler through the vertical incision, b. Open ends of the proximal and distal ilea, closed using another linear stapler load, c and d. The anastomotic edge was strengthened by interrupted Lembert sutures Zhong et al. BMC Surgery (2019) 19:9 Page 3 of 7 Zhong et al. BMC Surgery Fig. 2 The anti-reflux nipple valve for ileal ureter substitution Fig. 3 The combination of ileal ureter replacement and Boari flap-psoas hitch technique Results An anti-reflux nipple valve was created during ileocystostomy in 21 patients, and only 2 patients did not use the anti-reflux design because of the retained stone in the urinary tract. One patient required blood transfusion owing to severe adhesion around the lesion, which resulted in more blood loss (1000 mL). The median length of postopera- tive hospital stay was 15.1 d (range: 5–35). bilateral ileal ureter replacement. Each type of ureteral replacement is shown in Fig. 4. An anti-reflux nipple valve was created during ileocystostomy in 21 patients, and only 2 patients did not use the anti-reflux design because of the retained stone in the urinary tract. One patient required blood transfusion owing to severe adhesion around the lesion, which resulted in more blood loss (1000 mL). The median length of postopera- tive hospital stay was 15.1 d (range: 5–35). defects, including transureteroureterostomy, renal auto- transplantation, combined Boari flap and psoa hitch, and ileal ureteral replacement. However, ileal interposition is occasionally utilized as the last resort for long ureteral obstructed segment. Long segment ureteral defects can be caused by multiple insults, such as iatrogenic trauma, urinary tuberculosis, bilharziasis, recurrent renal calculi, retro- peritoneal fibrosis, and so on [11]. The etiology of ureteral defects in the present series, compared with the earlier series, has evolved considerably. Armatys et al. [11] reported on 91 patients who underwent ureteroileoplasty. The main indication for ureteral replacement was iatrogenic injury involving genitouri- nary surgery in 29 cases (31.9%), nonurological sur- gery in 16 cases (17.6%), and radiation induced stricture in 17 cases (18.7%). Romero et al. [12] demon- strated that the most common procedure associated with iatrogenic ureteral injury was ureteroscopic stone removal. Similarly, in our study, iatrogenic injuries caused by endourological procedures, such as ureteroscopic lithotripsy, have become the main cause of long segment ureteral defect. With regard to the postoperative complications (Table 2), 6 minor complications (grade 2) according to the Clavien–Dindo classification developed within 30 d after surgery [10]. Urinary infection was identified as the most common complication, with 3 patients (13.0%) suf- fering from urinary infection. A small bowel-related complication occurred in only 1 patient with incomplete ileus, which was resolved by conservative treatment. Two patients presented incisional hernia and underwent hernia repair surgery. Only 1 patient was found to have metabolic acidosis treated with a long-term application of oral sodium bicarbonate. Results During a mean follow-up period of 45 months, no postoperative reflux was reported. The average creatin- ine levels before surgery, 1 month after surgery, and at the last follow-up visit were 1.18 (0.79–1.92), 1.07 (0.57– 2.60), and 1.03 (0.76–1.69) mg/dL, respectively. Renal function in 22 patients increased or remained stable. Only 1 patient with a solitary kidney, who subsequently developed urinary infection, experienced renal deterior- ation after surgery. The infection was successfully man- aged with oral antibiotics, and the serum creatinine decreased to the normal level without major sequelae. Contemporary series have shown promising results after prudent selection of patients in ileal interposition. Renal insufficiency before ileal interpositon is generally understood to increase the risk of metabolic acidosis. Several previous studies have emphasized that preopera- tive renal function is a crucial prognostic factor for ileal interposition [5, 6, 13–15]. In 1979, Boxer reported on their single-institution experience with ileal interpositon, advocating for the first time that patients with serum creatinine concentration higher than 2 mg/dL should undergo ureteral replacement cautiously [5]. Chung et al. [13] indicated that 50% of patients with serum creatinine > 2.0 mg/dL developed worsening azotemia after ureteral replacement. Wolff et al. [15] further showed a success rate > 90% when focused on patients with preoperative serum creatinine < 1.7 mg/dL. Results A total of 23 patients, including 13 males and 10 females, underwent ileal ureter replacement by the same surgeon between January 2010 and January 2015. The patients’ characteristics are shown in Additional file 1: Table S1. Mean age was 37.1 y (range: 16–63). The main indication for ureteral replacement in the current series was the presence of iatrogenic injuries in 15 patients (65.3%). Of these 15 patients, 11 (47.8%) had undergone urological procedures, including endoscopic uretero- lithotomy in 9 patients (39.1%) and ureteral reimplanta- tion in 2 patients (8.7%). The remaining indications are listed in Table 1. Notably, 3 patients had a solitary kidney. Fig. 3 The combination of ileal ureter replacement and Boari flap-psoas hitch technique Table 1 Indication for ileal ureter replacement Etiology N (%) Iatrogenic injury 15 (65.3) Urologic surgery 11 (47.8) URSL 9 (39.1) Others 2 (8.7) Gynecologic surgery 2 (8.7) Orthopedic surgery 1 (4.3) General surgery 1 (4.3) Car crash 2 (8.7) Congenital obstruction 2 (8.7) Fibrosis after radiotherapy 2 (8.7) Tuberculosis 1 (4.3) Ureteral TCC 1(4.3) URSL ureteroscopic lithotripsy; TCC transitional cell carcinoma Table 1 Indication for ileal ureter replacement Table 1 Indication for ileal ureter replacement Etiology N (%) Table 1 Indication for ileal ureter replacement Etiology N (%) Iatrogenic injury 15 (65.3) Urologic surgery 11 (47.8) URSL 9 (39.1) Others 2 (8.7) Gynecologic surgery 2 (8.7) Orthopedic surgery 1 (4.3) General surgery 1 (4.3) Car crash 2 (8.7) Congenital obstruction 2 (8.7) Fibrosis after radiotherapy 2 (8.7) Tuberculosis 1 (4.3) Ureteral TCC 1(4.3) URSL ureteroscopic lithotripsy; TCC transitional cell carcinoma y Among the 23 patients, 8 developed ureteral injuries in the proximal mid-ureter, 2 in the mid-ureter, 8 in the distal or mid-distal ureter, and 5 in the full-length. The mean length of the injury segment was 18.6 cm (range: 5–30). All patients underwent nephrostomy prior to sur- gery, with a mean time of 5.5 months (range: 2–15). Among the 23 patients, 20 patients received a unilateral ileal ureter replacement, 2 received combined ileal ureter substitution and Boari flap–psoas hitch, and 1 received Zhong et al. BMC Surgery (2019) 19:9 Zhong et al. BMC Surgery (2019) 19:9 Page 4 of 7 Zhong et al. BMC Surgery (2019) 19:9 Page 4 of 7 bilateral ileal ureter replacement. Each type of ureteral replacement is shown in Fig. 4. Discussion The surgical principle in managing long ureteral injury is to construct a non-refluxing and non-obstructive urin- ary outflow as soon as possible, thereby restoring or sta- bilizing the renal function. As we know, several options have been used for the reconstruction of long ureteral Fig. 4 Different types of ureteral replacement. a unilateral ileal ureter replacement, b. bilateral ileal ureter replacement, c. combined ileal–ureter substitution and Boari flap–psoas hitch Fig. 4 Different types of ureteral replacement. a unilateral ileal ureter replacement, b. bilateral ileal ureter replacement, c. combined ileal–ureter substitution and Boari flap–psoas hitch Page 5 of 7 Zhong et al. BMC Surgery (2019) 19:9 Table 2 Postoperative complications Complication Patients (n) Therapy Early postoperative complications Urinary infection (Grade 2) 3 Antibiotics ureteroileal anastomosis leakage (Grade 2) 2 Open the percutaneous nephrostomy tube for draining Incomplete ileus (Grade 2) 1 Conservative therapy, including fast, decompression, anti-infection and balance of electrolytes. Late postoperative complications Recurrent urinary infection 2 Antibiotics and sodium bicarbonate Incisional hernia 2 Surgical repair Metabolic acidosis 1 Oral sodium bicarbonate replacement Stone formation 1 ESWL on ileal ureteral replacement side ESWL extracorporeal shock wave lithotripsy Table 2 Postoperative complications There have been concerns regarding postoperative reflux. Tanagho et al. [16] reported on 5 patients who experienced progressive renal deterioration after persist- ent reflux. The study suggested that refluxing uropathy resulted from excessively high ileal pressure, which could lead to metabolic derangements and renal impair- ment. Several studies have shown that anti-reflux surgery can effectively prevent progressive loss of renal function [17–20]; regardless, whether an antireflux technique is necessary remains undetermined. Verduyckt et al. [4] compared refluxing and anti-reflux procedures in a retrospective study, concluding that the reflux rate between the two methods only slightly differed. Waldnerd et al. [21] postulated that ileal peristalsis could provide a dampening effect to retrograde reflux, and antireflux procedures were not always necessary given an ileal graft of sufficient length; they recommended the use of ≥15 cm of ileum to prevent the reflux from reaching the renal pelvis. We speculated that preoperative renal function evaluation, patient selection, and preparation are in- dispensable for this procedure. In our own series, after receiving internal ureteral stent or nephrostomy, patients with significant renal insufficiency were ex- cluded. In addition, patients with borderline renal function for the procedure underwent combined ileal replacement and Boari flap–psoas hitch to minimize the length of an ileal graft. Discussion Consequently, 22 patients (95.7%) had their renal function preserved after the procedure. Metabolic acidosis was reported in only 1 patient, who underwent bilateral ureteral replacement with an ileal graft longer than 30 cm. Several published studies have reported intestine-related complications after surgery [11, 15]. Re-establishment of small-bowel continuity presents a technical chal- lenge to an unskilled urologist. We illustrated a sim- ple and direct side-to-side anastomosis with the use of 2 Endo-GIA stapler loads. Small-bowel reconstruc- tion using our technique took 3–5 min to complete. Furthermore, the technique achieved good hemostasis and reduced the risk of anastomotic stricture and leakage. Among the 23 patients, few patients reported small bowel-related complications, except for one pa- tient who had an episode of ileus, which was resolved by conservative treatment. In our patient cohort, we used a distal anti-reflux nipple valve for the antireflux procedure in 21 patients. The remaining 2 patients with urinary stone underwent refluxing anastomosis to facilitate the expulsion of the renal stone. No cases of postoperative reflux were reported, as determined by a voiding cystogram. Despite the occurrence of potential complications, such as Table 3 Outcomes of this series compared with other studies Authors (year) Patients (n) Antireflux/reflux technique Follow-up (months) Renal function improved or stable(%) Minor complications (%) Major complications (%) Verduyckt et al. [13] (2002) 18 7/11 65a 88.8 77.8 50 Matlaga et al. [1] (2003) 16 0/16 18.6a 100 16.7 0 Chung et al. [12] (2006) 56 0/56 72a 89.5 17.9 10.5 Armatys et al. [10] (2009) 91 2/89 36a 74.7 60.4 82.4 Wolff et al. [14] (2011) 17 0/17 174b 64.7 82.3 58.8 Present series 23 21/2 45a 95.7 43.5 8.7 aMean bMedian Table 3 Outcomes of this series compared with other studies Zhong et al. BMC Surgery (2019) 19:9 Page 6 of 7 Consent for publication f d b Informed consent was obtained from all individual participants included in the study. And all the authors agreed to publish this article. With the advance of laparoscopic surgery, more and more cases of laparoscopic ileal ureteric replacement have been performed in recent decades [23–27]. Stein et al. [25] presented a review of 7 patients undergoing lap- aroscopic interposition and 7 patients undergoing open ileal interposition. The comparative study demonstrated a significant benefit in narcotic requirement and conva- lescence for the laparoscopy group. In 2008, Wagner et al. [27] reported on the first case of robot-assisted lap- aroscopic ileal ureter, proving the feasibility of the pro- cedure in a robotic technique. However, no studies have been reported on either laparoscopic or robotic tech- niques in ileal ureter replacement in China. Competing interests Competing interests The authors report no conflict of interest. The authors report no conflict of interest. Funding No funding. No funding. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Acknowledgments We are grateful to the medical illustrator Madam Xinying Ma, who drawed figures for us. We are grateful to the medical illustrator Madam Xinying Ma, who drawed figures for us. Author details 1 1Department of Urology, Peking University First Hospital, Institute of Urology, National Urological Cancer Center, Peking University, No.8 Xishiku St, Xicheng District, Beijing 100034, China. 2Department of Urology, Lanzhou University Second Hospital, Lanzhou, Gansu, China. 3Department of Urology, Affiliated Hospital of Chifeng University, Chifeng, Inner Mongolia, China. 4Department of Urology, The General Hospital of Civil Aviation Administration of China, Beijing, China. 5Department of Urology, Qindao University Medical College Affiliated Yantai Yuhuangding Hospital, Yantai, Shandong, China. 6Department of Urology, China Meitan General Hospital, Beijing, China. 7Department of Urology, Heilongjiang provincial hospital, Harbin, Heilongjiang China 7Department of Urology, Heilongjiang provincial hospital, Harbin, Heilongjiang, China. Ethics approval and consent to participate All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. They were allowed by Peking University First Hospital ethics committee. And written, informed consent was obtained from all patients. Abbreviations CTU: Computed tomography urography; ESWL: Extracorporeal shock wave lithotripsy; F: Female; M: Male; MRU: Magnetic resonance urography; TCC: Transitional cell carcinoma; URSL: Ureteroscopic lithotripsy CTU: Computed tomography urography; ESWL: Extracorporeal shock wave lithotripsy; F: Female; M: Male; MRU: Magnetic resonance urography; TCC: Transitional cell carcinoma; URSL: Ureteroscopic lithotripsy 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. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Additional file stenosis, desussception, and stone formation secondary to the antireflux technique [22], the creation of an inverted nipple in patients without renal stones is recommended. Additional file 1: Table S1. Patients’ characteristics. (DOCX 20 kb) Compared with the results in other recent series (Table 3) [4, 11, 13–15], those of the present series seem encouraging. On the basis of our experience, the following recommendations with respect to surgi- cal technique are presented: (1) The ureter should be carefully isolated to diminish the risk of damage to the blood supply. Considering that exposure of the injured ureter could be difficult, in some cases with heavy inflammatory adhesion, we used a metal probe and inserted it through the nephrostomy tube to identify the position of the renal pelvis. (2) As long as a tension-free and water-tight anastomosis could be ensured, minimizing the length of ileal graft is crucial. (We divided an extra 5 cm of ileum for re- placement). However, for patients with improved renal function, combined ileal interposition and Boari flap– psoas hitch may be an option. (3) Side-to-side anastomosis can increase the ease and reduce the intestine-related complications during small-bowel reconstruction. (4) The ileal segment should be maintained in an isoperistaltic orientation; alternatively, a distal antireflux procedure may be performed to prevent postoperative reflux. However, a proximal antireflux technique is unnecessary because the outflow resistance of the ureter should be eliminated before anastomosing. (5) Appropriate stenting in the ileal segment is crucial to prevent anastomotic leakage. The ureter stent should be fixed to the proximal and distal ends of the ileal graft to prevent dislocation. Received: 13 December 2017 Accepted: 4 January 2019 Received: 13 December 2017 Accepted: 4 January 2019 With increasing utilization of endoscopic technology, iatrogenic long segment ureteral injury has become the main indication for ileal ureteral replacement. Despite the difficult and risky process of ileal ureter, the proced- ure can be carried out effectively and safely by skilled surgeons after careful patient selection. Anthors’ contributions WLZ and PH reviewed literatures and wrote the manuscript. GPD reviewed literature and revised the manuscript. KLY reviewed literatures and revised the manuscript. XSL and LQZ helped to revise and submit the manuscript. Besides, all the surgery operations were performed by XSL. JSB collected literatures. GCB and LC collected the clinical data and pictures. CPM and NC carried out data analysis and prepared manuscript. ZL and PZ carried out statistical analysis. All authors read and approved the final manuscript. Conclusion h Received: 13 December 2017 Accepted: 4 January 2019 References 1. Martínez-Sagarra JM, Amón Sesmero JH, Santos Largo J, et al. ileal ureteroplasties. Arch Esp Urol. 1992;45:961–6. 2. Shoemaker GE. Viii. Removal of the ureter with a tuberculous kidney. Ann Surg. 1911;53:696–8. Page 7 of 7 Zhong et al. BMC Surgery (2019) 19:9 Zhong et al. BMC Surgery (2019) 19:9 3. GOODWIN WE, WINTER CC. TURNER RD. replacement of the ureter by small intestine: clinical application and results of the ileal ureter. J Urol. 1959;81: 406–18. 4. Verduyckt FJ, Heesakkers JP, Debruyne FM. Long-term results of ileum interposition for ureteral obstruction. Eur Urol. 2002;42:181–7. 5. Boxer RJ, Fritzsche P, Skinner DG, et al. 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https://openalex.org/W2992641871	http://jurnal.unidha.ac.id/index.php/jteksis/article/download/45/38	Indonesian	null	PENGARUH SISTEM E-BILLING DAN KUALITAS PELAYANAN TERHADAP KEPATUHAN WAJIB PAJAK	Jurnal Teknologi Dan Sistem Informasi Bisnis/Jurnal Teknologi dan Sistem Informasi Bisnis	2,019	cc-by	2,562	ISSN : 2655-8238 ISSN : 2655-8238 ISSN : 2655-8238 Jurnal Teknologi Dan Sistem Informasi Bisnis Vol. 1 No. 2 Juli 2019 DOI: https://doi.org/10.47233/jteksis.v1i2.45 Jurnal Teknologi Dan Sistem Informasi Bisnis Vol. 1 No. 2 Juli 2019 DOI: https://doi.org/10.47233/jteksis.v1i2.45 Program Studi Akuntansi, Fakultas Ekonomi dan Bisnis, Universitas Putra Indonesia “YPTK”. Jalan Raya Lubuk Begalung, Padang 25221 Indonesia am Studi Akuntansi, Fakultas Ekonomi dan Bisnis, Universitas Putra Indonesia “YPTK”. Jalan Raya Lubu Begalung, Padang 25221 Indonesia rindycitradewi@gmail.com y g Abstrak : Penelitian ini bertujuan untuk mengetahui bagaimana Pengaruh Sistem E-Billing dan Kualitas Pelayanan dengan Pengetahuan Perpajakan sebagai variable control Terhadap Kepatuhan Wajib Pajak. Sampel pada penelitian ini diambil dengan metode incidential sampling pada wajib pajak yang sudah bekerja yang terdaftar di KPP Pratama Padang Satu.Teknik pengumpulan data yang digunakan adalah dengan menyebar kuesioner/angket kepada wajib pajak yang sudah bekerja yang terdaftar di KPP Pratama Padang Satu.Hasil penelitian ini menunjukkan bahwa Sistem E-Billing berpengaruh signifikan terhadap Kepatuhan wajib Pajak dengan nilai sig 0,000, sedangkan Kualitas Pelayanan tidak berpengaruh signifikan terhadap terhadap Kepatuhan Wajib Pajak dengan nilai sig 0,245. Dan secara bersama-sama sistem e-billing da kualitas pelayanan berpengaruh terhadap kepatuhan wajib pajak dengan nilai sig 0,000. Kata kunci: e-Billing, Kualitas Pelayanan, Kepatuhan Wajib Pajak Direktorat Jenderal Pajak melakukan beberapa perubahan dalam sistem pelayanannya untuk mengikuti teknologi informasi yang berkembang di masyarakat. Direktorat Jendral Pajak meluncurkan e- billing untuk kemudahan pembayaran pajak secara elektronik. E-billing adalah pembayaran pajak melalui media elektronik dengan memanfaatkan kode billing sebagai kode transaksi. Transaksi pembayaran atau penyetoran pajak secara elektronik, dilakukan melalui bank atau pos persepsi dengan menggunakan kode billing. Direktorat Jendral Pajak mengemukakan bahwa manfaat dari adanya e-billing yaitu system pembayaran menjadi lebih mudah, lebih cepat dan lebih akurat. Menurut hasil penelitian (Pusparesmi, 2016), menyimpulkan bahwa penggunaan e-billing berpengaruh positif terhadap kepatuhan wajib pajak. Sistem yang menerbitkan kode billing untuk pembayaran atau penyetoran penerimaan negara secara elektronik, tanpa perlu membuat surat setoran (SPP) manual akan meningkatkan kepatuhan wajib pajak. Direktorat Jenderal Pajak melakukan beberapa perubahan dalam sistem pelayanannya untuk mengikuti teknologi informasi yang berkembang di masyarakat. Direktorat Jendral Pajak meluncurkan e- billing untuk kemudahan pembayaran pajak secara elektronik. E-billing adalah pembayaran pajak melalui media elektronik dengan memanfaatkan kode billing sebagai kode transaksi. Transaksi pembayaran atau penyetoran pajak secara elektronik, dilakukan melalui bank atau pos persepsi dengan menggunakan kode billing. Direktorat Jendral Pajak mengemukakan bahwa manfaat dari adanya e-billing yaitu system pembayaran menjadi lebih mudah, lebih cepat dan lebih akurat. Menurut hasil penelitian (Pusparesmi, 2016), menyimpulkan bahwa penggunaan e-billing berpengaruh positif terhadap kepatuhan wajib pajak. Sistem yang menerbitkan kode billing untuk pembayaran atau penyetoran penerimaan negara secara elektronik, tanpa perlu membuat surat setoran (SPP) manual akan meningkatkan kepatuhan wajib pajak. y Citra Dewi1, Hanna Pratiwi2,Avezrima Rahmamuthi3, Berta Agus Petra4 Agung Ramadhan Rindy Citra Dewi1, Hanna Pratiwi2,Avezrima Rahmamuthi3, Berta Agus Petra4 Agung Ramadhanu5 Program Studi Akuntansi, Fakultas Ekonomi dan Bisnis, Universitas Putra Indonesia “YPTK”. Jalan Raya Lubuk Begalung, Padang 25221 Indonesia 2. Tinjauan Pustaka 2.1 Pajak y Sistem pemungutan pajak berdasarkan self assessment yang memberikan kesempatan kepada wajib pajak untuk menghitung, membayar dan melaporkan kewajiban perpajakannya sendiri.Hal ini berarti kesadaran dan kepatuhan wajib pajak dalam memenuhi kewajiban perpajakannya berada pada wajib pajak itu sendiri.Salah satu bentuk upaya meningkatkan kepatuhan wajib pajak dapat dilakukan melalui peningkatan kualitas pelayanan. Kualitas adalah suatu kondisi dinamis yang berhubungan dengan produk, jasa manusia, proses dan lingkungan yang memenuhi atau melebihi harapan pihak yang menginginkannya. Pelayanan yang berkualitas adalah pelayanan yang dapat memberikan kepuasan kepada pelanggan serta dilakukan secara terus-menerus. j Pajak menurut Pasal 1 ayat 1 Undang- Undang Nomor 16 Tahun 2009 tentang Perubahan Keempat Atas Undang-Undang Nomor 6 Tahun 1983 Tentang Ketentuan Umum dan Tata Cara Perpajakan, disebutkan bahwa pajak adalah kontribusi wajib pajak kepada negara yang terutang oleh orang pribadi atau badan yang bersifat memaksa berdasarkan Undang-Undang, dengan tidak mendapatkan imbalan secara langsung dan digunakan untuk keperluan Negara bagi sebesar-besarnya kemakmuran rakyat. Menurut pernyataan Prof. Dr. Rochmat Soemitro,S.H. dalam Siti Resmi (2014:1) bahwa, “pajak adalah iuran peralihan kekayaan dari rakyat kepada kas Negara untuk membiayai pengeluaran rutin dan surplusnya digunakan untuk public saving yang merupakan sumber utama untuk membiayai public investment”. 2.3 Sistem e-Billing e-Billing pajak menurut Direktorat Jendral Pajak (DJP) adalah sistem bayar pajak online (elektronik) dengan cara pembuatan kode billing atau ID billing terlebih dahulu. e- billing pajak ini hadir untuk mewujudkan komitmen DJP dalam pengalihan sistem manual menuju sistem elektronik perpajakan. Billing system merupakan serangkaian proses yang meliputi kegiatan pendaftaran peserta billing, pembuatan kode billing, pembayaran berdasarkan kode billing dan rekonsiliasi billing dalam sistem modul penerimaan negara. 1. Pendahuluan Pajak merupakan unsur penting bagi setiap negara, bahkan penting dalam rangka menopang anggaran penerimaan negara. Untuk meningkatkan penerimaan pajak, tentu diperlukan juga kerjasama dari wajib pajak, yang mana dibutuhkan kepatuhan dari wajib pajak baik pribadi maupun badan dalam melaporkan dan membayar pajak.Menurut Pratami dkk (2017:2) Kepatuhan Wajib Pajak (Tax Compliance) dapat diidentifikasi dari kepatuhan Wajib Pajak dalam mendaftarkan diri, kepatuhan untuk melaporkan kembali Surat Pemberitahuan (SPT), kepatuhan dalam menghitung dan membayar pajak terhutang. Dalam pelaksanaanya, seluruh kewajiban ini berkaitan dengan pemberian pelayanan pada Wajib Pajak. Pelayanan yang diberikan kepada Wajib Pajak diharapkan dapat diberikan secara cepat, aman dan murah. Dengan adanya kemudahan ini, Wajib Pajak diharapkan dapat lebih meningkatkan kewajiban perpajakannya. Kepatuhan wajib pajak merupakan ketaatan wajib pajak untuk melakukan ketentuan- ketentuan atau aturan-aturan perpajakan yang diwajibkan (Noviantari dan Setiawan 2018). Adapun faktor-faktor yang mempengaruhi kepatuhan wajib pajak diantaranya yaitu : sistem e-billing dan kualitas pelayanan. menyimpulkan bahwa penggunaan e-billing berpengaruh positif terhadap kepatuhan wajib pajak. Sistem yang menerbitkan kode billing untuk pembayaran atau penyetoran penerimaan negara secara elektronik, tanpa perlu membuat surat setoran (SPP) manual akan meningkatkan kepatuhan wajib pajak. Pelayanan fiskus juga merupakan hal penting dalam menggali penerimaan negara dimana fiskus seharusnya melayani wajib pajak dengan jujur, professional dan bertanggungjawab tetapi faktanya para fiskus Copyright@2019 Prodi Sistem Informasi Universitas Dharma Andalas 93 ISSN : 2655-8238 Jurnal Teknologi Dan Sistem Informasi Bisnis Vol. 1 No. 2 Juli 2019 DOI: https://doi org/10 47233/jteksis v1i2 DOI: https://doi.org/10.47233/jteksis.v1i2.45 didalam pemenuhannya dilakukan secara sukarela. tidak semuanya bersih dan ada juga yang menyalahgunakan kewenangannya untuk memanipulasi data yang terkait dengan SPT wajib pajak seperti contoh kasus Dirjen Pajak memecat pegawai pajak Ambon pada tahun 2018. Hasil penelitian Susmita& Supadimi (2016), kualitas pelayanan fiskus yang diberikan oleh petugas pelayanan pajak terhadap wajib pajak juga dapat mempengaruhi tingkat kepatuhan wajib pajak orang pribadi. Penelitian ini berfokus pada kepatuhan wajib pajak orang pribadi (WPOP) di Kantor Pelayanan Pajak Pratama Padang Satu. KPP Pratama Padang satu adalah salah satu dari 9 (sembilan) KPP di wilayah kerja Dirjen Pajak Sumbar-Jambi. 3. Metode Penelitian 3.1 Objek Penelitian Penelitian ini dilakukan di Kantor Pelayanan Pajak Pratama Padang Satu yang beralamat di Jalan Bagindo Azis Chan No.26, Padang Timur, Padang.Penelitian ini mencakup dalam bidang perpajakan yaitu dengan mengumpulkan jurnal-jurnal, buku-buku yang berkaitan serta melalui studi pustaka dan data primer dengan memberikan kuesioner kepada wajib pajak yang terdaftar di Kantor Pelayanan Pajak Pratama Padang Satu. 3.3 Sumber Data Sumber data dan responden dalam penelitian ini adalah data primer dan data sekunder.Data primer diperoleh dengan menyebarkan kuesioner. Dan data sekunder diperoleh seperti media massa yang mendukung dan perpustakaan. 2.2 Kepatuhan Wajib Pajak p j j Abdul Rahman dalam Nurhidayah (2015) mengemukakan kepatuhan perpajakan dapat didefenisikan sebagai keadaan dimana wajib pajak memenuhi semua kewajiban perpajakan dan melaksanakan hak perpajakannya. Kepatuhan wajib pajak adalah pemenuhan kewajiban perpajakan yang dilakukan oleh pembayar pajak dalam rangka memberikan kontribusi bagi pembangunan negara yang diharapkan 3.2 Variabel Penelitian 3.2 Variabel Penelitian Copyright@2019 Prodi Sistem Informasi Universitas Dharma Andalas 94 Jurnal Teknologi Dan Sistem Informasi Bisnis Vol. 1 No. 2 Juli 2019 DOI htt //d i /10 47233/jt k i 1i2 DOI: https://doi.org/10.47233/jteksis.v1i2.45 ISSN : 2655-8238 Jurnal Teknologi Dan Sistem Informasi Bisnis Vol. 1 No. 2 Juli 2019 DOI htt //d i /10 47233/jt k i 1i2 DOI: https://doi.org/10.47233/jteksis.v1i2.45 Variabel-variabel yang digunakan dalam penelitian ini adalah: Variabel-variabel yang digunakan dalam penelitian ini adalah: Hasil Regresi Linear Berganda 1. Variabel dependen yaitu kepatuhan wajib pajak. 2. Variabel dependen yaitu sistem e- billing dan kualitas pelayanan. 3. Variabel kontrol yaitu pengetahuan perpajakan. Berdasarkan tabel diatas maka dapat dilihat persamaan regresinya yaitu : 3.5 Metode Analisis Data Metode analisi data dalam penelitian ini menggunakan analisis regresi linear berganda. Yaitu untuk melihat sejauah mana kemampuan dari variabel independen dalam menjelaskan atau mempengaruhi variabel dependen dengan rumus : 3. Koefisien regresi variabel kualitas pelayanan sebesar 0,103 artinya jika kualitas pelayanan mengalami peningkatan satu (1) satuan dengan asumsi sistem e-billing diabaikan atau bernilai nol (0), maka kepatuhan wajib pajak akan mengalami peningkatan sebesar 0,103. Y= a+𝑏1𝑋1+𝑏2𝑋2 + 𝑒 Keterangan : Keterangan : Y = kepatuhan wajib pajak 𝑋1 = sistem e-billing 𝑋2 = kualitas pelayanan KWP= 4,614 + 0,586X1 + 0,103X2 + e Dari persamaan regresi berganda di atas dapat disimpulkan bahwa : 1. Nilai konstanta sebesar 4,614 artinya jika sistem e-billing dan kualitas pelayanan diabaikan atau bernilai nol (0), maka nilai kepatuhan wajib pajak (KWP) nilainya adalah tetap sebesar 4,614. 3.4 Populasi dan Sampel Populasi dalam penelitian ini adalah seluruh wajib pajak orang pribadi di KPP Pratama Padang Satu yang menggunakan e-billing dalam melakukan pembayaran pajak. Sampel dalam penelitian ini menggunakan teknik random sampling yang dipilih secara acak tanpa memperhatikan strata yang ada dalam populasi itu (Sugiyono, 2012) 2. Koefisien regresi variabel sistem e-billing sebesar 0,586 artinya jika sistem e-billing mengalami peningkatan satu (1) satuan dengan asumsi kualitas pelayanan diabaikan atau bernilai nol (0), maka nilai kepatuhan wajib pajak akan mengalami peningkatan sebesar 0,586. 3.6 Uji Hipotesis Adapun pengujian hipotesis dalam penelitian ini yaitu : uji t, uji F dan koefisien determinasi Uji t bertujuan untuk menguji pengaruh satu variabel penjelas/independen secara individual dalam menerangkan variasi variabel dependen, dengan tingkat signifikansi 0,1 (Ghozali,2011). Hasil uji t dapat dilihat tabel berikut: 4.2 Uji Hipotesis 1. Uji t 3.6 Uji Hipotesis 4.1 Analisis Regreai Berganda Analisis regresi digunakan untuk melihat pengaruh variabel independen : sistem e- billing dan kualitas pelayanan secara simultan terhadap variable dependen : kepatuhan wajib pajak dengan dengan asumsi variable independen lain dianggap konstan. Tabel 4.1 Tabel 4.1 Copyright@2019 Prodi Sistem Informasi Universitas Dharma Andalas Copyright@2019 Prodi Sistem Informasi Universitas Dharma Andalas 95 ISSN : 2655-8238 Jurnal Teknologi Dan Sistem Informasi Bisnis Vol. 1 No. 2 Juli 2019 h //d i / /j k i i DOI: https://doi.org/10.47233/jteksis.v1i2.45 Tabel 4.2 Hasil Uji t Koefisien Determinasi (R) menunjukkan mengenai besarnya pengaruh variabel independen terhadap variabel dependen dan hasilnya disajikan pada tabel 4.26 berikut ini: hasilnya disajikan pada tabel 4.26 berikut ini: Tabel 4.4 Hasil Koefisien Determinasi Dari tabel 4.4 dapat dilihat bahwa koefisien determinasi (Adjust R Square) sebessar 0.458 yang berarti bahwa variabel-variabel independen mampu menerangkan kepatuhan wajib pajak sebesar 45.8%.Sedangkan sisanya 54.2% dipengaruhi oleh faktor-faktor lain yang tidak dijelaskan dalam penelitian ini. Dari tabel 4.4 dapat dilihat bahwa koefisien determinasi (Adjust R Square) sebessar 0.458 yang berarti bahwa variabel-variabel independen mampu menerangkan kepatuhan wajib pajak sebesar 45.8%.Sedangkan sisanya 54.2% dipengaruhi oleh faktor-faktor lain yang tidak dijelaskan dalam penelitian ini. Berdasarkan tabel 4.2 hasil uji t dinyatakan bahwa: Berdasarkan tabel 4.2 hasil uji t dinyatakan bahwa: 1. Nilai variabel Sistem e-Billing (X1) t- hitung > t-tabel sebesar 6.405 > 1.663 dan memiliki nilai sig 0,000 < 0,1 maka H1 diterima. Hal ini menunjukkan bahwa variabel Sistem e-Billing berpengaruh terhadap Kepatuhan Wajib Pajak. 2. Nilai variabel kualitas pelayanan (X2) t- hitung < t-tabel sebesar 1.171 <1.663dan memiliki nilai sig 0,245 > 0,1 maka H2 ditolak. Hal ini menunjukkan bahwa variabel kualitas pelayanan tidak berpengaruh terhadap Kepatuhan Wajib Pajak. j 2. Uji F j p j 2. Kualitas pelayanan tidak berpengaruh terhadap kepatuhan wajib pajak j Uji F bertujuan menunjukkan apakah dalam model regresi semua variabel independen atau bebas yang dimasukkan dalam model mempunyai pengaruh secara bersama-sama terhadap variabel dependen, yang diuji pada tingkat signifikan 0,1 (Ghozali, 2011). Hasil uji F dapat dilihat pada tabel berikut: j p j 3. .Sistem e-Billing dan kualitas pelayanan secara simultan atau bersama-sama berpengaruh signifikan terhadap kepatuhan wajib pajak Tabel 4.3 Hasil Uji F Tabel 4.3 Hasil Uji F 5. Kesimpulan p Berdasarkan dari hasil analisis dan pembahasan diatas, maka dapat disimpulkan bahwa yaitu : y 1. Sistem e-Billing berpengaruh positif dan signifikan terhadap kepatuhan wajib pajak j 2. Uji F DAFTAR PUSTAKA Direktorat Jendral Pajak. 2014. “Peraturan Direktorat Jendral Pajak Nomor PER-26/PJ/2014 Tentang Sistem Pembayaran Pajak Secara Elektronik”. Direktorat Jendral Pajak. 2014. “Peraturan Direktorat Jendral Pajak Nomor PER-26/PJ/2014 Tentang Sistem Pembayaran Pajak Secara Elektronik”. Tabel 4.3 dalam model ANOVA dapat diperoleh F hitung sebesar 34.777 lebih besar dari Ftabel sebesar 2.77 dengan tingkat signifikan 0,000. Oleh karena itu tingkat signifikan lebih kecil dari 0,1 maka variabel Sistem e-Billing dan kualitas pelayanan secara simultan atau bersama-sama berpengaruh signifikan terhadap variable dependen yaitu Kepatuhan Wajib Pajak. Tabel 4.3 dalam model ANOVA dapat diperoleh F hitung sebesar 34.777 lebih besar dari Ftabel sebesar 2.77 dengan tingkat signifikan 0,000. Oleh karena itu tingkat signifikan lebih kecil dari 0,1 maka variabel Sistem e-Billing dan kualitas pelayanan secara simultan atau bersama-sama berpengaruh signifikan terhadap variable dependen yaitu Kepatuhan Wajib Pajak. Tabel 4.3 dalam model ANOVA dapat diperoleh F hitung sebesar 34.777 lebih besar dari Ftabel sebesar 2.77 dengan tingkat signifikan 0,000. Oleh karena itu tingkat signifikan lebih kecil dari 0,1 maka variabel Sistem e-Billing dan kualitas pelayanan secara simultan atau bersama-sama berpengaruh signifikan terhadap variable dependen yaitu Kepatuhan Wajib Pajak. Direktorat Jendral Pajak Kementerian Keuangan. 2016. E-Billing. http://www.pajak.go.id/e-billing. Dilihat pada Maret 2019. Ghozali, Imam. 2011. “Aplikasi Analisis Multivariate Dengan Program IBM SPSS 19. Semarang: Badan Penerbitan Universitas Diponegoro. 3. Koefisien Determinasi (𝑹𝟐 ) 3. Koefisien Determinasi (𝑹𝟐 ) Copyright@2019 Prodi Sistem Informasi Universitas Dharma Andalas 96 ISSN : 2655-8238 Jurnal Teknologi Dan Sistem Informasi Bisnis Vol. 1 No. 2 Juli 2019 DOI: https://doi org/10 47233/jteksis v1i2 DOI: https://doi.org/10.47233/jteksis.v1i2.45 Jurnal Teknologi Dan Sistem Informasi Bisnis Vol. 1 No. 2 Juli 2019 DOI: https://doi org/10 47233/jteksis v1i2 DOI: https://doi.org/10.47233/jteksis.v1i2.45 Jurnal Teknologi Dan Sistem Informasi Bisnis Vol. 1 No. 2 Juli 2019 DOI: https://doi org/10 47233/jteksis v1i2 DOI: https://doi.org/10.47233/jteksis.v1i2.45 Suandy, Erly. 2014. Hukum Pajak. Jakarta: Salemba Empat. Sugiyono. 2011. Statistika Untuk Penelitian. Bandung: Alfabeta. Sugiyono. 2012. Memahami Penelitian Kualitatif. Bandung: Alfabeta Sugiyono. 2013. Metode Penelitian Bisnis. Bandung: Alfabeta. Suandy, Erly. 2014. Hukum Pajak. Jakarta: Salemba Empat. Sugiyono. 2011. Statistika Untuk Penelitian. Bandung: Alfabeta. Sugiyono. 2012. Memahami Penelitian Kualitatif. Bandung: Alfabeta Sugiyono. 2013. Metode Penelitian Bisnis. Bandung: Alfabeta. Handayani, Ni Putu Milan Novita dan Naniek Noviari. 2016. “Pengaruh Persepsi Manajemen Atas Keunggulan Penerapan E-Billang dan E-SPT Pajak Pertambahan Nilai Pada Kepatuhan Perpajakan”. Jurnal Akuntansi Universitas Udayana, Vol 15(2): hal 1001-1028. Bandung: Alfabeta. Mardiasmo. 2009. Perpajakan Edisi Revisi 2009. Yogyakarta: Andi Offset. Mardiasmo. 2010. Perpajakan Edisi Revisi 2009. Yogyakarta: Andi Offset. Mardiasmo. 2011. Perpajakan. Edisi Revisi. Yogyakarta: Andi Offset. Mardiasmo. 2016. Perpajakan Edisi Terbaru 2016. Yogyakarta: Andi Offset. Noviantri, Putri dan Putu Ery Setiawan. 2018. “Pengaruh Persepsi Kualitas Pelayanan, Pemahaman, Persepsi Sanksi Perpajakan, dan Lingkungan Terhadap Kepatuhan Wajib Pajak”, Jurnal Akuntansi Universitas Udayana, Vol 22(3): hal 1711-1740. Peraturan Direktur Jendral Pajak Nomor PER 26/PJ/2014 tentang Sistem Pembayaran Pajak Secara Elektronik.http://ditkeu.unair.ac.id/do wnload/PER_26_PJ_2014.pdf. Dilihat April 2019. Dilihat April 2019. Pratami, Sulindawati dan Wahyuni, 2017. “Pengaruh Penerapan E-system Perpajakan Terhadap Tingkat Kepatuhan Wajib Pajak Orang Pribadi Dalam Membayar Pajak Pada Kantor Pelayanan Pajak (KPP) Pratama Singaraja”, e-Jurnal S1 Ak Universitas Pendidikan Ganesha, Vol 7, No.1. Pusparesmi, A. 2016. “Pengaruh Penggunaan Sistem Administrasi Berbasis Internet Terhadap Tingkat Kepatuhan Wajib Pajak Orang Pribadi Pada KPP Pratama Surakarta”, Skripsi Universitas Islam Batik Surakarta. Resmi, Siti. 2011. Perpajakan: Teori dan Kasus. Jakarta: Salemba Empat. Resmi, Siti. 2014. Perpajakan Teori dan Kasus Edisi 8. Jakarta: Salemba Empat. Copyright@2019 Prodi Sistem Informasi Universitas Dharma Andalas 97
https://openalex.org/W2074229704	https://bmcmusculoskeletdisord.biomedcentral.com/counter/pdf/10.1186/1471-2474-12-177	English	null	Activity pacing for osteoarthritis symptom management: study design and methodology of a randomized trial testing a tailored clinical approach using accelerometers for veterans and non-veterans	BMC musculoskeletal disorders	2,011	cc-by	8,186	* Correspondence: sumurphy@umich.edu 1Department of Physical Medicine and Rehabilitation, University of Michigan, Ann Arbor, MI, USA Full list of author information is available at the end of the article Activity pacing for osteoarthritis symptom management: study design and methodology of a randomized trial testing a tailored clinical approach using accelerometers for veterans and non-veterans Susan L Murphy1,2*, Angela K Lyden1, Maria Clary2, Michael E Geisser1, Raymond L Yung2,3, Daniel J Clauw4 and David A Williams4 Murphy et al. BMC Musculoskeletal Disorders 2011, 12:177 http://www.biomedcentral.com/1471-2474/12/177 Murphy et al. BMC Musculoskeletal Disorders 2011, 12:177 http://www.biomedcentral.com/1471-2474/12/177 © 2011 Murphy 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. STUDY PROTOCOL Open Access Activity pacing for osteoarthritis symptom management: study design and methodology of a randomized trial testing a tailored clinical approach using accelerometers for veterans and non-veterans Background Osteoarthritis (OA) is a prevalent and chronic disease. It affects over 27 million adults in the U.S. and is expected to increase as the population ages [1]. OA is more pre- valent among females, however, male veterans are also significantly affected, with 1/3 of males of all ages diag- nosed and nearly half of those who use the Veteran’s Affairs (VA) healthcare system [2]. OA causes substan- tial physical and psychosocial disability. By 2020, it is expected that 12 million people in the U.S. will report activity limitations due to arthritis [3]. A primary reason people with OA seek treatment is for pain relief. Whereas people with OA often receive pharmacological treatment for pain reduction, non- pharmacological treatment for symptoms is considered the foundation of OA management [4,5]. Rehabilitation professionals, such as occupational therapists (OTs), can tailor non-pharmacological symptom management inter- ventions so as to promote adherence and behavior change. To date, however OTs have had only limited involvement in tailoring such interventions for people with OA. More research is needed on the effectiveness of these tailored symptom management interventions when delivered by rehabilitation professionals within the context of clinical care. Well-established community- based programs on self-management strategies (such as the Arthritis Self-Management Program) have been shown to have small but positive effects on pain and arthritis self-efficacy [6-8]. Although community-based programs are an important tool for OA management, such programs do not optimize the use of rehabilitation professionals in the delivery or tailoring of these programs. In this study, the intervention is designed to assist people in enhancing their arthritis self-efficacy by indivi- dually tailoring the use of activity pacing strategies to the context of where and when the strategies may be most useful. According to Social Cognitive Theory, self- efficacy is enhanced by building competency through practice (and by the rewards associated with success) in specific situations or conditions [22]. By tailoring activity pacing instruction to individuals’ real-life routines and activities, there is a greater chance for learning to occur and for self-efficacy to be enhanced given the greater proximity of the pacing schedule to real-life and person- ally relevant activities. Pain, Fatigue, and Their Relationship to Physical Activity Both pain and fatigue symptoms are linked to various functional problems in OA. Abstract Background: Osteoarthritis (OA) is a prevalent chronic disease and a leading cause of disability in adults. For people with knee and hip OA, symptoms (e.g., pain and fatigue) can interfere with mobility and physical activity. Whereas symptom management is a cornerstone of treatment for knee and hip OA, limited evidence exists for behavioral interventions delivered by rehabilitation professionals within the context of clinical care that address how symptoms affect participation in daily activities. Activity pacing, a strategy in which people learn to preplan rest breaks to avoid symptom exacerbations, has been effective as part of multi-component interventions, but hasn’t been tested as a stand-alone intervention in OA or as a tailored treatment using accelerometers. In a pilot study, we found that participants who underwent a tailored activity pacing intervention had reduced fatigue interference with daily activities. We are now conducting a full-scale trial. Methods/Design: This paper provides a description of our methods and rationale for a trial that evaluates a tailored activity pacing intervention led by occupational therapists for adults with knee and hip OA. The intervention uses a wrist accelerometer worn during the baseline home monitoring period to glean recent symptom and physical activity patterns and to tailor activity pacing instruction based on how symptoms relate to physical activity. At 10 weeks and 6 months post baseline, we will examine the effectiveness of a tailored activity pacing intervention on fatigue, pain, and physical function compared to general activity pacing and usual care groups. We will also evaluate the effect of tailored activity pacing on physical activity (PA). Discussion: Managing OA symptoms during daily life activity performance can be challenging to people with knee and hip OA, yet few clinical interventions address this issue. The activity pacing intervention tested in this trial is designed to help people modulate their activity levels and reduce symptom flares caused by too much or too little activity. As a result of this trial, we will be able to determine if activity pacing is more effective than usual care, and among the intervention groups, if an individually tailored approach improves fatigue and pain more than a general activity pacing approach. Trial Registration: ClinicalTrials.gov: NCT01192516 Murphy et al. BMC Musculoskeletal Disorders 2011, 12:177 http://www.biomedcentral.com/1471-2474/12/177 Murphy et al. Abstract BMC Musculoskeletal Disorders 2011, 12:177 http://www.biomedcentral.com/1471-2474/12/177 Page 2 of 10 behavior, the central determinants include 1) knowledge of the risks and benefits, 2) outcome expectations about costs and benefits for engaging in health behaviors, 3) the health goals people set for themselves and plans for goal achievement, 4) the perceived facilitators and bar- riers (such as external social or work demands), and 5) self-efficacy, the belief in the ability to perform under a certain set of conditions [21,22]. Self-efficacy is often a central focus in self-management interventions because it influences behavior both directly and indirectly through the other determinants above [22]. Numerous studies have found that improvements in pain coping skills are related to improvements in arthritis self-effi- cacy [19,23,24]. Background Pain in OA affects the abil- ity to engage in activities of daily living, work, and other meaningful activities and is associated with a reduced quality of life [25-27]. Fatigue is not as well-studied in OA, however, it is one of the most frequently reported OA symptoms [28,29]. Attention to the clinical impor- tance of fatigue is growing. In large studies, substantial fatigue was reported by 41% of patients with OA [28], and problematic fatigue or tiredness was reported in moderate levels [30]. Fatigue in OA is one of the stron- gest predictors of functional disability [28,31] and work dysfunction [28]. Fatigue has also been associated with pain, sleep problems, functional disability, and depres- sion [28,30-32]. Qualitative studies have further charac- terized the fatigue experience in OA as debilitating, causing activity restriction, and greatly impacting life [29,33]. Behavior Change and Tailoring Interventions g g A tailored approach to interventions is being increas- ingly recommended in order to make the content per- sonally relevant and to promote long-term behavior change. Several interventions are individually tailored on key variables identified in theoretical health behavior models, such as motivational readiness, decisional bal- ance, or self-efficacy [9-11]. Interventions also have been tailored on personal characteristics (e.g., gender [12], depression [13], pain level [14]). There is growing evi- dence that tailoring enhances the effectiveness of clinical interventions designed to alter health risk behaviors such as smoking [15,16] and low physical activity [17,18]. A better understanding of how symptoms affect activ- ity on a more micro level, such as over a day, week, or month, can help refine and tailor OA interventions. For example, tailored interventions may target timing of behaviors such as medication usage or variability in symptoms by time [34]. Although OA symptoms are believed to be less variable than other rheumatic For arthritis management interventions, Social Cogni- tive Theory has often been used as the theoretical foun- dation of intervention development [19,20]. It posits that people learn through an interaction of social and environmental influences that contribute to their own motivations and behaviors [21]. To adopt a new health Murphy et al. BMC Musculoskeletal Disorders 2011, 12:177 http://www.biomedcentral.com/1471-2474/12/177 Murphy et al. BMC Musculoskeletal Disorders 2011, 12:177 http://www.biomedcentral.com/1471-2474/12/177 Page 3 of 10 been effective at reducing pain [23,24]; however, it is impossible to disentangle the unique effects of activity pacing. Due to differences in how activity pacing is understood as a strategy, there is variability in activity pacing instruction and its delivery by therapists [42,43]. This trial addresses these limitations by using a standar- dized approach to teaching activity pacing to adults with knee or hip OA. This approach is structured around the overall goal of activity pacing, which is the balance between activity and rest in order to accomplish neces- sary and valued activities [44]. In this study, we will also be able to examine the unique effects of tailored activity pacing on fatigue for knee or hip OA in which our pre- liminary results showed significant improvements in fati- gue compared to a general activity pacing intervention [41]. The conceptual model that guided intervention development is shown in Figure 1. Methods/Design This is a three-arm randomized controlled trial that will examine outcomes at baseline, posttest (approximately 10 weeks post baseline), and 6 months. Ethical approval for this study was obtained by the University of Michigan Medical School Institutional Review Board and the Sub- committee on Human Studies in the Veteran’s Affairs Ann Arbor Healthcare System. The overall goal of the study is to examine the effectiveness of a tailored activity pacing intervention for adults with symptomatic knee or hip OA. The primary outcomes are fatigue and pain. Behavior Change and Tailoring Interventions conditions such as rheumatoid arthritis (RA), an emer- ging body of research has shown that there is important variability in OA pain [35-38] across days or even within a day. These pain fluctuations have been associated with limited daily activities, days of missed work, sleep inter- ference, and mood [36], and increased pain variability may be associated with personal factors [e.g., body mass index (BMI)] [35]. Fewer studies have examined fatigue variability in OA. Given the strong relationship between pain and fatigue [28,31,32] in OA and other conditions, it is not surpris- ing that fatigue is often considered a by-product of pain. However, when pain and fatigue are measured daily or within-day, differences emerge. For example, compared to samples of persons with RA and fibro- myalgia, daily fatigue fluctuations were not as strongly associated with daily pain fluctuations in persons with OA [39]. In our work, we found that within-day fatigue was experienced at greater intensities than was pain [40]. Additionally, in one of the few studies to examine the symptom-activity relationship in OA, we found that fatigue was more highly associated with objective physi- cal activity (PA; measured by accelerometer) compared to pain [40]. Lastly, we found differential effects for pain and fatigue by group when we tailored activity pacing [41], providing support for addressing pain and fatigue separately. Participant Recruitment Community-living veteran and non-veteran participants are recruited from the University of Michigan and VA clinics, senior housing sites, and through public adver- tisements. Based on our previous studies, our optimal recruitment methods are ad placement at clinics and through community ads and fliers. As in our previous studies, areas with higher concentrations of minorities are particularly targeted for recruitment. Minorities such as African Americans are particularly important to include in this study because they not only have higher rates of knee and hip OA than Caucasians [45], they also report more severe OA symptoms and more physi- cal disability even when controlling for demographics and other factors [46]. In addition, African Americans are less likely than Caucasians to opt for hip or knee replacements [47] and more likely to manage OA through home remedies that often have little to no evi- dence of effectiveness (such as copper bracelets or her- bal medicines) [48]. return materials in person or by mail to the study team. Participants are randomly stratified by age and gender into the tailored activity pacing intervention, general activity pacing intervention, or usual care group. The usual care group participants undergo all outcome assessments and have maintained contact through monthly emails between outcome assessments similar to the intervention groups. The contact period between Weeks 11 - 23 is being undertaken to better ensure par- ticipation in the 6 month assessment for all groups. Each intervention arm involves 3 individual sessions with an OT. In the tailored activity pacing group, data collected from the home monitoring period are used to generate reports for participants about how symptoms relate to physical activity and are used by the occupa- tional therapist. The general activity pacing group does not use the information from the home monitoring per- iod during OT sessions. Eligibility Criteria The following inclusion criteria were designed to iden- tify a cohort of community-living adults who are likely experiencing symptoms specifically due to their OA. Prior to enrollment, each prospective participant is eval- uated for inclusion. Table 1 lists the inclusion and exclusion criteria. Inclusion Criteria Eligible individuals are randomly stratified by age and gender into one of three arms: tailored activity pacing intervention, general activity pacing intervention, or usual care. The tailored and general activity pacing interventions are equivalent in session number and length. Participants are involved in study procedures over 6 months. For the intervention groups, involvement consists of 3 individual sessions, 3 testing visits, 3 home monitoring periods, 3 weekly follow-up phone calls, and monthly contact phone calls until the outcome assess- ment at 6 months. The usual care group participates in all activities described above except for the intervention sessions and the 3 weekly follow-up phone calls. • Age ± 50 years old • Age ± 50 years old • Pain for at least 3 months duration • Mild to moderate severity on the WOMAC pain scale • Radiographic evidence of knee or hip OA • Community-living (i.e, own home, senior residence, apartment) • Adequate cognitive status (score of > 5 on the 6-Item Screener) [66] • Ambulatory either with or without an assistive device • Reliable operation of the Actiwatch-Score accelerometer • English-speaking • Community-living (i.e, own home, senior residence, apartment) • Adequate cognitive status (score of > 5 on the 6-Item Screener) [66] Exclusion Criteria • Medical conditions that may interfere with pain and fatigue reporting or activity monitoring (multiple sclerosis, lupus, rheumatoid arthritis, Parkinson’s disease, peripheral neuropathy) • Cancer diagnosis in the last year (other than skin cancer) or currently undergoing treatment for cancer • Medicine changes within last 2 weeks • Anemia or unmanaged thyroid dysfunction (from blood work result) • 2 or more days of complete bed rest within last month • Limb hemiplegia or amputation • Knee arthroscopic procedure within the last 2 months • Replacement of any hip or knee within the last 6 months • Knee joint injection within the last 3 months • Current receipt of physical or occupational therapy for OA symptoms or knee/hip problems • Current attendance or attendance in the past 12 months in a cognitive behavioral program or other self-management program that includes activity pacing instruction. • Medical conditions that may interfere with pain and fatigue reporting or activity monitoring (multiple sclerosis, lupus, rheumatoid arthritis, Parkinson’s disease, peripheral neuropathy) • Medical conditions that may interfere with pain and fatigue reporting or activity monitoring (multiple sclerosis, lupus, rheumatoid arthritis, Parkinson’s disease, peripheral neuropathy) p p p y • Cancer diagnosis in the last year (other than skin cancer) or currently undergoing treatment for cancer y g g • Medicine changes within last 2 weeks g • Anemia or unmanaged thyroid dysfunction (from blood work result) Pacing Intervention Effectiveness The specific aims are: The specific aims are: Although activity pacing is a recommended non-phar- macological intervention for OA management based on consensus guidelines [4], its effectiveness as a stand- alone intervention is understudied. Typically, activity pacing is combined with other programs (e.g., coping skills training, cognitive behavioral therapy). In these studies of knee or hip OA, the overall programs have Aim 1: To examine the effectiveness of a tailored activity pacing intervention on fatigue, pain, and physi- cal function. Aim 2: To determine if increased arthritis self-efficacy post intervention is related to improvements in symp- tom severity and function. Aim 3 Aim 1 OUTCOMES Tailored Activity Pacing Pain Fatigue Physical Function Physical Activity Mechanism of Change Arthritis Self-efficacy Aim 2 Figure 1 Model of Symptoms, Self-Efficacy, Physical Function and PA in Adults with Osteoarthritis. Physical Activity Tailored Activity Pacing Pain Fatigue Physical Function Mechanism of Change Arthritis Self-efficacy Figure 1 Model of Symptoms, Self-Efficacy, Physical Function and PA in Adults with Osteoarthritis. Figure 1 Model of Symptoms, Self-Efficacy, Physical Function and PA in Adults with Osteoarthritis. Page 4 of 10 Murphy et al. BMC Musculoskeletal Disorders 2011, 12:177 http://www.biomedcentral.com/1471-2474/12/177 Murphy et al. BMC Musculoskeletal Disorders 2011, 12:177 http://www.biomedcentral.com/1471-2474/12/177 Aim 3: To evaluate the effect of tailored activity pacing on physical activity. Study Arms Usual Care Group The first session for both the tailored activity pacing and general activity pacing includes a brief presentation by the OT on the nature of Regardless of whether people are assigned to the tai- lored activity pacing or general activity pacing interven- tion groups, participants meet with an OT for 3 Murphy et al. BMC Musculoskeletal Disorders 2011, 12:177 http://www.biomedcentral.com/1471-2474/12/177 Murphy et al. BMC Musculoskeletal Disorders 2011, 12:177 http://www.biomedcentral.com/1471-2474/12/177 Murphy et al. BMC Musculoskeletal Disorders 2011, 12:177 http://www.biomedcentral.com/1471-2474/12/177 Page 6 of 10 OA and how physical demand and mood can affect the symptom experience. This is followed by group-specific instruction on activity pacing as a tool for symptom management which includes printed materials that are a take-home reference for the participant. In the tailored activity pacing intervention, the OT discusses how to incorporate activity pacing based on the individual’s pro- file of symptoms and activities determined from the baseline home monitoring period. In the general activity pacing intervention, the OT uses generic examples and does not mention the individual symptom experience from the baseline home monitoring period. By the end of the first OT session for both the tailored and general activity pacing groups, the participants develop a time- based activity plan with goals related to activity pacing. The second session focuses on individual progress with activity pacing, goal setting, and addressing barriers to using the recommended strategies. The third session focuses on reviewing activity pacing tenets, evaluating the use of time based activity pacing and planning for the future. After the 3 individual sessions have con- cluded, the OT makes 3 brief follow-up phone calls (1 call per week) to discuss activity pacing progress, symp- toms, and any barriers. series of didactic sessions with the principal investigator (PI) and study team members. The initial training will also involve role playing in pairs and an observer who will rate the mock treatment session for key concepts to ensure consistency across therapists. Any discrepancies in how the intervention is delivered across therapists will be discussed and a consensus will be reached about intervention delivery. To assess therapist adherence to the intervention pro- tocol, they are asked to log the actual treatment time per session with each individual. The therapist also has a checklist of items for each visit to facilitate consistent presentation of the protocol. Strategies for Ensuring Treatment Fidelity We will also assess participant factors related to treat- ment fidelity, i.e., how the treatment is received by the participant. This involves an assessment of individual factors such as comprehension of information, and the ability to use and perform skills associated with both the data collection and behavioral elements of the study. We have a standardized interactive learning module to teach the participant how to input symptoms on the wrist- worn accelerometer. Because the validity of the symp- tom information is central to our tailoring methods, all participants will undergo this learning module prior to their first session with the OT. In addition, both the tai- lored and general activity pacing interventions include strategies to ensure comprehension of the material. For example, participants are asked to reiterate main pacing concepts back to the therapist (e.g., importance of plan- ning, resting before symptoms get worse, etc.). Partici- pants also receive homework assignments that reinforce the lessons presented during the session and are asked to complete a daily activity logbook. Both of these homework items are used to assess personal compliance and overall protocol adherence. Each subsequent session involves a review of previously presented topics, a review of goals, and time for discussion of any barriers. Session 3 is focused on relapse-prevention, and con- tinues the theme of setting goals and problem-solving to minimize barriers that impede the use of activity pacing. Lastly, we conduct debriefing interviews with each Treatment fidelity is defined as “the procedures used to monitor and enhance the reliability and validity of beha- vioral interventions [49],” and is an essential component of this study. Per recommendations from the National Institutes of Health (NIH) behavior change consortium [49,50], several strategies are being used to ensure treat- ment fidelity in this study. These strategies target fidelity of treatment delivery (standardized modules for the intervention and interventionist training and monitor- ing) and treatment receipt (i.e., participant involvement in treatment). Study Arms Usual Care Group In addition, we are audio- taping sessions (as long as each individual consents to that process) and an independent rater listens to the tapes shortly after the sessions to determine if original criteria for intervention delivery in each treatment arm are being met. Any discrepancies in the audiotapes by therapists in a particular treatment arm will be resolved in a face-to-face meeting through consensus to reduce any variability in delivery. To prevent protocol ‘drift’, the PI and therapists in each arm have monthly tele- phone contact to discuss any issues and additional train- ing sessions among therapists occurs as needed. Study Arms Usual Care Group Figure 2 shows a flow chart of the study protocol. In brief, trained research personnel screen individuals for eligibility, administer performance-based assessments, and train participants in the use of the wrist-worn accel- erometer and accompanying logbook. Participants pro- vide written consent to participate in the study. They wear the accelerometer for a 7 day period and then This group participates in all outcome measures and monthly outcomes are tracked during the study period using a survey measure to determine if any new OA pharmacological or non-pharmacological treatments are undertaken (e.g., injection, physical therapy, medication change) to treat symptoms. Murphy et al. BMC Musculoskeletal Disorders 2011, 12:177 http://www.biomedcentral.com/1471-2474/12/177 Page 5 of 10 General and Tailored Activity Pacing Interventions Regardless of whether people are assigned to the tai- lored activity pacing or general activity pacing interven- tion groups, participants meet with an OT for 3 individual sessions (total treatment time: 3 hours over Weeks 4 - 9 of the study). The first session for both the tailored activity pacing and general activity pacing includes a brief presentation by the OT on the nature of Monthly Phone Calls Clinic Visit Functional Performance Outcome Assessment Clinic Visit Functional Performance Outcome Assessment Home Monitoring Period 7-day Activity & Symptom Monitoring Home Monitoring Period 7-day Activity & Symptom Monitoring Weeks 4-9 Week 10 Week 24 Session 1 with OT Session 2 with OT Session 3 with OT Telephone Interview: Level I Eligibility Clinic Visit Level II Eligibility & Baseline Functional Performance Home Monitoring Period 7-day Activity & Symptom Monitoring RANDOMIZATION General Weeks 1-3 Tailored Pacing Usual Care Figure 2 Study Flow Chart. Telephone Interview: Level I Eligibility Home Monitoring Period 7-day Activity & Symptom Monitoring RANDOMIZATION Usual Care General Clinic Visit Functional Performance Outcome Assessment Home Monitoring Period 7-day Activity & Symptom Monitoring General and Tailored Activity Pacing Interventions General and Tailored Activity Pacing Interventions Regardless of whether people are assigned to the tai- lored activity pacing or general activity pacing interven- tion groups, participants meet with an OT for 3 individual sessions (total treatment time: 3 hours over Weeks 4 - 9 of the study). Procedure for the Home Monitoring Periods The accelerometer is mounted on each participant’s non-dominant wrist. Participants are instructed on how to enter responses into the accelerometer using a stan- dardized interactive learning module that we have devel- oped and used in previous studies. Participants are given the opportunity to practice rating their symptoms and using the accelerometer’s input button to record the information. If, for some reason, participants fail the learning module (e.g., inability to rate symptom experi- ence on a numeric rating scale, inability to press the input button) they are excluded. Although we have not encountered exclusion for this reason in any of our past studies, this reporting is central to the tailoring portion of the intervention and therefore is an important criter- ion for study inclusion. Participants also become familiar with the logbook that accompanies the accelerometer and serves to cross-validate the items. The logbook is used as a back-up if there are missing data from the accelerometer. Momentary pain and fatigue severity are measured on 0 -10 numerical rating scales that will be directly input into the accelerometer five times a day (at rise time in the morning, 3 times during waking hours, and at bed- time). The accelerometer is worn for 7 days at baseline and at the outcome assessment periods. Pain is rated on a scale from 0 = no pain to 10 = pain as bad as you can imagine. Fatigue, defined as tiredness or weariness [28], is rated on a scale from 0 = no fatigue to 10 = fatigue as bad as you can imagine. Momentary ratings are used in the study for the sole purpose of generating tailored reports on individual symptom-activity relationships during the baseline week for the tailored activity pacing intervention. Other secondary outcomes and potential moderators include physical function, assessed by the 6 Minute Walk Test [57], a commonly used, validated measure of physical performance in adults; arthritis self-efficacy, the perceived ability to manage arthritis and pain and fati- gue symptoms, measured by the Arthritis Self Efficacy Scale [58]; and other variables such as knee extension strength (as measured by a hand-held dynamometer), and general health status variables (e.g., BMI, self- reported chronic conditions, depression using the Geria- tric Depression Scale [59], medication use). In addition, PA will be assessed by a wrist-worn accelerometer [Acti- watch-Score, Phillips Respironics-Mini Mitter Co, Bend Measures The primary outcome measures are fatigue and pain. Fatigue is assessed in two ways. First, fatigue is mea- sured by the Brief Fatigue Inventory (BFI) [51], a reliable and valid measure of fatigue in adults with cancer. There is no gold standard measure of fatigue for adults with OA; however, the BFI is of particular relevance because it assesses fatigue by asking about interference in daily life, which is our primary interest. This scale is usually scored by calculating an overall score based on an average of 9 of the 10 items. In samples with rheu- matic conditions, the BFI has recently been divided into two fatigue subscales (fatigue severity and fatigue inter- ference) [52]. Although no studies have examined the clinical significance of these subscales in OA or in other samples, we are expecting similar or greater improve- ments in fatigue severity and fatigue interference as seen in our pilot study (8% and 15% improvement respec- tively) [41]. Second, because there is currently no com- monly-used fatigue measure in OA across studies, an additional method of assessing fatigue from the Patient- Reported Outcomes Measurement (PROMIS) assess- ment tools will be used. PROMIS is a collection of care- fully constructed item banks that can be used to assess multiple domains of relevance to complex and chronic illnesses. Due to the methods used to construct these item banks, such measures are expected to be more pre- cise and less burdensome to patients to complete [53]. We are using the 8-item short form PROMIS fatigue scale [54]. Pain, the other primary outcome, is assessed by the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC). This is a validated, dis- ease-specific questionnaire for adults with knee or hip OA in which participants rate their currently experi- enced pain and physical disability [55]. Based on pre- vious studies, we will consider a 2 point decrease on the WOMAC pain scale to be clinically significant [56]. Fidelity of Treatment Delivery We have designed the study such that both intervention groups are equivalent in dose and administration of treatment (i.e., the number and length of sessions), including the manner in which participants are con- tacted from 10 weeks through 6 months. There are sev- eral procedures in place to ensure fidelity in how treatment is delivered. It is likely that there will be mul- tiple therapists per active intervention arm (i.e., tailored activity pacing and general activity pacing). We decided that extra therapists per group could reduce the possibi- lity of treatment effect due to therapist characteristics and ensure that we can deliver the intervention in the event that any interventionists drop out of the study. All therapists will undergo an initial training period which will involve structured education via a manual and a Murphy et al. BMC Musculoskeletal Disorders 2011, 12:177 http://www.biomedcentral.com/1471-2474/12/177 Page 7 of 10 Murphy et al. BMC Musculoskeletal Disorders 2011, 12:177 http://www.biomedcentral.com/1471-2474/12/177 participant to examine their experiences in the interven- tion including adherence with activity pacing in daily life at the end of their involvement in the study. OR] that measures changes in acceleration. Although it is worn on the wrist, it is highly associated with whole- body movement [60,61]. Changes in acceleration are recorded as activity counts and saved every 15 seconds. Higher activity counts reflect participation in higher intensity activities [62,63]. PA from the accelerometer will be aggregated in different ways. We will look at average activity counts and total activity counts that occur: 1) in the time between symptom reporting peri- ods, 2) over each day, and 3) over the entire home mon- itoring period similar to our previous studies [40,41,64]. Data Analysis The target sample size in this study is 156 participants. In our recruitment plans, we have factored in a 30% attrition rate to achieve this number. Although there are three arms to the study: tailored activity pacing, general activity pacing, and usual care group, the study was powered to find effects between the two active Murphy et al. BMC Musculoskeletal Disorders 2011, 12:177 http://www.biomedcentral.com/1471-2474/12/177 Murphy et al. BMC Musculoskeletal Disorders 2011, 12:177 http://www.biomedcentral.com/1471-2474/12/177 Page 8 of 10 Page 8 of 10 and Development Service (IRX000410A). We thank Kathy Welch for her statistical consultation on this project. intervention groups (tailored activity pacing and general activity pacing). Specifically, the power analysis was based on our pilot data in which we compared the tai- lored activity pacing intervention with the general activ- ity pacing intervention and detected large group differences using the effect size Cohen’s d [65] on BFI fatigue severity and interference (d = .79 and 1.1 respec- tively) at 10 week-follow-up. Based on a 2 group t-test at a significance level of p = .05, we will have greater than 90% power to detect an effect size of .74 or larger with 40 people in each of the two active intervention groups. Because most people with OA do not receive this type of intervention, it is important to compare the active intervention groups to a group receiving usual care. The study was powered to detect treatment effects between the active intervention groups; therefore we will also have sufficient power to detect differences between the active intervention groups and usual care group. and Development Service (IRX000410A). We thank Kathy Welch for her statistical consultation on this project. Author details 1 1Department of Physical Medicine and Rehabilitation, University of Michigan, Ann Arbor, MI, USA. 2VA Ann Arbor Healthcare System, GRECC, Ann Arbor, MI, USA. 3Department of Internal Medicine, Division of Geriatric Medicine, University of Michigan, Ann Arbor, MI, USA. 4Department of Anesthesiology, University of Michigan, Ann Arbor, MI, USA. References 1. Lawrence R, Felson D, Helmick C, Arnold L, Choi H, Deyo R, Gabriel S, Hirsch R, Hochberg M, Hunder G, Jordan JM, Katz JN, Kremers HM, Wolfe F: Estimates of the prevalence of arthritis and other rheumatic conditions in the United States. Part II. Arthritis Rheum 2008, 58:26-35. To examine the effectiveness of a tailored activity pacing intervention on fatigue, pain, and physical func- tion, we will use repeated measures analysis of variance (RM ANOVA) models to analyze the responses (pain, fatigue, and 6 minute walk test) at baseline, 10 weeks, and 6 months for the three treatment groups (tailored activity pacing, general activity pacing, and usual care group). This will allow us to estimate the effects of treatment group, time point and the treatment × time interaction. Similarly, we will use RM ANOVA models to determine if increased arthritis self-efficacy post intervention is related to improvements in symptom severity and function and to evaluate the effect of tai- lored activity pacing on physical activity. 1. Lawrence R, Felson D, Helmick C, Arnold L, Choi H, Deyo R, Gabriel S, Hirsch R, Hochberg M, Hunder G, Jordan JM, Katz JN, Kremers HM, Wolfe F: Estimates of the prevalence of arthritis and other rheumatic conditions in the United States. Part II. 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This study is examining the effect of a brief, individually-tailored, OT-led activity pacing intervention on pain and fatigue in adults with knee and hip OA. Given the positive effects of the tai- lored activity pacing approach compared to general activity pacing on fatigue in a small pilot study, we hypothesize that fatigue in particular will be improved with the tailored activity pacing intervention. We will also explore arthritis self-efficacy as a potential mechan- ism of change and the effects of pacing on PA at the 6 month follow-up period in an exploratory analysis. 7. Warsi A, Wang P, LaValley M, Avorn J, Solomon D: Self-management education programs in chronic disease: a systematic review and methodological critique of the literature. Arch Intern Med 2004, 164:1641-1649. 7. Warsi A, Wang P, LaValley M, Avorn J, Solomon D: Self-management education programs in chronic disease: a systematic review and methodological critique of the literature. Arch Intern Med 2004, 164:1641-1649. 8. Zhang W, Nuki G, Moskowitz RW, Abramson S, Altman RD, Arden NK, Bierma-Zeinstra S, Brandt KD, Croft P, Doherty M, Dougados M, Hochberg M, Hunter DJ, Kwoh K, Lohmander LS, Tugwell P: OARSI recommendations for the management of hip and knee osteoarthritis: Part III: changes in evidence following systematic cumulative update of research published through January 2009. Osteoarthritis Cartilage 2010, 18:476-499. 9. Kerns RD, Rosenberg R: Predicting responses to self-management treatments for chronic pain: application of the pain stages of change model. Pain 2000, 84:49-55. 9. Kerns RD, Rosenberg R: Predicting responses to self-management treatments for chronic pain: application of the pain stages of change model. Pain 2000, 84:49-55. 10. Marcus BH, Forsyth LH: Tailoring interventions to promote physically active lifestyles in women. Womens Health Issues 1998, 8:104-111. 10. Marcus BH, Forsyth LH: Tailoring interventions to promote physically active lifestyles in women. Womens Health Issues 1998, 8:104-111. 11. Shin Y, Hur H, Pender N, Jang H, Kim MS: Exercise self-efficacy, exercise benefits and barriers, and commitment to a plan for exercise among Korean women with osteoporosis and osteoarthritis. Int J Nurs Stud 2006, 43:3-10. 12. Theis K, Helmick C, Hootman J: Arthritis burden and impact are greater among U.S. women than men: intervention opportunities. J Womens Health 2007, 16:441-453. 10. Marcus BH, Forsyth LH: Tailoring interventions to promote physically active lifestyles in women. Womens Health Issues 1998, 8:104-111. Competing interests Th h d l h The authors declare that they have no competing interests. The authors declare that they have no competing interests. The authors declare that they have no competing interests. Received: 14 July 2011 Accepted: 2 August 2011 Published: 2 August 2011 Received: 14 July 2011 Accepted: 2 August 2011 Published: 2 August 2011 Murphy et al. BMC Musculoskeletal Disorders 2011, 12:177 http://www.biomedcentral.com/1471-2474/12/177 13. Damush T, Wu J, Bair M, Sutherland J, Kroenke K: Self-management practices among primary care patients with musculoskeletal pain and depression. J Behav Med 2008, 31:301-307. 36. Affleck G, Tennen H, Keefe FJ, Lefebvre JC, Kashikar-Zuk S, Wright K, Starr K, Caldwell DS: Everyday life with osteoarthritis or rheumatoid arthritis: independent effects of disease and gender on daily pain, mood, and coping. Pain 1999, 83:601-609. 14. Turk DC, Rudy TE, Kubinski JA, Zaki HS, Greco CM: Dysfunctional patients with temporomandibular disorders: evaluating the efficacy of a tailored treatment protocol. J Consult Clin Psychol 1996, 64:139-146. 37. Hutchings A, Calloway M, Choy E, Hooper M, Hunter DJ, Jordan JM, Zhang Y, Baser O, Long S, Palmer L: The longitudinal examination of arthritis pain (LEAP) study: relationships between weekly fluctuations in patient-related joint pain and other health outcomes. J Rheumatol 2007, 34:2291-2300. 15. Strecher V, Wang C, Derry H, Wildenhaus K, Johnson C: Tailored interventions for multiple risk behaviors. Health Educ Res 2002, 17:619-626. 16. Prochaska JO, DiClemente CC, Velicer WF, Rossi JS: Standardized, individualized, interactive, and personalized self-help programs for smoking cessation. Health Psychol 1993, 12:399-405. 38. Keefe F, Affleck G, France C, Emery C, Waters S, Caldwell D, Stainbrook D, Hackshaw K, Fox L, Wilson K: Gender differences in pain, coping, and mood in individuals having osteoarthritic knee pain: a within-day analysis. Pain 2004, 110:571-577. 17. Greaney M, Riebe D, Garber C, Rossi J, Lees F, Burbank P, Nigg C, Ferrone C Clark P: Long-term effects of a stage-based intervention for changing exercise intentions and behavior in older adults. Gerontologist 2008, 48:358-367. 39. Zautra AJ, Fasman R, Parish BP, Davis MC: Daily fatigue in women with osteoarthritis, rheumatoid arthritis, and fibromyalgia. Pain 2007, 128:128-135. 18. van Sluijs EMF, van Poppel MNM, van Mechelen W: Stage-based lifestyle interventions in primary care: are they effective? Am J Prev Med 2004, 26:330-343. 40. Murphy SL, Smith DM, Clauw DJ, Alexander NB: The impact of momentary pain and fatigue on physical activity in women with osteoarthritis. Arthritis Rheumatism 2008, 59:849-856. 41. Murphy S, Lyden A, Smith D, Dong Q, Koliba J: Effects of a tailored activity pacing intervention on pain and fatigue for older adults with osteoarthritis. Am J Occup Ther 2010, 64:869-876. 19. Lorig KR, Mazonson PD, Holman HR: Evidence suggesting that health education for self-management in patients with chronic arthritis has sustained health benefits while reducing health care costs. Murphy et al. BMC Musculoskeletal Disorders 2011, 12:177 http://www.biomedcentral.com/1471-2474/12/177 Arthritis Rheum 1993, 36:439-446. 42. Gill J, Brown C: A structured review of the evidence for pacing as a chronic pain intervention. Eur J Pain 2009, 13:214-216. 20. Hughes SL, Seymour RB, Campbell R, Pollak N, Huber G, Sharma L: Impact of the fit and strong intervention on older adults with osteoarthritis. Gerontologist 2004, 44:217-228. 43. Beissner K, Henderson C, Papaleontiou M, Olkhovskaya Y, Wigglesworth J, Reid MC: Physical therapists’ use of cognitive-behavioral therapy for older adults with chronic pain: a nationwide survey. Phys Ther 2009, 89:456-469. 21. Bandura A: Self-Efficacy: The Exercise of Control.Edited by: Freeman WH 1997. 22. Bandura A: Health promotion by social cognitive means. Health Educ Behav 2004, 31:143-164. 44. Murphy SL, Clauw DJ: Activity pacing: what are we measuring and how does it relate to treatment? Pain 2010, 149:582-583. 45. Golightly YM, Dominick KL: Racial variations in self-reported osteoarthritis symptom severity among veterans. Aging Clin Exp Res 2005, 17:264-269. 23. Keefe FJ, Caldwell DS, Williams DA, Gil KM, Mitchell D, Robertson C, Martinez S, Nunley J, Beckham JC, Crisson JE, Helms M: Pain coping skills training in the management of osteoarthritic knee pain: a comparative study. Behav Ther 1990, 21:49-62. 46. Satish S, Postigo LG, Ray LA, Goodwin JS: Chronic rheumatologic symptoms in a tri-ethnic sample of men and women aged 75 and older. J Gerontol A Biol Sci Med Sci 2001, 56:M471-M476. 24. Keefe FJ, Caldwell DS, Williams DA, Gil KM, Mitchell D, Robertson C, Martinez S, Nunley J, Beckham JC, Helms M: Pain coping skills training in the management of osteoarthritic knee pain-II: follow-up results. Behav Ther 1990, 21:435-447. 47. Skinner J, Weinstein J, Sporer S, Wennberg J: Racial, ethnic, and geographic disparities in rates of knee arthroplasty among Medicare patients. N Engl J Med 2003, 349:1350-1359. 25. Hill CL, Parsons J, Taylor A, Leach G: Health related quality of life in a population sample with arthritis. J Rheumatol 1999, 26:2029-2035. 48. Ibrahim SA, Siminoff LA, Burant CJ, Kwoh CK: Variation in perceptions of treatment and self-care practices in elderly with osteoarthritis: a comparison between African American and white patients. Arthritis Rheum 2001, 45:340-345. 26. Boutron I, Rannou F, Jardinaud-Lopez M, Meric G, Revel M, Poiraudeau S: Disability and quality of life of patients with knee or hip osteoarthritis in the primary care setting and factors associated with general practitioners’ indication for prosthetic replacement within 1 year. Osteoarthritis Cartilage 2008, 16:1024-1031. 49. Murphy et al. BMC Musculoskeletal Disorders 2011, 12:177 http://www.biomedcentral.com/1471-2474/12/177 Bellg A, Borrelli B, Resnick B, Hecht J, Minicucci D, Ory M, Ogedegbe G, Orwig D, Ernst D, Czajkowski S: Enhancing treatment fidelity in health behavior change studies: best practices and recommendations from the NIH Behavior Change Consortium. Health Psychol 2004, 23:443-451. 27. Grotle M, Hagen K, Natvig B, Dahl F, Kvien T: Prevalence and burden of osteoarthritis: results from a population survey in Norway. J Rheumatol 2008, 35:677-684. 50. Borrelli B: The assessment, monitoring, and enhancement of treatment fidelity In public health clinical trials. J Public Health Dent 2011, 71: S52-S63. 28. Wolfe F, Hawley DJ, Wilson K: The prevalence and meaning of fatigue in rheumatic disease. J Rheumatol 1996, 23:1407-1417. 51. Mendoza TR, Wang XS, Cleeland CS, Morrissey M, Johnson BA, Wendt JK, Huber SL: The rapid assessment of fatigue severity in cancer patients: use of the Brief Fatigue Inventory. Cancer 1999, 85:1186-1196. 29. Power JD, Badley E, French M, Wall A, Hawker G: Fatigue in osteoarthritis: a qualitative study. BMC Musculoskelet Disord 2008, 9:63. 30. Sale JEM, Gignac M, Hawker G: The relationship between disease symptoms, life events, coping and treatment, and depression among older adults with osteoarthritis. J Rheumatol 2008, 35:335-342. 52. Wolfe F: Fatigue assessments in rheumatoid arthritis: comparative performance of visual analog scales and longer fatigue questionnaires in 7760 patients. J Rheumatol 2004, 31:1896-1902. 53. Cella D, Riley W, Stone A, Rothrock N, Reeve B, Yount S, Amtmann D, Bode R, Buysse D, Choi S, Cook K, Devellis R, DeWalt D, Fries JF, Gershon R, Hahn EA, Lai JS, Pilkonis P, Revicki D, Rose M, Weinfurt K, Hays R, PROMIS Cooperative Group: The Patient-Reported Outcomes Measurement Information System (PROMIS) developed and tested its first wave of adult self-reported health outcome item banks: 2005-2008. J Clin Epidemiol 2010, 63:1179-1194. 31. Wolfe F: Determinants of WOMAC function, pain and stiffness scores: evidence for the role of low back pain, symptom counts, fatigue and depression in osteoarthritis, rheumatoid arthritis and fibromyalgia. Rheumatology 1999, 38:355-361. 32. Fishbain D, Cole B, Cutler RB, Lewis J, Rosomoff HL, Fosomoff RS: Is pain fatiguing? A structured evidence-based review. Pain Med 2003, 4:51-62. 33. Gignac M, Davis A, Hawker G, Wright J, Mahomed N, Fortin P: “What do you expect? You’re just getting older": A comparison of perceived osteoarthritis-related and aging-related health experiences in middle- and older-age adults. Arthritis Rheum 2006, 55:905-912. 54. The Assessment Center. [http://www.assessmentcenter.net]. 55. Discussion 12. Theis K, Helmick C, Hootman J: Arthritis burden and impact are greater among U.S. women than men: intervention opportunities. J Womens Health 2007, 16:441-453. Acknowledgements and Funding All i i id i i f Acknowledgements and Funding All participants provide written informed consent to participate in this study.. This study was funded through a grant from the VA Rehabilitation Research Page 9 of 10 Page 9 of 10 Murphy et al. BMC Musculoskeletal Disorders 2011, 12:177 http://www.biomedcentral.com/1471-2474/12/177 Murphy et al. BMC Musculoskeletal Disorders 2011, 12:177 http://www.biomedcentral.com/1471-2474/12/177 Murphy et al. BMC Musculoskeletal Disorders 2011, 12:177 http://www.biomedcentral.com/1471-2474/12/177 57. 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Int J Obes Relat Metab Disord 1999, 23:S45-S49. 62. Swartz AM, Strath SJ, Bassett DR, O’Brien WL, King GA, Ainsworth BE: Estimation of energy expenditure using CSA accelerometers at hip and wrist sites. Med Sci Sports Exerc 2000, 32:S450-S456. 63. Murphy SL: Review of physical activity measurement using accelerometers in older adults: considerations for research design and conduct. Prev Med 2009, 48:108-114. 64. Murphy SL, Strasburg DM, Lyden AK, Smith DM, Koliba JF, Dadabhoy DP, Wallis SM: Effects of activity strategy training on pain and physical activity in older adults with knee or hip osteoarthritis: a pilot study. Arthritis Rheum 2008, 59:1480-1487. 65. Cohen J: Statistical Analyses for the Behavioral Sciences. Hillsdale: Lawrence Erlbaum Associates;, 2 1988. 65. Cohen J: Statistical Analyses for the Behavioral Sciences. Hillsdale: Lawrence Erlbaum Associates;, 2 1988. 66. Callahan C, Unverzagt F, Hui S, Perkins A, Hendrie H: Six-item screener to identify cognitive impairment among potential subjects for clinical research. Med Care 2002, 40:771-781. 66. Callahan C, Unverzagt F, Hui S, Perkins A, Hendrie H: Six-item screener to identify cognitive impairment among potential subjects for clinical research. Med Care 2002, 40:771-781. Murphy et al. BMC Musculoskeletal Disorders 2011, 12:177 http://www.biomedcentral.com/1471-2474/12/177 Bellamy N, Buchanan WW, Goldsmith CH, Campbell J, Stitt LW: Validation study of WOMAC: a health status instrument for measuring clinically important patient relevant outcomes to antirheumatic drug therapy in patients with osteoarthritis of the hip or knee. J Rheumatol 1988, 15:1833-1840. 34. Allen K: The value of measuring variability in osteoarthritis pain. J Rheumatol 2007, 34:2132-2133. 35. 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Page 10 of 10 Page 10 of 10 Pre-publication history The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2474/12/177/prepub The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2474/12/177/prepub The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2474/12/177/prepub The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2474/12/177/prepub doi:10.1186/1471-2474-12-177 Cite this article as: Murphy et al.: Activity pacing for osteoarthritis symptom management: study design and methodology of a randomized trial testing a tailored clinical approach using accelerometers for veterans and non-veterans. BMC Musculoskeletal Disorders 2011 12:177. 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
https://openalex.org/W54832784	https://europepmc.org/articles/pmc4328319?pdf=render	English	null	The prospective study of cardiac MRI in the diagnosis and treatment of cardiac sarcoidosis associated with atrioventricular block	Journal of cardiovascular magnetic resonance	2,015	cc-by	793	© 2015 Kunimoto et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Kunimoto et al. Journal of Cardiovascular Magnetic Resonance 2015, 17(Suppl 1):P373 Kunimoto et al. Journal of Cardiovascular Magnetic Resonance 2015, 17(Suppl 1):P373 http://www.jcmr-online.com/content/17/S1/P373 Open Access Open Access Background Sixty-six AVB cases of all fourteen hundred eighty-seven CMR cases underwent CMR for five years. On the other hand, in one hundred fifty-four cases, a pacemaker was newly implanted for AVB and one hundred twenty-five were under 85 years old. Among these cases, CMR scan were performed in fifty (40% of one hundred fifty-five) cases. Thirteen cases (20% of sixty-six) indicate the pat- tern of CS with late Gd enhancement (LGE). Nine of thirteen cases were diagnosed as CS with criteria and FDG-PET scan. Five cases received steroid therapy and three cases showed improvement of AVB. Especially, pacemaker implantation was able to avoid in one case. Unfortunately one case did not receive steroid therapy at early phase and developed congestive heart failure. In addition, FDG-PET is more useful than Ga scintigram for assessment activity of CS. Sarcoidosis is systemic granulomatous disease of unknown etiology. Most patients present with pulmon- ary involvement, but association of cardiac sarcoidosis (CS) is critical factor determining prognosis. Recently, cardiac magnetic resonance imaging (CMR) and 18F- fluoro-2-deoxyglucose positron emission tomography (18F-FDG PET) can detect cardiac lesions in asympto- matic sarcoidosis patients. On the other hand, Conduc- tion abnormalities and ventricular tachycardia are the most common arrhythmias with CS. With the experi- ence of CS pointed out accidentally in PET medical examination at earlier stage got us to recognize the use- fulness of CMR, we conducted a study to figure out real-world prevalence of CS in advanced atrioventricular block (AVB) cases and to assess the usability of “aggres- sive” diagnosis of CS by cardiac MRI. The prospective study of cardiac MRI in the diagnosis and treatment of cardiac sarcoidosis associated with atrioventricular block The prospective study of cardiac MRI in the diagnosis and treatment of cardiac sarcoidosis associated with atrioventricular block Satoshi Kunimoto*, Kensuke Yamamoto, Toshiko Nakai, Yasuo Okumura, Mahoto Kato, Tadateru Takayama, Takafumi Hiro, Ichiro Watanabe, Atsushi Hirayama From 18th Annual SCMR Scientific Sessions Nice, France. 4-7 February 2015 Methods Between June 2009 and May 2013, one thousand four hundred eighty-seven patients (nine hundred fifty-two males) underwent CMR. We underwent CMR to advanced AVB cases that have been the adaptation of pacemaker implantation of five years if at all possible. CMR was performed by using 1.5-T magnet MR sys- tems (Intera Achieva, Philips Medical Systems) with 5- element cardiac coils. In cases with temporary pacing, CMR scan were performed just behind extraction tem- porary pacing lead with confirmation of spontaneous beat, which followed by pacemaker implantation. We excluded cases over 85 years old or in severe circulatory failure with AVB. CMR in advanced AVB has been accompanied with dif- ficulty because of the bradycardia. Even if only few cases were found as CS in all cases newly pacemaker implanted, its impact on the therapeutic strategy is enormous. In diagnosis of CS, sign of positive LGE and positive PET have important implications. CMR is useful as screening tool for CS and should be performed as far as possible in advanced AVB cases. Division of Cardiology, Nihon University School of Medicine, Tokyo, Japan Funding Division of Cardiology, Nihon University School of Medicine, Tokyo, Japan Page 2 of 2 Kunimoto et al. Journal of Cardiovascular Magnetic Resonance 2015, 17(Suppl 1):P373 Kunimoto et al. Journal of Cardiovascular Magnetic Resonance 2015, 17(Suppl 1):P373 http://www.jcmr-online.com/content/17/S1/P373 Figure 1 The effect of PSL administration to cardiac sarcoidosis, which prevent pacemaker implantation. Figure 1 The effect of PSL administration to cardiac sarcoidosis, which prevent pacemaker implantation. Figure 1 The effect of PSL administration to cardiac sarcoidosis, which prevent pacemaker implantation. Published: 3 February 2015 doi:10.1186/1532-429X-17-S1-P373 Cite this article as: Kunimoto et al.: The prospective study of cardiac MRI in the diagnosis and treatment of cardiac sarcoidosis associated with atrioventricular block. Journal of Cardiovascular Magnetic Resonance 2015 17(Suppl 1):P373. 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:
https://openalex.org/W4361005938	https://www.mdpi.com/2075-4698/13/4/80/pdf?version=1679816106	English	null	Keeping the Knives Sharp: Socioeconomic Innovation in the Artisan Sector of Butchery in Italy	Societies	2,023	cc-by	10,747	Michele Filippo Fontefrancesco 1 Michele Filippo Fontefrancesco 1 1 University of Gastronomic Sciences, 12042 Pollenzo, Italy 2 Department of Anthropology, Durham University, DH1 3DE Durham, UK * Correspondence: m.fontefrancesco@unisg.it 1 University of Gastronomic Sciences, 12042 Pollenzo, Italy 2 Department of Anthropology, Durham University, DH1 3DE Durham, UK * Correspondence: m.fontefrancesco@unisg.it Abstract: This paper investigates the socioeconomic and cultural transformation in food artisan entrepreneurship due to the complexiﬁcation of the food sector and ongoing globalisation through a case study conducted among the artisan butchers of Genoa, Italy. The butcher’s trade has enjoyed centrality and social and cultural promotion that made butcher shops key places in the urban foodscape. However, this centrality is challenged by both new consumption trends and the imposition of large-scale organised distribution as the fulcrum of mass food trade. These changes raise the question about the future of the profession and its knowledge. This paper addresses this question by investigating the structure of the butcher’s business and the practices involved in knowledge creation and transmission, exploring the factor of change and its effects on the butcher’s profession, as well as the very foundational aspects of the artisanship. The research suggests that artisanship should be read as a form of entrepreneurship capable of placing and moving the craftsman within the global social hierarchy of a community. Therefore, the preservation of artisanship and its sociocultural complexity cannot be limited to the mere preservation of gastronomic forms and techniques. Keywords: artisanship; food sector; meat sector; retail sector; Italy; ethnography Article Keeping the Knives Sharp: Socioeconomic Innovation in the Artisan Sector of Butchery in Italy Michele Filippo Fontefrancesco 1,2,* and Andrea Costa 1 societies societies In the past 20 years, the retail food sector has undergone substantial changes [1]. The global increased mobility of goods and people has led to an expansion of the array of foods available in the markets thanks to the introduction of new, foreign products sold alongside more traditional ones, as well as the invention of new foods combining different culinary traditions [2–4]. Moreover, while the 20th century marked the advent of supermarkets [5], recent decades have seen the success of new forms of online commerce [6]. All these new forms of retailing have furthered and strengthened the food global value chains, thereby transforming them [7–10]. On the one hand, they favoured a global standardisation of agrifood production and food consumption patterns [5]. On the other hand, they promoted industrial mass production, strengthening the sociocultural prestige of these goods [11]. Finally, both the birth of shopping malls in the outskirts of the urban centre and the success of e-groceries moved food purchase away from the traditional locations of the artisan shops in the city centres and showed the fragility of the traditional retail system based on small business and food artisan professions, such as bakers, greengrocers, and butchers [12,13]. Hence, the last 20 years were a period of strong transformation in food artisanship. Citation: Fontefrancesco, M.F.; Costa, A. Keeping the Knives Sharp: Socioeconomic Innovation in the Artisan Sector of Butchery in Italy. Societies 2023, 13, 80. https:// doi.org/10.3390/soc13040080 Citation: Fontefrancesco, M.F.; Costa, A. Keeping the Knives Sharp: Socioeconomic Innovation in the Artisan Sector of Butchery in Italy. Societies 2023, 13, 80. https:// doi.org/10.3390/soc13040080 Academic Editor: Gregor Wolbring Received: 8 March 2023 Revised: 23 March 2023 Accepted: 24 March 2023 Published: 26 March 2023 Received: 8 March 2023 Revised: 23 March 2023 Accepted: 24 March 2023 Published: 26 March 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/). y p g p Artisanship is a widely debated concept. It is generally opposed to mechanical re- production based on an ideal contraposition between a non-alienating form of production (crafts-based) and an alienated one (machine-based), and it has often been associated with concepts such as design, manual work, small production, originality, oral lore, and traditions [14]. Since the late 20th century, this concept has been increasingly used in the study of the food sector [15] to identify those small-scale productions mostly based on manual work [16–18]. Sennet indicates artisanship as “a special human condition of https://www.mdpi.com/journal/societies Societies 2023, 13, 80. https://doi.org/10.3390/soc13040080 Societies 2023, 13, 80 2 of 13 2 of 13 being engaged” [19] that “suggests ways of using tools, organising bodily movements, thinking about materials that remain alternative, viable proposals on how to conduct life with skill” [19] against a model of life and production based on modern detachment of body and hand. This deﬁnition resonates with Ingold’s indication of artisanship as a complete expression of all forms of knowledge underpinning human making [20], or Marchand’s [21] reading of artisanship as an expression of embodied knowledge that underpins material manipulation. In this respect, the concept of artisanship refers also to food professions, speciﬁcally those that require the manipulation of raw materials to prepare and create new food products. While chefs achieved unprecedented popularity in recent decades [22,23], other professions did not enjoy the same success, being in the margin of a global hierarchy of value [24]. One of these is the profession of butchers. Butchery is the process of cutting and preparing animal carcasses for consumption. This practice enjoyed particular prestige given the strong symbolic value of meat as a food marker of social status, prestige, and prowess [25,26]. The butcher’s trade has enjoyed centrality and social and cultural promotion that made butcher shops key places in the urban foodscape. However, this centrality is challenged by both new consumption trends critical of meat consumption [27], and the imposition of large-scale organised distribution as the fulcrum of mass food trade [28]. These changes, which are reducing the proﬁtability and possibility of continuing the business as it used to be, raise a question about the future of the profession and its knowledge—a question that is shared by other artisan food professions. This paper addresses this question by investigating the sociocultural transformation that affects business in Italy through a case study [29] focused on the city of Genoa. Speciﬁ- cally, it investigates the structure of the butcher’s business and the practices involved in knowledge creation and transmission, exploring the factor of change and its effects on the profession, as well as the very foundational aspects of the artisanship. The article presents the methodology and the activities conducted during the research, then presents the speciﬁcities of the business in Genoa and its recent development. In so doing, it highlights the elements needed to further explore the characteristics of food artisan knowledge and its reaction to social and economic change. 2. Materials and Methods Although in the past decades, the slaughterhouses closed down, new ones were built in more external areas of Genoa (e.g., Piazzale Bigny in the northern periphery of the city), and although the number of butcher shops has substantially reduced to a handful, the Figure 1. Localisation of the field (map based on OpenStreetMap cartography). Figure 1. Localisation of the ﬁeld (map based on OpenStreetMap cartography). area is still associated with meat retailing by the locals. Figure 1. Localisation of the field (map based on OpenStreetMap cartography). gure 1. Localisation of the field (map based on OpenStreetMap cartograph Figure 1. Localisation of the ﬁeld (map based on OpenStreetMap cartography). Figure 1. Localisation of the field (map based on OpenStreetMap cartography). The research moved from this area and expanded to the city (Figure 2). Based on methodologies of street ethnography [41] and business ethnography [42], the research used on butcher shops in the city, exploring their operations and the dynamics that elop there. Following Healy et al. [43], the research conducted its analysis within pace of shops. Extensive in-depth interviews were conducted with the butchers runn he businesses (Carey, 2013; Guala, 2003). Based on the life story approach [44], the in iews explored the modern history of butchery in the city, how the profession develo d h h i li l The research moved from this area and expanded to the city (Figure 2). Based on the methodologies of street ethnography [41] and business ethnography [42], the research focused on butcher shops in the city, exploring their operations and the dynamics that develop there. Following Healy et al. [43], the research conducted its analysis within the space of shops. Extensive in-depth interviews were conducted with the butchers running the businesses (Carey, 2013; Guala, 2003). Based on the life story approach [44], the interviews explored the modern history of butchery in the city, how the profession developed, and the changes in clientele. The research moved from this area and expanded to the city (Figure 2). Based on the methodologies of street ethnography [41] and business ethnography [42], the research fo- cused on butcher shops in the city, exploring their operations and the dynamics that de- velop there. Following Healy et al. [43], the research conducted its analysis within the space of shops. Extensive in-depth interviews were conducted with the butchers running the businesses (Carey, 2013; Guala, 2003). 2. Materials and Methods The article is the result of a case study [29] conducted in Genoa between March and December 2022. Italy is one of the countries most renowned for its gastronomic tradition, a speciﬁcity that is the result of the strong regionalism and geographical diversity of the peninsula [30]. In Italian gastronomy, meat has played a crucial role as one of the key symbols of prosperity and afﬂuence since the Middle Ages [31,32]. With it, butchers have played a key role in the food profession, a cultural prominence that reached its peak in the 1980s [33]. In the face of this success, the past decades have been marred by a decline caused by the transformation of the retail sector due to the proliferation of supermarkets, epidemiological occurrences, and a new attitude of consumers toward the consumption of meat [34]. The dynamics that affect Genoa are framed within this context. Genoa is one of the 10 largest cities in Italy, with over 500,000 inhabitants. Since the Middle Ages, the economy of the city has been based on its port, one of the most important in the country [35]. During the 20th century, it met a fast industrialisation that made Genoa one of the three main poles of the so-call Economic Boom of the 1960–1970s [36]. Despite the de-industrialisation of the past 30 years, Genoa still represents one of the key economic centres in northwestern Italy and a thriving place of commerce. As a result of its prosperity, Genoa has been a centre of important gastronomic cre- ativity, capable of developing a distinct cuisine [37]. Unlike other port cities of which gastronomy focuses on the primary use of ﬁsh resources, Genoese cuisine is characterised by the substantial presence of meat dishes [38]. This refers to the use of red meat, mainly beef, as well as white meat, poultry, and small animals [39]. In this respect, butcher shops played a crucial role in the life of the city, as well as in the expression of its gastronomy. Societies 2023, 13, 80 3 of 13 cher sh astrono 3 of 13 cher sh astrono They were concentrated in the area of Macelli di Soziglia (tr. Soziglia slaughterhouse), where the city’s main slaughterhouse was located (Figure 1). The Macelli are in the middle of the historic centre, in the area commonly called the “Vicoli” (tr. the alleys, due to the narrowness and tortuosity of the streets). 2. Materials and Methods Beginning in the 13th century and for over 500 years, this area, and speciﬁcally nearby Piazza Soziglia (tr. Soziglia square) and via Macelli di Soziglia (tr. Soziglia slaughter houses alley), has housed the city’s slaughter houses and butcher shops as well as the stables where cows, pigs, and other animals were kept [40]. Although in the past decades, the slaughterhouses closed down, new ones were built in more external areas of Genoa (e.g., Piazzale Bigny in the northern periphery of the city), and although the number of butcher shops has substantially reduced to a handful, the area is still associated with meat retailing by the locals. here the city’s main slaughterhouse was located (Figure 1). The Macelli are in the mi the historic centre, in the area commonly called the “Vicoli” (tr. the alleys, due to arrowness and tortuosity of the streets). Beginning in the 13th century and for over ars, this area, and specifically nearby Piazza Soziglia (tr. Soziglia square) and via Ma Soziglia (tr. Soziglia slaughter houses alley), has housed the city’s slaughter houses utcher shops as well as the stables where cows, pigs, and other animals were kept lthough in the past decades, the slaughterhouses closed down, new ones were bui ore external areas of Genoa (e.g., Piazzale Bigny in the northern periphery of the c nd although the number of butcher shops has substantially reduced to a handful ea is still associated with meat retailing by the locals. , p y, [ ] p , p played a crucial role in the life of the city, as well as in the expression of its gastronomy. They were concentrated in the area of Macelli di Soziglia (tr. Soziglia slaughterhouse), where the city’s main slaughterhouse was located (Figure 1). The Macelli are in the middle of the historic centre, in the area commonly called the “Vicoli” (tr. the alleys, due to the narrowness and tortuosity of the streets). Beginning in the 13th century and for over 500 years, this area, and specifically nearby Piazza Soziglia (tr. Soziglia square) and via Macelli di Soziglia (tr. Soziglia slaughter houses alley), has housed the city’s slaughter houses and butcher shops as well as the stables where cows, pigs, and other animals were kept [40]. 2. Materials and Methods While the older informants (over 65) completed only their junior high school education (9), all the other informants completed a high school education (3). None have completed university studies. The training was completed through apprenticeship from older artisans (only one informant completed formal training in a school). The trainers were often kin-related (6), and this pattern is not signiﬁcantly related to the age of the informants. All the informants are men, since no woman butcher owning and running a business was identiﬁed in Genoa. The average age is over 50 years old. The sample includes two informants under 35 (in their late 20s), six informants between 35 and 65 years old, and ﬁve over 65. While the interviews show that until the 1990s it was not uncommon for people in their 20s to start their business (three informants shared this experience), the event is rare at the present, as portrayed by the last national census [46]. Field research was conducted by AC, under the supervision of MFF. A preliminary analysis of the data was produced by AC. Data were further analysed by MFF, who developed them in the present form of the paper. 3. Results 3.1. An Artisan Profession Sennet [19] identiﬁes the development of an artisan profession in a process of acquiring and testing creative skills and competence that leads the individual from apprenticeship to journeymanship, and from journeymanship to mastership. This is a path of social legitimation marked by speciﬁc rites of passage [47], but it is also and above all a process through which the individual embodies and practices knowledge [21]. This process is followed by the butchers in Genoa. Butchery is a craft that is acquired by direct experience under the guidance of an older Societies 2023, 13, 80 4 of 13 by respondents as relevant for research for their professional experience and knowledge. In this sense, the research has also extended beyond the original scope of research. Table 1. Summary of the informants involved in the research. Identiﬁcation Age Trainer Education Youth Entrepreneurship (under 25) Family Business Int. 1 20–34 Kin-related (father) Junior high school Yes Int. 2 Over 65 Non-kin-related Junior high school Int. 3 RC Over 65 Kin-related (father) Junior high school Int. 4 35–65 Kin-related (father) Junior high school Yes Yes Int. 5 ADC 35–65 Non-kin-related Junior high school Yes Int. 6 LS 35–65 Non-kin-related Junior high school Int. 2. Materials and Methods 7 EA Over 65 Non-kin-related Junior high school Int. 8 MT2 35–65 Non-kin-related High school Int. 9 IS 35–65 Kin-related (father) Junior high school Yes Int. 10 CF 35–65 Non-kin-related High school Int. 11 DP Under 35 Non-kin-related High school Int. 12 RN Over 65 Kin-related (great-grandfather) Junior high school Yes Int. 13 VB 35–65 Kin-related (father)School of Italian Butchers training (1984–1986) Junior high school Yes Yes Table 1. Summary of the informants involved in the research. The proﬁle of the informants corresponds to what is commonly recognised as a professional craftsman who has reached the necessary professional skills to manage the main stages of production [19]. While the older informants (over 65) completed only their junior high school education (9), all the other informants completed a high school education (3). None have completed university studies. The training was completed through apprenticeship from older artisans (only one informant completed formal training in a school). The trainers were often kin-related (6), and this pattern is not signiﬁcantly related to the age of the informants. All the informants are men, since no woman butcher owning and running a business was identiﬁed in Genoa. The average age is over 50 years old. The sample includes two informants under 35 (in their late 20s), six informants between 35 and 65 years old, and ﬁve over 65. While the interviews show that until the 1990s it was not uncommon for people in their 20s to start their business (three informants shared this experience), the event is rare at the present, as portrayed by the last national census [46]. Field research was conducted by AC, under the supervision of MFF. A preliminary analysis of the data was produced by AC. Data were further analysed by MFF, who developed them in the present form of the paper. 2. Materials and Methods Based on the life story approach [44], the inter- views explored the modern history of butchery in the city, how the profession developed, and the changes in clientele. g Figure 2. Localisation of the respondents (flagged in green) based on Google Map cartography. Figure 2. Localisation of the respondents (ﬂagged in green) based on Google Map cartography. Figure 2. Localisation of the respondents (flagged in green) based on Google Map cartography. Figure 2. Localisation of the respondents (ﬂagged in green) based on Google Map cartography. gure 2. Localisation of the respondents (flagged in green) based on Google Map cartography The sample includes 13 professional butchers (Table 1) who own and manage at l The sample includes 13 professional butchers (Table 1) who own and manage at least one butcher shop. The sample was built starting from the butchers now active in the area The sample includes 13 professional butchers (Table 1) who own and manage at least one butcher shop. The sample was built starting from the butchers now active in the area of Macelli di Sozziglia and identifying others through snowball sampling [45], recognised cieties 2023, 13, 80 4 of 13 by respondents as relevant for research for their professional experience and knowledge. In this sense, the research has also extended beyond the original scope of research. Table 1. Summary of the informants involved in the research. Identiﬁcation Age Trainer Education Youth Entrepreneurship (under 25) Family Business Int. 1 20–34 Kin-related (father) Junior high school Yes Int. 2 Over 65 Non-kin-related Junior high school Int. 3 RC Over 65 Kin-related (father) Junior high school Int. 4 35–65 Kin-related (father) Junior high school Yes Yes Int. 5 ADC 35–65 Non-kin-related Junior high school Yes Int. 6 LS 35–65 Non-kin-related Junior high school Int. 7 EA Over 65 Non-kin-related Junior high school Int. 8 MT2 35–65 Non-kin-related High school Int. 9 IS 35–65 Kin-related (father) Junior high school Yes Int. 10 CF 35–65 Non-kin-related High school Int. 11 DP Under 35 Non-kin-related High school Int. 12 RN Over 65 Kin-related (great-grandfather) Junior high school Yes Int. 13 VB 35–65 Kin-related (father)School of Italian Butchers training (1984–1986) Junior high school Yes Yes The proﬁle of the informants corresponds to what is commonly recognised as a professional craftsman who has reached the necessary professional skills to manage the main stages of production [19]. 3.1. An Artisan Profession My friend understood it and made me do prontocuoci as a ﬁrst assignment. That’s how I learned”. p g Learning takes place in the workshop, experiencing the everyday reality of the shop (only Int. 13 started his activity with participation in a vocational training course). Learning passes from the relationship with the elder butcher who becomes master and “almost father” (Int. 1) for the apprentice. In this sense, it is an all-encompassing experience which builds a strong personal and professional identity. Moreover, among those for whom butchery is a family occupation, the trade is permeated by a sense of legacy that is proudly di l d h hi f h i lif d b i i ld Thi i h f I 12 displayed once the history of their life and business is told. This is the case for Int. 12: “I did not choose to be a butcher. It was a stroke of unluck. I am joking. I was supposed to continue studying. My great-grandfather opened the shop over 110 years ago. When I was a boy, I was studying and, in the meantime, my grandfather was teaching me the profession. My dad sadly passed away, and I had to start working under my grandfather in the shop. The passion came along. After all, I really did like the profession; nonetheless, I was studying. I was a quick learner. As they would leave me alone in the shop very often, I had to manage the store and the clients all by myself. This is because I learned very quickly. We have this trade in the blood. My son now continues the activity after I ofﬁcially retired some years ago.” Entering the profession is linked to a sense of cultural intimacy [48] dictated by kinship or by the personal knowledge of the master butcher. Becoming a butcher is the result of a direct interpersonal relationship between trainer and trainee which is not mediated by other institutions, such as a school. As Int. 2 puts it, “It does not happen to become a butcher. Either you have it in your blood or you fall in love because someone makes you live it [butchery] before you even set foot in the shop”. The apprenticeship lasts several years and is completed when the butcher knows how to manage the entire carcass of the animal and prepare all the cuts from it. 3.1. An Artisan Profession 3.1. An Artisan Profession 3.1. An Artisan Profession Sennet [19] identiﬁes the development of an artisan profession in a process of acquiring and testing creative skills and competence that leads the individual from apprenticeship to journeymanship, and from journeymanship to mastership. This is a path of social legitimation marked by speciﬁc rites of passage [47], but it is also and above all a process through which the individual embodies and practices knowledge [21]. This process is followed by the butchers in Genoa. Butchery is a craft that is acquired by direct experience under the guidance of an older artisan. It is based on the knowledge that is “stolen with his eyes” (Int. 7) during everyday practice in the shop. All the butchers describe this process that starts with the completion Societies 2023, 13, 80 5 of 13 of the easiest task. As Int. 7 explains, “the ﬁrst thing I had to learn is how they behaved and handled things”. Int. 12 remembers: “It was in the ‘70s when I began working in a butcher’s shop. I began because I was passionate and wanted to undertake this profession. So, I got the ﬁrst opportunity I had to learn from a butcher. The ﬁrst thing he taught me was to clean the shop and all the tools”. After managing the tools, the apprentice starts learning how to use them. The ﬁrst cut that an apprentice learns to deal with is the belly. Int. 4 explains: “The ﬁrst thing you learn is how to debone and skin the belly of the cow, as this allowed apprentices to make more mistakes at the beginning as that cut isn’t as ﬁne as other fresh cuts, such as the posterior like the thigh”. Similarly, Int. 7 indicates: “The belly is one the least reﬁned cuts of the cow; therefore, you can allow yourself to make more mistakes. It is perfect for learning. Then, you worked from the belly up to the most reﬁned cuts of the cow”. Another easy preparation is linked with the so-called “prontocuoci” (ready cuts, such as hamburgers, roulades, skewers, or other preparation-ready and fast-to-cook cuts). As Int. 10 explains, “I wasn’t working, and a friend of his asked me to help him out in his shop. He was my ﬁrst teacher. I had been a baker; therefore, I was used to doing things manually. 3.1. An Artisan Profession However, in the words of my informants, it is only ofﬁcial when the butcher becomes able to also manage the shop and its clientele, thus being able to run the commercial activity proﬁtably. As put by Int. 3, “In the shop, we all [butchers of Genoa] always had some apprentices. The most talented and enterprising either took over the shop or opened their own and, at some point, started teaching a new generation the craft. The others remained to work as employees, giving strength to the business”. The company structure of the individual butcher shop followed a usual model that is at the same time spatial, occupational, and gendered. Similarly to what was highlighted by Simpson et al. [49], it hinges on the presupposition that a butcher should be a man and develops as a family business that involves husband (butcher) and wife, as well as the next generations (children, grandchildren), especially when they are boys. The shop accordingly develops a precise construction of the space that distinguishes between the shop ﬂoor and the back shop. The latter includes laboratories and warehouses. The shop ﬂoor is marked by the presence of a meat counter, on which meat is displayed, and of a checkout. The Societies 2023, 13, 80 6 of 13 space for the butcher and employees is located behind these structures, and from there they serve the customers, who are in front of them. The space behind the meat counter as well as the back shop are managed by the butcher and his men assistants, whereas the checkout is run by women, who are also in charge of bookkeeping. This structure is attested in the memory of the informants and, in some cases, is still what occurs in the present day. It was also the model reproduced when a new butcher shop opened, still based on the centrality of the butcher-shop owner around which a community of practitioners orbited, making the shop run. However, the informants highlight the impossibility or difﬁculty of maintaining the employment levels of the past, the failing intergenerational presence, and the shrinking of their businesses and, above all, the sector. “We were many, and nowadays we are few butcher shops. People buy less meat, and supermarkets are taking away shares of our market and the sector have to change to keep going,” commented Int. 3.2. Changes in Consumption Pattern During the 20th century, meat consumption experienced a radical transformation. In the ﬁrst half of the century, meat, especially red meat, was rarely found on the tables of the popular classes [50]. Meat was mostly food for special occasions and festivity, consumed sparingly and with the utmost care to avoid waste, also through the reuse of leftovers in preparations such as sauces, meatballs, stufﬁng, and fried food. For the popular classes, access to red meat was limited to the use of second-grade cuts, such as muscle, tail, or offal, and often of older animals. They were cuts that required culinary care and long cooking to cope with the strong ﬂavour and the ﬁbrous and tough consistency. In a social context in which it was common for women, once married, to play the role of family caregivers under whose responsibility the purchase of food was placed, women had a central role in the choice as well as in the purchase and preparation of meat. The informants emphasise the role of women, massaie (tr. Housewife). “They would handle all the house chores along with grocery shopping, and they were the ones that would pick meat cuts and cook for the family”, explains Int. 2, “Massaie were a fundamental part of the clientele still in the ‘60s. They had the knowledge of meat cuts, and unlike the clientele of today, they wouldn’t linger on noble cuts; rather, they would pick out anterior cuts which would require long cooking times, as the anterior part of the animal is the most muscular one”. This is due to the morphology of the cow, as it rests the most on its anterior paws, causing these cuts to be harder texture-wise. Today, it is unlikely that younger customers would order these types of cuts. yp Behind this choice, the interviews identify two main motivations: on the one hand, the preference for simpler and faster cuts to prepare, and on the other hand, the lack of gas- tronomic knowledge among modern consumers, as well as the facilities and time available to prepare the cuts. The butchers indicate the change occurred starting in the 1980s. Spinelli [33] describes how the 1980s in Italy marked a radical change in consumer food habits. Italians appeared to fall in love with new industrial products and especially with cooking preparations and dishes closer to those of American tables seen in movies or teleﬁlms. 3.1. An Artisan Profession 9, “We and the sector have to change to keep going . . . ”. 3.3. New Names and New Cuts Old culinary consumption practices faded away, and new ones came along. This dynamic sheds light on the transformation that affected the quotidian reality of butchers’ artisanship. This change touches both the terminology of the trade as well as its materiality. The second half of the 20th century marks the demise of dialects in Italy, the local Italic languages that were spoken by communities as their primary languages before the success of Italian as the primary language of Italy [52]. The interviewees indicated that the 1960s were the turning point of the process, after which the use of dialectal terms became less and less frequent, as customers started using Italian ones. An example is the muscles of the beef shoulder. This cut was locally called “punta du belin” (tr. tip of the dick). This term slowly lost use in favour of Italian alternatives. These could be more local terms, such as “sottopaletta” (tr. under slice) or “punta del codino” (tr. tip of the tail), or more widespread in Italy, such as “cappello del prete” (tr. priest’s hat). The lexical evolution did not stop, however. In the past 20 years, with the popularisa- tion of international cooking broadcasting, such as MasterChef, as well as the success of international cuisines and the proliferation of international restaurants, such as Brazilian, Argentinian, and Peruvian ones, consumers became accustomed to and mesmerised by new recipes and preparations. This led to the use of terminologies borrowed from international culinary traditions, which thus led to the popularisation of the term “Picanha” to identify the cut. The globalisation of taste and forms of meat consumption led the butchers to learn new terminology as well as new ways of cutting meat. In this respect, Int. 10′s shop is an example. There, the owner decided to marginalise the traditional cuts in favour of ones taken from the American BBQ tradition, including new products such as dry rubs (e.g., gin rubs, whisky rubs, rumshine rubs, trufﬂe shufﬂe rubs, lemon pepper garlic rubs). Int. 10 has contacts all over the USA and managed to establish a “dictionary” for meat cuts with his colleagues, presented with the American deﬁnition and the Italian deﬁnition next to it. 3.2. Changes in Consumption Pattern Moreover, in the same decade, the Italian family structure transformed. Speciﬁcally, the decade marked the decline of the single-income family model in favour of realities in which both partners or consorts actively participated in the job market [51]. This led to the disappearance of someone who could care for long food preparations every day. Consequentially, a new demand for food based on preparations or cuts that are quick to prepare became dominant. Butchers had to change their product, expanding the range of products to include more of “delicacies”. “The ’80s saw an increase in requests for ‘prontocuoci’, and we butchers needed to adapt and keep up with the customers’ requests”, remembers LS, who owned a butcher shop that also served delicatessen such as ﬁlled zucchini ﬂowers, octopus, potatoes, and Russian salad back in the 1980s. “There was a demand for more elaborate proposals, not just the classic breaded slices ‘Milanesi’ [beefsteak], but rather cuts such Societies 2023, 13, 80 7 of 13 7 of 13 as tartare seasoned with different condiments, stew, and many more”. In the face of this change, in a few years, informants also noticed a progressive thinning of the gastronomic knowledge of customers, now not only women. As explained by Int. 6: “If I compare my clients born in the ‘70s and their parents . . . the younger seems to be the least interested in cooking I have ever met . . . on a general basis they can barely distinguish a slice of liver from a beefsteak”. However, in the face of this rarefaction, over the past 15 years, the increased media attention, described by Corvo [1], has brought new consumers closer to new recipes, traditional cuts disregarded in the wake of the 1980s consumption change, and more complex preparations, imposing a new global imagination of meat cuisine. As put by Int. 6: p y “There is a difference between women born in the ‘70s who experienced the event of globalisation and rise of fast foods compared to a woman born at the beginning of the ’90s. On a general basis, the people born in the ‘70s due to the event of industrialisation and the rise of fast food may have less knowledge in regards to cooking, whereas 35-year-olds seem to have more interest in regards to cooking. 3.2. Changes in Consumption Pattern These clients, when they come to my shop, seem to have more knowledge and more curiosity about which cuts serve which dish compared to 50-year-olds. I believe it is also due to the rise of mass media engaging customers with TV shows such as MasterChef, Bake-Off, etc... which engaged with the public and reinvigorated interest towards meal preparations.” 3.4. The Perceived Future The need for change and a strong entrepreneurial capacity indicated by the respon- dents shows a perception of an uncertain future. Butchers predict increasing competition and a market jeopardised by new competitors, among which are supermarkets. While the overall consumption per capita of meat has almost tripled in 50 years (from 27 kg in 1961 to 79 kg in the 2010s; see Marchi, 2019), butcher shops turned out to be only one source of meat supply for urban consumers. “In the ’80s, an Italian would buy around 43 kg of meat a year, whereas now we hardly reach 13 kg”, highlighted Int. 2. However, consumers look at the traditional butcher shop for high-quality meat and reﬁned meat products, which are not available in the other retail outlets, for which they are willing to spend more. y g p Additionally, the butcher shop retains the role of social venue, although having lost the centrality it still had in the 1970s. “In the ‘60s and ‘70s, there was a different routine adopted by the clients. They attended the 5:30 a.m. mass to then carry on to grocery shopping. We had different opening times and stayed open on Sundays because of the clients’ routine. This is not relevant anymore, as people began to go to church less throughout the years. However, people come here in person. They like to physically purchase meat as an excuse to have a chat and shop simultaneously”, explains Int. 8, “The relationship between the client and the artisan is the strong point of artisanship”. These assumptions—the importance of socialisation in the shop and the increasingly strong identiﬁcation of butcher shops as places for (only) ﬁne cuts—fuel the ongoing trans- formation of the shops. In the 1980s, with the introduction and growing space given to the sale of prepared cuts, butcher shops changed to assume a role closer to that of delicatessen shops. Nowadays, the changes have led to the restaurant sector. “In the 1990s, there was the ﬁrst successful attempt to make a butcher shop as a small restaurant”, remembers RC. Taking from the model of the American steak houses, since the ﬁrst experiences of the 1990s, other butchers are experimenting with forms of catering that link the preparation of meat, its cooking, and service. “The future goes in that direction, unfortunately”, remarks Int. 1, “The butcher’s profession will become increasingly complex. 3.3. New Names and New Cuts In this way, butchers from Italy are able to understand the request of customers and prepare the exact cut requested because, for example, “the top blade cut that could potentially become tenderloin, if it is cut horizontally, it becomes a cut called ﬂat iron, and these are the only deﬁnitions established for these cuts”, explains Int. 10. Innovations in consumer practices led butchers to innovate their assortments. On the one hand, butchers inserted new products, as seen with the prontocuoci or the rubs of Int. 10. On the other hand, butchers experimented with new and alternative uses of the Societies 2023, 13, 80 8 of 13 8 of 13 traditional cuts. This is the case of the punta du belin used for BBQ. The same cut was the subject of other experimentations. Int. 1 reinvented its use by curing it and slicing away the fat from the cut in order to obtain raw meat to use for tartare or carpaccio. Similarly, Int. 8 works the neck, originally used for long-time cooking soups, to transform it into tartare, which is meat chopped with the knife. “Whether we like it or not”, commented Int. 8, presenting his products and innovations, “we [butchers] have to evolve, even if this means that being a butcher today is deﬁnitely different from what it meant 20, 30, 50 years ago”. 3.4. The Perceived Future It is no longer about opening up the shop in the morning and preparing the work table, selecting the meat, and serving it. It means the butcher of tomorrow should be an artisan, an entrepreneur, and a cook”. Alongside business complexity, the biggest uncertainty in the industry is the recruit- ment of new apprentices. “To this day there are not enough young adults in the full-time profession, and if there are, they are highly interested in the trends of butchery, rather than learning the profession from the bottom to the top”, suggests Int. 5. The interviewees indicate as an example the trend of dry ageing: “They come here to acquire the knowledge on how to treat dry-aged meats rather than learning the basics. Thus, they are never become butchers, but just well . . . TikTokers!?!” (Int. 11). “In the long run it is a profession that requires consistency and devotion, and the income at the beginning is quite mediocre. I had to give up a lot of my projects and dreams in order to keep up with this store”, Int. 6 explains. “I had a young 20-year-old working with me and he was really good, but he felt like he was not moving forwards in this job, in comparison to the jobs that are offered today and the income they offer. You can understand that a 20-year-old wants to change path. The passion is fundamental; it’s the ﬁre that keeps you curious and going”, explained Int. 10. Societies 2023, 13, 80 9 of 13 Recruitment concerns the interviewees also for the maintenance of their butchery knowledge. They highlight the substantial difference in the learning process and resulting knowledge carried out outside the shops. “When young apprentices are employed in supermarkets to work in the butchery section, they do not follow the same learning path as they do in a butcher shop. They learn to do one thing and only one thing, as in an assembly line. When a butcher teaches you the profession, he doesn’t leave a single piece of information to do the job properly. Nothing is ruined, and you don’t get hurt”, Int. 4 explained. “Deboning is an example. Often in the butcher’s section of the supermarket, we notice that certain cuts are not as ﬁne as those found in butchers, for example, the amount of skin that remains on a certain cut of meat. 3.4. The Perceived Future If I leave a little skin on a cut, I’ll be scolded by my master, and that’s how it should be. This is not what happens there, where the ﬁrst goal is to have ready things and full cabinets”, Int. 12 remarked. g y g The model described by the informants focuses on the most extreme scenario in which the holistic approach that characterises artisan apprenticeship is substituted by a form of training in which knowledge is partitioned and its dissemination is functionalised according to the actual role of the employee in the chain of production [19]. The image reﬂects the archetypical distinction between artisan and factory worker, which is foundational to the contemporary understanding of artisanship [53]. Despite these limits, it conveys the fear concerning the loss of trade knowledge as well as one of the limitations of butchers’ agency and entrepreneurship. In other words, it is the fear of marginalisation and decline. In the face of this perspective, some of the informants, such as Int. 7 and Int. 13, are planning public initiatives to “preserve” the profession in Genoa, such as establishing new training courses for young butchers who want to undertake the profession and teaching the old techniques and gastronomic knowledge underpinning the profession, otherwise perceived as lost. Overall, the future of the trade appears to the butchers to be jeopardised, on the one hand, by the overall changes in consumption and, on the other, by the competition of the supermarkets and their approach to retailing. In this respect, butchers defend the distinction between their service and that provided by supermarkets as the only way forward, because “if butchers start competing with grand sale distributions, we lose at the start”, says Int. 9. “The butcher of the future is as far as possible from the ideology of the supermarket because the butcher is an artisan and a shopkeeper, which is the dimension in which this profession gives its best”, remarks Int. 10. 3.4. The Perceived Future While the role of the shop as a place where clients ﬁnd the best meat and also a place where they can acquire gastronomic knowledge and socialise is key, the butchers point out the need for them to learn from other sectors, primarily the restaurant sector, and expand the services offered by their shops: “We should go outside [the shop] because there is always something new to learn even though this is one of the most ancient professions in the world”, explains Int. 9. The butchers also indicate the need to build a stronger relationship with breeders and the farming sector in general to reinforce the quality and strength of the supply chain, secure the quality of the materials, and learn about the supply chain to explain the cut of meat and the history behind it to the client. 4. Discussion The shop is also the place of contact between the artisan and the world and thus the stage where they perform their artisanship and through which they can afﬁrm the sociocultural status of their profession, as Spinelli [33] illustrates. The shop, however, is also the expression of the economic and social success of the individual butcher, who ﬁnds material expression in the increasing number of customers, the volume of business, and the size and fame of the venue. The shop is so important and central in the narrative that a butcher outside this space is no longer recognised by his colleagues as a proper butcher. As the descriptions of butchers employed in supermarkets show, a butcher without a shop is not recognised for his professional status, and his skills are described as partial or limited in their expression. The interviews outline two main innovation dynamics of the butcher shop. On the one hand is the dynamic properly linked to the evolution of the forms of what Pye famously deﬁned as the workmanship of risk, which is any “workmanship using any kind of technique or apparatus, in which the quality of the result is not predetermined, but depends on the judgment, dexterity and care which the maker exercises as he works” [57]. The interviews show this ability has evolved over the last few decades. The ﬁeld of expression and application of artisanship expanded and shifted, experimenting with new preparations (e.g., the prontocuoci), drawing on and hybridising with different traditions of butchery, and taking from other sectors, such as the catering sector, to empower and revamp the business. In this sense, food artisanship becomes adaptable to the socioeconomic context in which it is expressed. The displacement of the ﬁeld of practice brings with it a change of language, retail practices, business management forms, and relations with the city and the ﬁeld of commerce. Precisely because of this constant dialogue with the contingent, it can be said that craftsmanship is the daughter and expression of a particular historical and geographical assembly [58], always current and constantly changing. On the other hand, the transformation is linked to the shops and their size, mainte- nance, and vitality. The transformation outlines a slow erosion of the sociocultural and economic centrality of the shops in the city. This shrinks, as we have seen, the very space where the butchers afﬁrm their social status. 4. Discussion The research shows the change in the butcher’s profession. The craft has become the subject of socioeconomic and cultural innovation faced with the profound transformations of consumption and purchasing practices, as well as the change in the areas in which meat retail is carried out. The interviewees clearly indicate that the butcher’s profession is an expression of embodied knowledge acquired through a process. This does not fully correspond to the model of peripheral legitimation proposed by Lave and Wegner [54] and repeatedly taken up in the scholarship on artisanship, e.g. [14,24,55]. Lave and Wenger [54] have described learning a craft as a contextual social process that involves the trainee’s engagement with a community of practitioners. Through this form of quotidian interaction, a trainee learns Societies 2023, 13, 80 10 of 13 10 of 13 expert knowledge, skills, and competences and is legitimised by the other professionals. By performing increasingly complicated tasks and interacting with the others, the trainee learns the trade but, above all, embodies the vocabulary, values, and organising principles shared by the professional community. The trainee acquires the legitimacy and prestige required to be considered a full-ﬂedged professional both within the community of practitioners and within the wider context of the society. Thus, where this model places the acquisition of prestige within the community of practitioners at the centre of professional growth, the butchers suggest that individual prestige takes on a secondary role, while great emphasis is placed on the individual’s ability to acquire techniques, correctly apply them, and potentially innovate them. In this respect, what the butchers describe appears a path more traditionally associated with vocational training [56], which is built around the acquisition and application of professional know-how. Butchery does not encompass only gastronomic know-how; it also involves commercial and management know-how. This conﬁguration expands the traditional understanding of professional artisanship to include the economic and entrepreneurial aspects. In so doing, it makes artisanal knowledge ﬁt the French expression “savoir, savoir-faire, et faire savoir” (to know, to know how to do, to do what others know). This knowledge is embedded within the person of the artisan; however, the research suggests the artisan is always embedded and can be understood only in the framework of the microcosm of the shop. The shop is the silent protagonist of the stories of the butchers. In the shop, they learn butchery and practice it. 5. Conclusions The conﬁguration of dynamics that this research has explored highlights the historical and territorial sense of gastronomic artisanship. It suggests reading the artisan techniques and practices as cultural expressions always embedded in a speciﬁc economic and food reality. At the same time, the research shows that the cultural signiﬁcance of artisanship is not limited to mere food preparations but must always be read in its socioeconomic dimension. The research suggests that artisanship should be read as a form of entrepreneur- ship capable of placing and moving the craftsman within the global social hierarchy of a community [24]. Therefore, the preservation of artisanship and its sociocultural complexity cannot be limited to the mere preservation of gastronomic forms and techniques. While training and Vocational Education and Training programmes aimed at the continuation of butchery’s know-how are becoming increasingly popular, the signiﬁcant aspects concerning small and family entrepreneurship are marginalised. This leads to the emptying of artisanship of the very worldview that underpins it, reducing artisanship to mere technique [61]. While the ongoing socioeconomic processes seem to lead to the closing of butcher shops and all other small shops that are the basis of proximity commerce, the preser- vation of artisanship as a form of entrepreneurship can support the maintenance of the forms of local commerce and thereby the vitality of urban centres. The case study of Genoa raises questions and opens the ground to new research about how to fully support the continuation of artisanship in urban contexts deeply touched by modernisation and ongoing globalisation. Author Contributions: Conceptualization, M.F.F. and A.C.; methodology, M.F.F.; validation, M.F.F.; formal analysis, M.F.F.; investigation, A.C.; resources, M.F.F. and A.C.; data curation, A.C.; writing—original draft preparation, M.F.F. and A.C.; writing—review and editing, M.F.F.; visu- alization, M.F.F.; supervision, M.F.F.; project administration, M.F.F.; funding acquisition, M.F.F. All authors have read and agreed to the published version of the manuscript. Funding: The research is part of the project NODES, which has received funding from the MUR-M4C2 1.5 of PNRR with grant agreement no. ECS00000036, speciﬁcally for what concerns the modelling of the socio-cultural dynamics concerning the ending part of meat agrifood chain considering how Piedmontese animals (such as cows, chickens and pigs) are commonly traded and sold in Genoa, making the dynamics of the local market relevant for understanding the possibility of innovation for the entire meat sector of NW Italy. The APC were fully waivered by MDPI. 4. Discussion The present commercial dynamics compress it in an increasingly limited space: in a red ocean [59] where the chances of economic success are weathered. Given the expectations of modernity [60], the hope for success felt by the butchers is obfuscated, forcing them to innovate their business to maintain their position. This condition affects the vision of the future. It becomes uncertain from an economic point of view and especially from the point of view of the continuity of the profession. Societies 2023, 13, 80 11 of 13 11 of 13 The absence of a new generation of apprentices, who have moved away from the profes- sion in light of the difﬁculties and uncertainty, strengthens the sense of uncertainty and precariousness, causing butchers to question the sociocultural signiﬁcance of their art. 5. Conclusions Institutional Review Board Statement: The study was conducted in accordance with the Declaration of Helsinki, and the AAA’s Anthropological Ethics Guidelines. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: Relevant data were included in the text. Full data set available in: Costa, A. Butchery in Genoa. Thesis (BA), University of Gastronomic Sciences, 2022. Data Availability Statement: Relevant data were included in the text. Full data set available in: Costa, A. Butchery in Genoa. Thesis (BA), University of Gastronomic Sciences, 2022. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. p y g 2. Haugerud, A.; Stone, P.; Little, P.D. Commodities and Globalization: Anthropological Perspectives; Rowm Lanham, MD, USA, 2000. 1. Corvo, P. Food Culture, Consumption and Society; Palgrave Mcmillan: London, UK, 2015. 4. 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https://openalex.org/W3095237804	https://ieeexplore.ieee.org/ielx7/6287639/8948470/09247946.pdf	English	null	Ensemble Learning With Attention-Integrated Convolutional Recurrent Neural Network for Imbalanced Speech Emotion Recognition	IEEE access	2,020	cc-by	8,441	Received October 26, 2020, accepted November 2, 2020, date of publication November 4, 2020, date of current version November 13, 2020. Received October 26, 2020, accepted November 2, 2020, date of publication November 4, 2020, date of current version November 13, 2020. Digital Object Identifier 10.1109/ACCESS.2020.3035910 Digital Object Identifier 10.1109/ACCESS.2020.3035910 Ensemble Learning With Attention-Integrated Convolutional Recurrent Neural Network for Imbalanced Speech Emotion Recognition XUSHENG AI 1, VICTOR S. SHENG 2, (Senior Member, IEEE), WEI FANG3, CHARLES X. LING4, AND CHUNHUA LI 1 1Software and Service Outsourcing College, Suzhou Vocational Institute of Industrial Technology, Suzhou 215104, China 2Department of Computer Science, Texas Tech University, Lubbock, TX 79409, USA 3Jiangsu Engineering Center of Network Monitoring, School of Computer and Software, Nanjing University of Information Science and Technology, Nanjing 210044, China partment of Computer Science, Western University, London, ON N6A 5B7, Corresponding authors: Xusheng Ai (00754@siit.edu.cn) and Victor S. Sheng (victor.sheng@ttu.edu) responding authors: Xusheng Ai (00754@siit.edu.cn) and Victor This work was supported in part by the National Natural Science Foundation of China under Grant 61702351, in part by the Natural Science Foundation of the Jiangsu Higher Education Institutions of China under Grant 17KJB520036, in part by the Foundation of Key Laboratory in Science and Technology Development Project of Suzhou under Grant SZS201609, in part by the Suzhou Science and Technology Plan Project under Grant SYG201903, and in part by the Computer Basic Education Teaching Research Project under Grant 2018-AFCEC-328, 2019-AFCEC-288. ABSTRACT This article addresses observation duplication and lack of whole picture problems for ensemble learning with the attention model integrated convolutional recurrent neural network (ACRNN) in imbalanced speech emotion recognition. Firstly, we introduce Bagging with ACRNN and the observation duplication problem. Then Redagging is devised and proved to address the observation duplication problem by gen- erating bootstrap samples from permutations of observations. Moreover, Augagging is proposed to get oversampling learner to participate in majority voting for addressing the lack of whole picture problem. Finally, Extensive experiments on IEMOCAP and Emo-DB samples demonstrate the superiority of our proposed methods (i.e., Redagging and Augagging). INDEX TERMS Imbalance learning, ensemble learning, convolutional neural network, recurrent neural network, speech emotion recognition. This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0 I. INTRODUCTION In 2020, Kwon and Mustaqeem [20] proposed a deep-step convolutional neural network (DSCNN) to improve the accuracy of prediction by focusing on the salient and descriptive features of speech signals. Researchers have proposed methods or algorithms to address the imbalance issue for standard machine learning algorithms, and some of them are also valid for deep learning algorithms [11], [12]. Among these solutions, Bagging [13] is one of widely-used methods for its simplicity and effec- tiveness. But when Bagging subsamples a training set with replacement to construct bootstrap samples, some training examples may appear more than once in a sample. As a result, duplicate training examples in a bootstrap sample may prevent a good generalization of the base learner. We call the problem ‘‘observation duplication’’. Correspondingly, this article devises a Random Evenly Distributed Aggrega- tion (Redagging) method to address the observation dupli- cation problem. Additionally, the Redagging base learners trained on partial data may be prevented from seeing the whole picture captured in the entire data. We call the prob- lem ‘‘lack of whole picture’’. This article also proposes an Oversampling-Undersampling aggregation (Augagging) method to deal with the lack of whole picture problem. However, those work does not consider imbalanced speech emotion recognition. Classical deep learning algo- rithms iteratively update weights of models to reduce CE loss [10]. When the label distribution of a train data set is uneven, the ﬁnal model easily biases toward the majority class [21], [22]. Hensman and Masko [11] empirically stud- ied the impact of imbalanced data on CNN training. They concluded that oversampling is a viable way to counter the negative impact. Later, Buda et al. [12] systematically inves- tigated the impact of class imbalance of CNNs in terms of the classiﬁcation accuracy. They stated that the effect of class imbalance on classiﬁcation performance is detrimental and oversampling should be applied to eliminate the imbalance. Zheng et al. [23] suggested generating local features, compre- hensive information in local data, and global features from speech signals, and then they used an ensemble to perform speech emotion recognition. This article focuses on data-level methods that address class imbalance by changing data dis- tribution. Feature augmentation or algorithm enhancement is beyond the scope of this article. The rest of the paper is organized as follows. In Section II, we introduce related work. In Section III, we describe the base classiﬁer ACRNN and Bagging. I. INTRODUCTION In recent years, convolutional recurrent neural net- work (CRNN) is widely used for SER [5]–[7]. At an early stage, CRNN simply assumes that a sequence of frames share the same emotion-relevant weight in an utterance. Later an attention model integrated into convolutional recurrent neu- ral network (ACRNN) employs an attention layer to score weights of a sequence of frames [8], [9]. As a result, ACRNN can focus on emotion-relevant parts and produce discrimina- tive utterance-level representations for SER. However, stan- dard deep learning algorithms based on CE loss [10] does not consider the impact of uneven label distribution on the training set. That is, the number of the training examples labeled with an emotion is smaller than the number of exam- ples labeled with another emotion. The former label is called a minority class and the latter one is a majority class. Although standard deep learning algorithms obtains a model with a minimum of train error, the resulting model is often biased toward the majority class. Consequently, many examples of Emotion is important paralinguistic information in human communication. Emotion directs non-linguistic social sig- nals (such as body language and facial expression) to express wants, needs and desires [1]. There are many appli- cations of speech emotion recognition in different ﬁelds such as healthcare [2], services [3], and telecommunica- tion [4]. In the healthcare ﬁeld, speech emotion recognition can help clinicians assess patients’ psychological disorders online. In the industry of customer call centers, speech emo- tion recognition (SER) can be used to detect customers’ satisfaction. Speech emotion recognition can be also used to route 911 emergency call services for high priority emergency calls. The associate editor coordinating the review of this manuscript and approving it for publication was Shuping He . 199909 X. Ai et al.: Ensemble Learning With Attention-Integrated Convolutional Recurrent Neural Network the minority class are misclassiﬁed and the overall accuracy of SER is low. compared with CRNN, ACRNN can more precisely recog- nize the target emotion. In 2018, Chen et al. [18] proposed to feed Mel-spectrogram, Mel-spectrogram with deltas and Mel-spectrogram with delta-deltas into an attention model integrated CNN. In 2019, Latif et al. [19] devised parallel convolution layers with multiple ﬁlter widths to achieve good experimental results by directly capturing various contextual information. I. INTRODUCTION In Section IV, we intro- duce our Redagging and Augagging. In Section V, we show our experimental results on an English database of emo- tional speech IEMOCAP and a German database of emo- tional speech Emo-DB. Finally, we conclude our study in Section VI. II. RELATED WORK In this section, we introduce imbalance issue on deep learning techniques and ensemble learning methods. Ensemble learning methods are one of the most popular methods of addressing class imbalance for its simplicity and easy-migration. So far, there is no systematic research on ensemble learning methods to address the class imbalance of CNN. Actually, Ensemble learning is a good way to counter the class imbalance of CNN for the independence of algo- rithms or features. This article also proposes two ensemble learning methods to improve the performance of CNN. A. IMBALANCE ISSUE ON DEEP LEARNING TECHNIQUES A. IMBALANCE ISSUE ON DEEP LEARNING TECHNIQUES At an early stage, traditional machine learning methods were used for speech emotion recognition. Speciﬁcally, after speech signals are transformed into statistical fea- tures, speech emotion recognition can be transformed into multi-classiﬁcation problems [1], [14]–[17]. Recently, with the success in image recognition, CRNNs [5]–[7] have been adopted for SER tasks. Generally, CRNN includes three main parts. Firstly, convolutional layer transforms input features into high-dimension feature representations. Then BiLSTM layer generates a sequence of low-dimension feature repre- sentations. Finally, full connected (FC) layer outputs a prob- ability array. B. ENSEMBLE LEARNING METHODS There are three popular classes of ensemble learning meth- ods: Bagging [13], Boosting [24], and Stacking [25]. Bagging repeatedly subsamples a training set with replacement to construct bootstrap samples, on which a learning algorithm trains a sequence of base learners. After obtaining the base learners, Bagging combines them by majority voting and the most-voted class is predicted. Boosting is in fact a family of algorithms in that there are many variants. In Boost- ing algorithms, each learner takes into account its previ- ous learner’s success. After each training step, the weights are redistributed. Misclassiﬁed data increases its weights to emphasize the most difﬁcult cases. In this way, subse- quent learners will focus on them during their training. The ﬁnal learner is derived by weighted majority voting of the base learners. Stacking ﬁrst generates a number of ﬁrst-level In CRNN, BiLSTM layer assumes that each frame has the same inﬂuence on the target emotion. However, some scholars argued that the most inﬂuence often comes from a few frames. Then some attention mechanisms are pub- lished [8], [9]. An attention model integrated convolutional recurrent neural network assigns each frame with a dif- ferent weight. With the weights of a sequence of frames, ACRNN can focus on emotion-relevant parts and produce discriminative utterance-level representations for SER. Thus, 199910 199910 VOLUME 8, 2020 VOLUME 8, 2020 X. Ai et al.: Ensemble Learning With Attention-Integrated Convolutional Recurrent Neural Network Then, the log-Mel ﬁlterbank energies (log-Mels) mi, the delta features, and the delta-deltas features are produced according to (1), (2), (3). individual learners on a training set by employing differ- ent learning algorithms. Those individual learners are then combined by a second-level learner which is called as meta learner. Eventually, the meta learner makes the prediction. mi = log(qi) (1) (1) Boosting improves the generalization error by reducing the training error. However, a neural network (i.e, ACRNN) may be set up with thousands of parameters. A deep learning based learner can be easily over-trained on the training set and lose its generalization properties. For instance, when we used ACRNN as the base learner, we often saw that the training error of the based learner is near zero, but the generation error is still high. Therefore, Boosting is difﬁcult to improve generalization by reducing the training error of the deep learning based learner. Stacking needs to construct base learners using different learning algorithms. B. ENSEMBLE LEARNING METHODS However, the algorithm selection in Stack is not a simple problem. When inappropriate algorithms are selected to train stacking learners, the performance may be not as good as we expect. Hence, this article concentrates on the improvement of Bag- ging for SER tasks. where qi is the Mel ﬁlterbank energy through the ith Mel ﬁlterbank. where qi is the Mel ﬁlterbank energy through the ith Mel ﬁlterbank. mi′ = PN n=1 n(mi+n −mi−n) 2 PN n=1 n2 (2) (2) where n is an incremental number and N is the number of the preceding (or following) frames that are calculated. mi′′ = PN n=1 n(mi+n′ −mi−n′) 2 PN n=1 n2 (3) (3) (3) With mi, mi′, and mi′′, x ∈RS×F×3 is produced according to (4). Then x is used as the input of the aftermentioned ACRNN. x = [ϕ(mi, 3), ϕ(mi′, 3), ϕ(mi′′, 3)]3 (4) (4) Bagging builds heterogeneous base learners by random drawing examples with replacement from a training set. A learning algorithm can generate multiple base learners in a parallel style. But the observation duplication problem of the base learners may cause overﬁtting [26], [27]. Thus, this article devises Redagging to prevent from observation duplication in bootstrap samples and proves the superiority of the proposed method. However, Bagging-based Redagging utilizes undersampling of the source data set to produce the multiple samples and hence, the learners on these multiple samples may be prevented from seeing the ‘‘whole picture’’ captured by the entire data set. So this article also propose Redagging-based Augagging to combine oversampling and Redagging by majority voting for addressing the lack of whole picture problem. III. BAGGING WITH ACRNN In this section, we introduce Bagging using ACRNN as the base classiﬁer and observation duplication problem. Firstly, we brieﬂy overview the construction of the network input. Then we describe the architecture of ACRNN. After that, we introduce Bagging using ACRNN as the base classiﬁer. Finally, we analyze the observation duplication problem in Bagging with ACRNN. αs = exp(W · hs) PS s=1 exp(W · hs) (5) B. ACRNN In the paper, ensembles use an attention model integrated convolutional recurrent neural network (ACRNN) as the base classiﬁer. The architecture of ACRNN is shown in Fig. 1. Firstly, three ConvPool layers transform input feature x into 3-D high-level representations. Secondly, the ﬁrst full-connected (FC) layer transforms the 3-D representations into 2-D space. Thirdly, BiLSTM layer obtains a sequence of high-level representations hs = (−→ hs , ←− hs ). Fourthly, the sec- ond FC layer calculates weight ratio of each frame αs accord- ing to (5). Fifthly, Fun layer performs the weighted sum of the high level features to get utterance-level features g according to (6). Sixthly, the third and fourth FC layer reduce the number of dimensions. Finally, Softmax layer outputs the probability of T class labels p = (p0, p1, . . . , pT−1) where pt represents the probability of emotion et. In convention, the position of the maximal element of p is obtained as the label of x. Note that the three ConvPool layers, the ﬁrst FC layer, and the third FC layer are all followed by a BatchNorm layer [28] and a LeakyLeRU [29] activation layer. Since the BatchNormalization layer and the LeakyLeRU layer do not change the dimension, they are combined into the preceding layer to save space. Alg. 1 Bagging Alg. 1 Bagging Input: Division of training set D∗; Number of labels T; ACRNN classiﬁer L; Number of learning rounds K. for k = 1 to K do for t = 0 to T −1 do Dt k ←observations of drawn from Dt with replace- ment end for Dk ←D0 k ∪D1 k ∪· · · ∪DT−1 k hk ←L(Dk) end for Let X and Y denote the instance space and the set of class labels, respectively, assuming Y = {0, 1, . . . , T −1}. Giving A training set D = {(x1, y1), (x2, y2), . . . , (xm, ym)}, D can be divided into D∗= {D0, D1, . . . , DT−1}, where yi = t if (xi, yi) ∈Dt. For convenience, suppose \|D0\| ≤\|D1\| ≤· · · ≤\|DT−1\|. \|Dt\| is the number of elements in Dt. At the k-th round, Bagging subsamples each subset Dt with replacement to generate T subsamples. Then T subsamples are united to construct the k-th bootstrap sample Dk. Next, ACRNN classiﬁer L is trained on the bootstrap sample Dk to obtain the base learner hk. After the base learners are all ready, the most-voted emotion y is obtained from the outputs of the base learners. The pseudo-code of Bagging is shown in Alg. 1. Dt k ←observations of drawn from Dt with replace- nt ment end for 0 end for 0 end for Dk ←D0 k ∪D1 k ∪· · · ∪DT−1 k hk ←L(Dk) d f H(x) ←argmax((h1(x) + h2(x) + · · · + hK(x))/K) ▷ argmax((p0, p1,. . . , pT−1)) returns the position t of the maximal element pt A. NETWORK INPUT (5) To train a model on audio data, we extracted spectral features from the speech signal and then converted them into array. At ﬁrst, let us deﬁne a few notations or functions as follows. T: number of classes, where W represents network layer parameters. g = XS s=1 αshs (6) T: number of classes, (6) S: time (frame) length, However, some researchers [11], [12] have found that imbalanced training data potentially have a negative impact on classiﬁcation performance of CNN. In the following, we introduce Bagging to address the class imbalance by making data distribution even on bootstrap samples. F: number of Mel-ﬁlter bank, φ(m, i): inserts a new axis into m at position i, [v1, v2, . . . , vc]i: join a sequence of arrays {v1, v2, . . . , vc} along an axis at position i, qi: the Mel ﬁlterbank energies through a Mel ﬁlterbank i. VOLUME 8, 2020 199911 X. Ai et al.: Ensemble Learning With Attention-Integrated Convolutional Recurrent Neural Network FIGURE 1. An ACRNN with a specific configuration used in our experiments. FIGURE 1. An ACRNN with a specific configuration used in our experiments. D. OBSERVATION DUPLICATION IN BAGGING WITH ACRNN Output: hypothesis H Output: hypothesis H In Alg. 1, for any t ∈Y, we have set a constraint \|Dt k\| = \|D0\|. Thus, class labels are evenly distributed in Dk. According to Theorem 1, if \|Dt\| ≈\|D0\|, then the probability that an example appears more than once is approximately 0.264. Since Since P(A0) = (1 −1 dt )d, P(A1) = d 1 dt (1 −1 dt )d−1 = d dt −1(1 −1 dt )d Theorem 1: At the k-th round, d elements are drawn from Dt to construct Dt k. For any x ∈X, if d →+∞, \|Dt k\| ≈ d, then the probability that draws x at least two times is approximately 0.264 (1 - 2 e) Proof of Theorem 1: Let us deﬁne some notations as fol- low. C. BAGGING WITH ACRNN C. BAGGING WITH ACRNN IV. OUR METHODS Firstly, Redagging is devised and proved to deal with the observation duplication problem based on Bagging. Based on Redagging, Augagging is proposed to combine oversampling and Redagging by majority voting to deal with the lack of whole picture problem. Dt,1 = Dt 1 ∪Dt 2∪, . . . , ∪Dt 3 Dt,2 = Dt 3+1 ∪Dt 3+2∪, . . . , ∪Dt 23 Dt,It = Dt (It−1)3+1 ∪Dt (It−1)3+2∪, . . . , ∪Dt It3 R(x) = arg max((r1(x) + r2(x) + · · · + rK(x))) (10) Theorem 2: For any t ∈Y, Redagging distributes Dt∗to K bootstrap samples in order. If \|Dt\|%\|D0\| = 0, for any x ∈Dt k, the number of occurrences of x in Dt k is 1. % returns the remainder left over when one operand is divided by a second operand. t Proof of Theorem 2: Let 3 = \|Dt\| \|D0\|. Given Dt, we have permutations Hence, Hence, P(A) = 1 −(1 −1 dt )d − d dt −1(1 −1 dt )d dt: Dt k, A: an event that draws x at least two times, Thus, if d →+∞, \|Dt k\| ≈d, we have Thus, if d →+∞, \|Dt k\| ≈d, we have A0: an event that draws x 0 times, A1: an event that draws x 1 times. P(A) ≈1 −1 e −1 e = 1 −2 e Then the probability that A occurs, P(A), can be obtained Then the probability that A occurs, P(A), can be obtained by P(A) = 1 −P(A0) −P(A1) 199912 X. Ai et al.: Ensemble Learning With Attention-Integrated Convolutional Recurrent Neural Network FIGURE 2. The flowchart of Redagging. FIGURE 2. The flowchart of Redagging. FIGURE 2. The flowchart of Redagging. where Dt k represents the subset of examples labelled with t at the kth round. Hence, the observation duplication problem occurs on bootstrap samples with a certain probability. Overﬁtting may happen owing to excessive duplicate examples in a bootstrap sample. In Section V (Experiments), experimental results will prove the impact of observation duplication. Additionally, heterogeneity is also critical. The homogeneous base learner cannot yield a good ensemble either. In the following section, our two methods take both the observation duplication and heterogeneity problems into consideration. R(x) = arg max((r1(x) + r2(x) + · · · + rK(x))) (10) (10) A. REDAGGING If \|Dt\|%\|D0\| = 0 is satisﬁed, for ∀k ∈{1, 2, . . . , K}, ∃! i ∈ {1, 2, . . . , It}, Dt k ⊆Dt,i. Since Dt,i is a permutation of Dt, for any x in Dt k, x appears only once in Dt k. If \|Dt\|%\|D0\| = 0 is satisﬁed, for ∀k ∈{1, 2, . . . , K}, ∃! i ∈ {1, 2, . . . , It}, Dt k ⊆Dt,i. Since Dt,i is a permutation of Dt, for any x in Dt k, x appears only once in Dt k. Our method Redagging includes three main steps. Firstly, Redagging randomly generates a number of different per- mutations of Dt(Dt,1, Dt,2, . . . , Dt,It) by the Mason rota- tion algorithm [30] and unions them to get Dt∗according to (7), (8). Then it distributesDt∗to K bootstrap samples D1, D2, . . . , DK in order, where Dk is deﬁned in (9). Finally, using ACRNN as the base classiﬁer, Redagging trains on bootstrap sample Dk to yield the base learner hk and com- bines an ensemble H according to (10). Because Redagging randomly generates permutations and then divides the het- erogeneous permutations into bootstrap samples, it addresses both the heterogeneity and observation duplication problems. The data ﬂow is shown in Fig. 2. Again, K is the number of bootstrap samples and \|DT−1\| represents the number of examples of the majority class. Actually, observation duplication only occurs in a small fraction of bootstrap samples even if \|Dt\|%\|D0\| = 0 is not satisﬁed. Compared with Bagging, Redagging constructs bootstrap samples with less observation duplications. Addi- tionally, various random seeds introduce the heterogeneity of the bootstrap samples. Thus, Redagging base learners are heterogeneous and address overﬁtting for observation duplication. Moreover, we will make further analysis of the performance impact of observation duplication in Section V (Experiments). Another straightforward method to address observation duplication is Sampling Without Replacement (SWOR), which samples without replacement on the level of the indi- vidual bootstrap samples. But SWOR changes the probability distribution. The probability that each example appears in K samples is not the same. As a result, some examples may never appear if K is small. Considering the computa- tion cost and model complexity, a high K is not practical It = K\|D0\| \|Dt\| (7) Dt∗= Dt,1 ∪Dt,2∪, . . . V. EXPERIMENTS In this section, we conduct speaker-independent experi- ments to investigate the performance of comparable meth- ods on both the Interactive Emotional Dyadic Motion Capture database (IEMOCAP) [31] and the Berlin Emotional Database Emo-DB [32]. The experiments run on a H3C G4900 server. The server is conﬁgured with a Tesla V100 independent 32G GPU graphics card, and installed Python 3.7.0, CUDA 10.01 acceleration platform and cuDNN 7.4.2.24 deep learning acceleration platform. A. REDAGGING , ∪Dt,It (8) Dk = D0 k ∪D1 k∪, . . . , ∪DT−1 k (9) It = K\|D0\| \|Dt\| (7) Dt∗= Dt,1 ∪Dt,2∪, . . . , ∪Dt,It (8) Dk = D0 k ∪D1 k∪, . . . , ∪DT−1 k (9) It = K\|D0\| \|Dt\| (7) Dt∗= Dt,1 ∪Dt,2∪, . . . , ∪Dt,It (8) Dk = D0 k ∪D1 k∪, . . . , ∪DT−1 k (9) (7) 199913 VOLUME 8, 2020 X. Ai et al.: Ensemble Learning With Attention-Integrated Convolutional Recurrent Neural Network FIGURE 3. The flowchart of Augagging. FIGURE 3. The flowchart of Augagging. of the original data. When oversampling does not cause over- ﬁtting, Augagging combines both the Redagging base learn- ers and OS learner by majority voting. Hence, theoretically Augagging is superior to Redagging. in many real-world applications. In contrast, Redagging not only reduce the probability of observation duplication but also maintains the same probability of observations. There- fore, Redagging is superior to SWOR by design. Redagging addresses the observation duplication problem, whereas the Redagging base learners lack of the whole pic- ture captured by the entire data. In the following, Augagging is proposed to address the observation duplication and lack of whole picture problems by combining oversampling and Redagging by majority voting. B. AUGAGGING From resampling perspective, a bootstrap sample is generated using an undersampled scheme. Thus, the knowledge of a Redagging base learner may be incomplete. Instead, over- sampling (OS) trains a learner on the oversampled data and acquires the complete knowledge of the source train set. Thus, the combination of Redagging and Oversampling can yield a stronger learner than Redagging ensemble if the OS learner does not introduce overﬁtting. Augagging just employs the combination of Redagging and Oversampling to address the lack of whole picture problem in Redagging. The data ﬂow of Augagging is shown in Fig. 3. The log-Mels are extracted by the Python speech features toolkit [33] with the window size of 25 ms, a 10 ms shift, and N set to 2. The training log-Mels, validation log-Mels and test log-Mels are all normalized by the global mean and the standard deviation of the training set. The NumPy [34] array is used to store features and perform matrix operations. Each layer of ACRNN is implemented by Keras [35]. The parameter of the model is optimized by cross entropy objec- tive function [10] with a mini-batch of 150 examples, using the Adam optimizer [36]. The initial learning rate is set to 10−3. Different from Redagging, Augagging constructs an extra OS learner ro learnt from the oversampled training set Do according to (12). Then r1, r2, . . . , rK, and ro together con- struct the undersampling-oversampling ensemble R∗accord- ing to (11). If the OS classiﬁer O based on (12) performs better than the Redagging ensemble R on the validation set V in term of unweighted average recall (UAR), R∗participates in majority voting; otherwise R does. On the IEMOCAP and Emo-DB databases, ACRNN is the benchmark method. Oversampling randomly clones the examples of the minority class until class distribution is even. Bagging, SWOR, Redagging and Augagging use ACRNN as the base classiﬁer. With different parameter initializations, we can obtain a wide range of results. Thus, we repeat each evaluation for 10 times with different random seeds and report the average to get more reliable results. R∗(x) = arg max((r1(x)+r2(x)+. . . B. EXPERIMENTS ON THE DATABASE IEMOCAP SRR = 1 K K X k=1 P (xi,yi)∈Dk,g((xi,yi),Dk)>1 g((xi, yi), Dk) \|Dk\| (13) IEMOCAP consists of ﬁve sessions, each session being com- pleted by a pair of speakers (female and male) in recitation lines and improvisation scenarios. The average sample length is 4.5 seconds and the sampling rate is 16 kHz. We con- duct experiments on four emotion samples: happy, angry, sad, and neutral. Each task employs 10-fold cross-validation technology. That is, we have 10 samples in all. The sample distribution of the ten samples are described in Table 1, where H = happy, A = angry, S = sad, N = neutral. (13) where g(a, Dk) is the number of occurrences of a in Dk. where g(a, Dk) is the number of occurrences of a in Dk. The right term of (13) represents the degree of observation duplication of bootstrap samples. A higher SRR means more observation duplications in samples. Then using ACRNN as the base classiﬁer, we conduct experiments on an Emo-DB data set to measure the impact of observation duplication. ppy g y We conduct experiments on the ten IEMOCAP samples. K = 5 when Bagging, Redagging, or Augagging is evaluated. Then the ACRNN models of the benchmark method, Over- sampling (OS), Bagging base learners, Sampling without replacement (SWOR) base learners, Redagging base learners, and Augagging base learners are all trained in 10 epochs. The experimental results are shown in Table 2. When ACRNN is used as the base classiﬁer, Augagging achieves the high- est average UAR (0.65), followed by Redagging (0.64) and SWOR (0.62). The average UARs of Oversampling (0.61) and Bagging (0.61) are both higher than the base method (0.60). In terms of UAF, Augagging (0.59) is the best, Redag- ging (0.58) and SWOR (0.56) are the second best, followed by Bagging (0.55). Oversampling (0.53) even performs worse than the benchmark method (0.54). To produce various SRRs on the data set, we choose some classes to apply Redagging and the rest classes to apply Bagging to construct different training data. That is, we ﬁrst choose all ﬁve classes of emotions to apply Redagging and obtain the lowest SRR (SRR = 0.12). And then, we choose the ﬁrst four classes to apply Redagging and the last one to apply Bagging and obtain the second lowest SRR (SRR = 0.19). We repeat the Bagging-Redagging aggregation three more times. B. AUGAGGING This indicates that Redagging can perform better than Bag- ging by reducing observation duplications. In the following subsections, we compare Redagging and Augagging with other imbalance learning methods. A. PERFORMANCE IMPACT OF OBSERVATION DUPLICATION To measure observation duplication, we deﬁne Samples Repetitive Rate (SRR) as deﬁned in (13). VOLUME 8, 2020 B. AUGAGGING + rK(x) + ro(x))) (11) O(x) = arg max((ro(x))) (12) (12) The Redagging base learners are trained on heterogeneous samples, whereas the OS learner can see the ‘‘whole picture’’ Since IEMOCAP and Emo-DB both contain 10 speak- ers, we employ a 10-fold cross-validation technique in our 199914 199914 VOLUME 8, 2020 VOLUME 8, 2020 X. Ai et al.: Ensemble Learning With Attention-Integrated Convolutional Recurrent Neural Network FIGURE 4. UARs and UAFs of Bagging-Redagging aggregation using ACRNN as the base classifier with various SRRs. TABLE 1. Sample distribution of the ten IEMOCAP samples. FIGURE 4. UARs and UAFs of Bagging-Redagging aggregation using ACRNN as the base classifier with various SRRs. TABLE 1. Sample distribution of the ten IEMOCAP samples. FIGURE 4. UARs and UAFs of Bagging-Redagging aggregation using ACRNN as the base classifier with various SRRs. evaluations. Speciﬁcally, for each evaluation, 8 speakers are selected as the training data and one speaker is select as the validation data, while the remaining one speaker is used as the test data. That is, we have 10 samples in all for each database. TABLE 2. Average UARs and UAFs of imbalance learning methods using ACRNN as the base classifier on the ten IEMOCAP samples. TABLE 2. Average UARs and UAFs of imbalance learning methods using ACRNN as the base classifier on the ten IEMOCAP samples. We save the optimal model which achieves the highest UAR. But when evaluating imbalance learning methods, (unweighted average F1-score) UAF is more comprehen- sive and objective in that F1-score is a weighted average of recall and precision. Thus, we measure the performance of imbalance learning methods in terms of both UAR and UAF. recognition in terms of UAR and UAF decreases consistently. When SRR = 0.49, we obtained the lowest UAR and UAF. This indicates that Redagging can perform better than Bag- ging by reducing observation duplications. In the following subsections, we compare Redagging and Augagging with other imbalance learning methods recognition in terms of UAR and UAF decreases consistently. When SRR = 0.49, we obtained the lowest UAR and UAF. This indicates that Redagging can perform better than Bag- ging by reducing observation duplications. In the following subsections, we compare Redagging and Augagging with other imbalance learning methods. recognition in terms of UAR and UAF decreases consistently. When SRR = 0.49, we obtained the lowest UAR and UAF. B. EXPERIMENTS ON THE DATABASE IEMOCAP Finally, we choose all the ﬁve classes to apply Bagging and obtained the highest SRR (SRR = 0.49). Our experimental results under the four different SRRs are shown in Fig. 4. From Fig. 4, we can see that when SRR = 0.12, we obtained the highest UAR (0.75) and UAF (0.78). With the increment of the SRR value, the accuracy of speech emotion 199915 VOLUME 8, 2020 X. Ai et al.: Ensemble Learning With Attention-Integrated Convolutional Recurrent Neural Network FIGURE 5. The average ranks of the five imbalance learning methods using ACRNN as the base classifier on the ten IEMOCAP samples (the lower, the better). FIGURE 6. SRR results of Bagging and Redagging on the ten IEMOCAP samples. Additionally, we rank these imbalance learning methods on each sample. The average rank of each method is shown in Fig. 5. From Fig. 5, we can see that Augagging maintains the leading position (the lower, the better) in terms of UAR and UAF. Redagging is the runner-up. SWOR comes higher FIGURE 7. A comparison of Bagging, Redagging, and Augagging using ACRNN as the base classifier on the 10 IEMOCAP samples, in terms of UAR. TABLE 3. Sample distribution of the ten Emo-DB samples. the ten IEMOCAP samples demonstrate the superiority of Augagging. C. EXPERIMENTS ON THE DATABASE EMO-DB FIGURE 7. A comparison of Bagging, Redagging, and Augagging using ACRNN as the base classifier on the 10 IEMOCAP samples, in terms of UAR. FIGURE 5. The average ranks of the five imbalance learning methods using ACRNN as the base classifier on the ten IEMOCAP samples (the lower, the better). FIGURE 7. A comparison of Bagging, Redagging, and Augagging using ACRNN as the base classifier on the 10 IEMOCAP samples, in terms of UAR. FIGURE 5. The average ranks of the five imbalance learning methods using ACRNN as the base classifier on the ten IEMOCAP samples (the lower, the better). TABLE 3. Sample distribution of the ten Emo-DB samples. TABLE 3. Sample distribution of the ten Emo-DB samples. FIGURE 6. SRR results of Bagging and Redagging on the ten IEMOCAP samples. FIGURE 6. SRR results of Bagging and Redagging on the ten IEMOCAP samples. Additionally, we rank these imbalance learning methods on each sample. The average rank of each method is shown in Fig. 5. From Fig. the ten IEMOCAP samples demonstrate the superiority of Augagging. the ten IEMOCAP samples demonstrate the superiority of Augagging. the ten IEMOCAP samples demonstrate the superiority of Augagging. B. EXPERIMENTS ON THE DATABASE IEMOCAP 5, we can see that Augagging maintains the leading position (the lower, the better) in terms of UAR and UAF. Redagging is the runner-up. SWOR comes higher than ACRNN in terms of UAF but degenerate ACRNN in terms of UAR. The average ranks of Oversampling and Bag- ging even falls behind the average rank of the benchmark method in terms of UAF. On the whole, Fig. 5 indicates that on the ten EMOCAP samples, Augagging and Redagging can improves performance of ACRNN, but Oversampling, Bagging and SWOR can’t. C. EXPERIMENTS ON THE DATABASE EMO-DB Average UARs and UAFs of imbalance learning methods using ACRNN as the base classifier on the ten Emo-DB samples. FIGURE 8. The average ranks of the six imbalance learning methods using ACRNN as the base classifier on the ten Emo-DB samples (the lower, the better). results are shown in Table 4. From Table 4, in terms of UAR, Augagging achieves the average highest UAR (0.71), f ll d b R d i (0 69) Th UAR f SWOR FIGURE 9. SRR results of Bagging and Redagging on the ten Emo-DB samples. TABLE 4. Average UARs and UAFs of imbalance learning methods using ACRNN as the base classifier on the ten Emo-DB samples. FIGURE 8. The average ranks of the six imbalance learning methods using ACRNN as the base classifier on the ten Emo-DB samples (the lower, the better). FIGURE 9. SRR results of Bagging and Redagging on the ten Emo-DB samples. TABLE 4. Average UARs and UAFs of imbalance learning methods using ACRNN as the base classifier on the ten Emo-DB samples. FIGURE 9. SRR results of Bagging and Redagging on the ten Emo-DB samples. FIGURE 8. The average ranks of the six imbalance learning methods using ACRNN as the base classifier on the ten Emo-DB samples (the lower, the better). results are shown in Table 4. From Table 4, in terms of UAR, Augagging achieves the average highest UAR (0.71), followed by Redagging (0.69). The average UARs of SWOR, Bagging and Oversampling are the same (0.68), which are higher than that of the base method (0.66). In terms of UAF, Augagging (0.66) is the best, followed by Redagging (0.65), followed by Bagging (0.64), followed by SWOR (0.63). Oversampling (0.62) performs better than the base method (0.61). FIGURE 10. A comparison of Bagging, Redagging, and Augagging using ACRNN as the base classifier on the 10 Emo-DB samples, in terms of UAR. perform better than Bagging by reducing observation duplica- tions of bootstrap samples. Moreover, Augagging combines the Redagging base learners and the OS learner by majority voting. The experimental results on the ten Emo-DB samples demonstrate the superiority of Augagging. Additionally, we rank these imbalance learning methods on each sample. The average rank of each method is shown in Fig. 8. Fig. 8 shows the average rank of Augagging is the best (the lower, the better) in terms of both UAR and UAF. Redagging ranks the second. [1] N. Yang, R. Muraleedharan, J. Kohl, I. Demirkol, W. Heinzelman, and M. Sturge-Apple, ‘‘Speech-based emotion classiﬁcation using multiclass SVM with hybrid kernel and thresholding fusion,’’ in Proc. IEEE Spo- ken Lang. Technol. Workshop (SLT), Miami, FL, USA, Dec. 2012, pp. 455–460. [2] M. S. Hossain, ‘‘Patient state recognition system for healthcare using speech and facial expressions,’’ J. Med. Syst., vol. 40, no. 12, pp. 1–8, Dec. 2016. C. EXPERIMENTS ON THE DATABASE EMO-DB SWOR ranks above Bagging in terms of UAR but does behind Bagging in terms of UAF. The average ranks of Bagging and OS (oversampling) are better than the rank of the base method. On the whole, Fig. 8 indicates that on the ten Emo-DB samples, these ﬁve imbal- ance learning methods all improves performance of ACRNN, whereas Augagging and Redagging perform the best. C. EXPERIMENTS ON THE DATABASE EMO-DB C. EXPERIMENTS ON THE DATABASE EMO-DB Emo-DB consists of 535 sentences from 10 professional actors, covering 7 emotions (neutral, fear, joy, anger, sadness, disgust and boredom). Speciﬁcally, the Emo-DB data set has ﬁve classes of emotions (containing 101 angry utterances, 66 bored utterances, 53 joyful utterances, 46 sad utterances, and 58 neutral utterances). The original audio is sampled at 44.1 kHz and later downsampled to 16 kHz. The number of anxious examples and the number of disgusting examples are no more than two on some validation sets so that the experiments results ﬂuctuate on the validation set. Thus, we only conduct experiments on ﬁve emotions (anger, bore- dom, joy, sadness and neutral). Each task employs the 10-fold cross-validation technology. The sample distribution of the 10 samples are described in Table 3, where A = anger, B = boredom, H = joy, S = sadness, and N = neutral. Furthermore, we conduct a comparison analysis on the ten IEMOCAP samples in terms of SRR and UAR. The SRR results and UAR results are shown in Fig. 6 and Fig 7, respectively. In Fig. 6, the average SRR of Redagging is about 0.05 while that of the SRRs of Bagging reaches 0.44 on aver- age. This indicates that Bagging introduces more observation duplications than Redagging. Moreover, as shown in Fig. 7, Redagging achieves higher UAR than Bagging on 9 out of 10 samples. That indicates that Redagging performs better than Bagging with high probability. Moreover, Augagging outperforms not only Bagging on 9 out of 10 samples but also Redagging on 5 samples (I1, I3, I5, I8, I10). Augagging constructs an ensemble combining both the Redagging base learners and the OS learner. The experimental results on We conduct experiments on the ten Emo-DB samples. K = 4 when Bagging, Redagging, or Augagging is evaluated. Then the ACRNN models of the benchmark method, Over- sampling (OS), Bagging base learners, Redagging base learn- ers, and Augagging base learners are all trained in 10 epochs. When ACRNN is used as the base classiﬁer, the experimental 199916 199916 VOLUME 8, 2020 VOLUME 8, 2020 X. Ai et al.: Ensemble Learning With Attention-Integrated Convolutional Recurrent Neural Network FIGURE 9. SRR results of Bagging and Redagging on the ten Emo-DB samples. FIGURE 10. A comparison of Bagging, Redagging, and Augagging using ACRNN as the base classifier on the 10 Emo-DB samples, in terms of UAR TABLE 4. VI. CONCLUSION This article studied the observation duplication and the lack of whole picture problems for ensemble learning with ACRNN in imbalanced speech emotion recognition. Redagging is proposed and proved to address the observation duplication problem in bootstrap samples by generating bootstrap sam- ples from permutations of observations. Furthermore, the pro- posed Augagging deals with the lack of whole picture prob- lem by making the OS learner participate in majority voting. Finally, extensive experiments on IEMOCAP and Emo-DB are given to demonstrate the superiority of the proposed methods. For the future research, our work can be extended to positive Markovian jumping neural networks [37], [38]. Furthermore, we conduct a comparison analysis of Bag- ging, Redagging, and Augagging on the ten Emo-DB samples in terms of SRR and UAR. The SRR results and UAR results are shown in Fig. 9 and Fig 10, respectively. In Fig. 9, the mean of the SRRs of Redagging is 0.14 while that of the SRRs of Bagging reaches 0.52. This indicates that Bagging introduces much more observation duplications than Redag- ging in bootstrap samples. Moreover, as shown in Fig. 10, Redagging achieves higher average UARs than Bagging on 8 out of 10 samples. Augagging outperforms not only Bag- ging on 9 out of 10 samples but also Redagging on 4 samples (I3, I4, I6, I10), These two ﬁgures indicate that when using ACRNN as the base classiﬁer, Redagging and Augagging REFERENCES His research inter- ests include machine learning, speech emotion recognition, and image recognition. [15] M. W. Bhatti, Y. Wang, and L. Guan, ‘‘A neural network approach for human emotion recognition in speech,’’ in Proc. IEEE Int. Symp. Circuits Syst., Vancouver, BC, Canada, May 2004, pp. 181–184. [16] A. Hassan and R. 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Ai et al.: Ensemble Learning With Attention-Integrated Convolutional Recurrent Neural Network CHUNHUA LI received the B.S. degree in detec- tion technology and instrument from the Wuhan University of Technology, Wuhan, China, in 2000, the M.S. degree in pattern recognition and intel- ligent system from the Huazhong University of Science and Technology, Wuhan, in 2003, and the Ph.D. degree in computer science and tech- nology from Soochow University, Suzhou, China, in 2017. Her research interests include machine learning, knowledge engineering, deep learning, and crowdsourcing. WEI FANG WEI FANG received the M.Sc. and Ph.D. degrees in computer science from Soochow University. He is currently an Associate Professor with the Department of Computer Science, Nanjing Uni- versity of Information Science and Technology, China, and the State Key Laboratory for Novel Software Technology, Nanjing University. His research interests include artiﬁcial intelligence, big data, deep learning, and meteorological infor- mation processing. He is a PC member for a num- ber of international conferences and a Reviewer for several international journals. CHARLES X. LING has been a Faculty Member for over 25 years. He is a Professor of computer science and the Distinguished Professor of sci- ence with Western University. His research inter- ests include (big) data analytics, machine learning, and data mining applications. He has published over 150 peer-reviewed research articles. Recently, he applies his research to mobile health, and cre- ated glucoguide, which won the First Prize in Diabetes Research Day with the Schulich School of Medicine and Dentistry of Western. He is the CEO and Founder of GlucoGuide Corporation. 199919 VOLUME 8, 2020
https://openalex.org/W2955611671	https://dialnet.unirioja.es/descarga/articulo/7007638.pdf	Portuguese	null	Ensino de Ciências Biológicas: materiais didáticos para alunos com necessidades educativas especiais	Ciência & Educação	2,019	cc-by	9,308	1 Universidade de São Paulo (USP), Escola Superior de Agricultura Luiz de Queiroz (ESALQ), Piracicaba, SP, Brasil. E-mail: massabni@usp.br doi: https://doi.org/10.1590/1516-731320190020006 doi: https://doi.org/10.1590/1516-731320190020006 Artigo publicado em acesso aberto sob uma licença Creative Commons Introdução Com o processo de evolução da sociedade e esclarecimentos morais, a mudança de comportamento tem influenciado no direito de muitas pessoas, dentre elas, as que possuem alguma deficiência, seja visual, auditiva, motora ou intelectual. Essas pessoas necessitam de oportunidades diferentes para serem incluídas na sociedade, motivo pelo qual é importante que haja maior sensibilidade, de maneira coletiva, de desenvolver capacidades em todos, mas principalmente, naqueles que necessitam maior apoio. Uma vez que “[...] a educação especial é uma modalidade que perpassa os níveis, etapas e modalidades da educação brasileira e atende a educandos com deficiência, transtornos globais do desenvolvimento e altas habilidades ou superdotação” (BRASIL, 2014b, p. 24), já instituída pela Constituição Federal de 1988 (BRASIL, 1988), esta modalidade se torna o caminho para a inclusão com efetiva aprendizagem, de tais alunos. Dentre todos os espaços em que a educação se faz presente no dia de uma pessoa, a escola, uma vez destinada ao ensino coletivo que garanta o direito à educação, mostra-se como espaço importante para a inclusão, desde os níveis iniciais. Para que a inclusão se efetive são necessárias reformas estruturais, inovação tecnológica e reestruturação do ensino, de modo que a prática pedagógica exercida pelos profissionais propicie a valorização da diversidade, por meio da participação e aprendizagem do aluno com deficiência, transtornos globais do desen- volvimento e altas habilidades, conforme previsto no Plano de Desenvolvimento da Educação (BRASIL, 2007). i Conforme Mantoan (2001), a temática da inclusão do grupo de alunos com defici- ência, transtornos globais do desenvolvimento e altas habilidades, integra-se a uma discussão mais ampla de educação, referente à inclusão de todos no processo educativo, fundamental para outros grupos discriminados, baseado no reconhecimento de sua condição econômica, raça, gênero ou opção sexual, entre outras É uma busca da redução das desigualdades e à valo- rização da diversidade das pessoas, conforme o Plano Nacional de Educação (PNE) (BRASIL, 2014a).i Segundo dados do Censo 2016 do Inep (figura 1), 57,8% das escolas brasileiras conta- vam com alunos com deficiência incluídos em classes comuns. Em 2008 esse percentual era de apenas 31%, o que indica aumento significativo nos últimos anos na matrícula de alunos com deficiência em escolas regulares. Esta constatação demanda melhor formação do professor e preparo das escolas para atender estes alunos, pois, segundo Laguna (2012, p. The teaching of Biological Sciences: teaching materials for students with special educational needs Larissa Ferreira Stella1 https://orcid.org/0000-0003-2559-2826 Vânia Galindo Massabni1 https://orcid.org/0000-0001-9917-7217 Resumo: A legislação e as metas educacionais, atualmente, valorizam a inclusão de alunos com necessi- dades educacionais especiais, especificamente alunos com deficiência, transtornos globais do desenvol- vimento e altas habilidades. Porém, para que a inclusão se efetive no que se refere às Ciências Biológicas, torna-se necessário aprimorar a formação inicial e continuada de professores, bem como a proposição e disponibilidade de recursos ou materiais didáticos apropriados para a aprendizagem da área, enfren- tando estas, entre outras questões que dificultam o processo educacional. Se metodologias e recursos diferenciados de ensino facilitam a inclusão destes alunos, precisam ser investigados e propostos. A partir disto, o presente estudo teve como objetivo analisar a diversidade dos materiais didáticos no ensi- no de Ciências Biológicas (ou Biologia) em revistas acadêmicas de plataformas de pesquisa na internet. Para o levantamento de trabalhos foram consultadas 16 bases de dados, sendo encontrados apenas 18 trabalhos. Constatou-se a necessidade de mais estudos relacionados à criação de recursos didáticos que possam ser utilizados em sala de aula na área de Biologia por alunos com deficiência. Palavras-chave: Educação inclusiva. Material didático. Ensino de biologia. Aluno com necessidades especiais. Pesquisa bibliográfica. Abstract: The legislation and the educational goals value the inclusion of students with special ed- ucational needs, specifically students with disabilities, global developmental disorders and high skills. However, with regard to Biological Sciences, there is inadequate initial and continuing training of science teachers, and a lack of funding in schools or of suitable teaching resources, among other issues, which hinders the educational process. If differentiated teaching methods and resources are necessary for the inclusion of these students, they need to be investigated and proposed. Considering that, the present study aimed to analyze the diversity of didactic materials for Biological Sciences in journals and research platforms on the Internet. In order to identify the publications, 16 databases were consulted, and 18 items were found. There is a need for more studies related to the creation of didactic resources that can be used in the classroom by students with disabilities, global developmental disorders and high skills. Keywords: Inclusive education. Teaching materials. Biology teaching. Student with special needs. Bibliographic search. 353 Ciênc. Educ., Bauru, v. 25, n. 2, p. 353-374, 2019 Artigo publicado em acesso aberto sob uma licença Creative Commons Stella, L. F.; Massabni, V. G. Ciênc. Educ., Bauru, v. 25, n. 2, p. 353-374, 2019 Introdução 15), “[...] no processo de inclusão não é o aluno que deve se adaptar na escola, mas ao contrário, a escola deve adaptar-se de acordo com a necessidade específica do aluno que está inserido no ambien- te escolar [...].” Percebe-se que estes estudantes podem estar na educação básica, frequentando a rede de ensino regular, e podem também estar em escolas exclusivas, que oferecem atendimento educacional especializado. Segundo a Lei de Diretrizes e Bases da Educação Nacional (LDB) (BRASIL, 1996), classes, escolas ou serviços especializados devem realizar atendimento edu- cacional sempre que as condições específicas dos alunos não possibilitarem a sua integração nas classes comuns de ensino regular. Com a aprovação da LDB em 1996, a educação especial passa a ser discutida na prática, porque consolidada como parte da educação da rede regular, dando a esses alunos o direito de permanecer dentro das escolas regulares com maior apoio. 354 Ciênc. Educ., Bauru, v. 25, n. 2, p. 353-374, 2019 Ensino de Ciências Biológicas: ... Figura 1 – Dados do censo escolar 2016 Figura 1 – Dados do censo escolar 2016 Fonte: Instituto Nacional de Estudos e Pesquisas Educacionais Anísio Teixeira (2019). Figura 1 – Dados do censo escolar 2016 Fonte: Instituto Nacional de Estudos e Pesquisas Educacionais Anísio Teixeira (2019). O avanço na inclusão, notado no aumento do percentual de matrículas em 2016 de- monstrado pelo Censo 2016 necessita ter continuidade. Conforme as propostas buscadas no Plano Nacional da Educação (PNE) (BRASIL, 2014a), previsto para ser colocado em prática a partir de 2014, os educadores distinguiram a educação especial como uma das metas da educa- ção brasileira considerando-a fundamental para que a educação do Brasil avance, pois há muito por fazer para a inclusão de todos na escola. O PNE reforça a importância de haver esforços do governo para atender às necessidades educacionais na perspectiva da inclusão educacional como ponto importante do direito dos alunos especiais uma vez que “[...] há ainda um grande desafio para promover a universalização, com acessibilidade ao ambiente físico e aos recursos didáticos e pedagógicos”. (BRASIL, 2014a, p. 25). Portanto, as escolas da rede regular de ensino devem matricular estes alunos, ofere- cendo serviços especializados para atender as peculiaridades de cada um, apresente ele defici- ência, transtornos ou altas habilidades, ou não. Introdução As dificuldades para conseguir atendimento es- pecializado existem, como necessidade de contratação de profissionais intérprete de LIBRAS (Língua Brasileira de Sinais) para deficientes auditivos, além de pedagogos, psicopedagogos ou outros profissionais que possam acompanhar os alunos, especialmente em casos de auxílio ao deslocamento de alunos com deficiência física e às necessidades básicas de cada pessoa. Além disto, desde o nascimento, a família pode buscar oportunidades de integrar a criança na sociedade, uma vez que o poder público tem possibilidades legais de oferecer apoio, 355 Ciênc. Educ., Bauru, v. 25, n. 2, p. 353-374, 2019 Stella, L. F.; Massabni, V. G. considerando dificuldades financeiras e de deslocamento, escolaridade baixa, entre outros pro- cessos relacionados à condição de vida da família. A falta de informação da família é uma das grandes dificuldades na inclusão e a participação da família na vida escolar deste aluno é de suma importância para o processo de inclusão. Já na escola, um aspecto relevante é a necessi- dade de se modificarem os métodos de ensino e de buscar recursos específicos que favoreçam o aprendizado, atendendo às diferentes necessidades destes alunos. A LDB (BRASIL, 1996) dedica um capítulo à Educação Especial e preconiza que: considerando dificuldades financeiras e de deslocamento, escolaridade baixa, entre outros pro- cessos relacionados à condição de vida da família. A falta de informação da família é uma das grandes dificuldades na inclusão e a participação da família na vida escolar deste aluno é de suma importância para o processo de inclusão. Já na escola, um aspecto relevante é a necessi- dade de se modificarem os métodos de ensino e de buscar recursos específicos que favoreçam o aprendizado, atendendo às diferentes necessidades destes alunos. A LDB (BRASIL, 1996) dedica um capítulo à Educação Especial e preconiza que: Art. 59. Os sistemas de ensino assegurarão aos educandos com deficiência, transtornos globais do desenvolvimento e altas habilidades ou superdotação: I – currículos, métodos, técnicas, recursos educativos e organiza- ção específicos, para atender às suas necessidades [...] (BRASIL, 1996). Ciênc. Educ., Bauru, v. 25, n. 2, p. 353-374, 2019 Introdução Assim, nota-se que na própria LDB está expressa preocupação com os recursos edu- cativos a serem utilizados no ensino, os quais podem conter ajustes como serem escritos em braile para alunos com necessidade visual, propiciarem o ensino com a Língua Brasileira de Sinais (LIBRAS), para as dificuldades dos não ouvintes ou, ainda, terem adaptações para a as necessidades intelectuais, entre outras, colaborando com o desenvolvimento de metodologias que facilitem o aprendizado. Ter estes recursos para as diferentes disciplinas que compõem o currículo escolar é um desafio, pois precisam ser elaborados ou propostos com criatividade, dedicação aos aspectos pedagógicos e específicos da área e atendimento das necessidades dos educandos na educação especial. Outra preocupação presente na prática educativa está relacionada à formação dos docentes. Por exemplo, o Capítulo 3 do Decreto nº 5.626/05 que regulamenta a Lei nº 10.436/02 (BRASIL, 2005) que reconhece como meio legal de expressão e comunicação a Língua Brasileira de Sinais (Libras) apresenta em seus artigos 4º e 5º a necessidade de forma- ção em nível superior de docentes para o ensino de Libras que atuarão na educação infantil, fundamental, média e na educação superior. Este decreto dispõe que a Libras deve ser inserida no currículo dos cursos de formação de professores na forma de disciplina obrigatória para o exercício do magistério, com a contratação de professores para este exercício, ou seja, a legis- lação impõe a necessidade de todas as instituições de ensino possuirem profissionais especia- lizados na área, preparando a escola para receber os alunos surdos ou com deficiência auditiva por direito, além de referir a necessidade de recursos didáticos para apoiar a educação de tais estudantes (BRASIL, 2005). A oferta de recursos didáticos variados nas escolas parece escassa e sua presença tor- na-se fundamental para que se efetive a inclusão. Assim, o objetivo da pesquisa relatada neste artigo foi investigar estudos acadêmicos que desenvolvam materiais didáticos voltados para o ensino de Ciências Biológicas, uma vez que, nesta área, o uso de modelos e outros recursos colabora para aprendizagem significativa dos alunos. Neste relato, as expressões “recursos didáticos” e “materiais didáticos” são usadas como sinônimos. É importante que os professores estejam capacitados a dar aula utilizando recursos variados para alunos por meio de cursos especializados e vivências, havendo atenção às necessidades específicas de cada aluno, ajudando-os em uma inclusão que se dedique à aprendizagem na escola, e na sociedade, por consequência. 356 Ciênc. Introdução Educ., Bauru, v. 25, n. 2, p. 353-374, 2019 Ensino de Ciências Biológicas: .. Logo, é tido como direito materiais e também métodos específicos para cada aluno deficiente, formação de professores especializados em atendê-los, integração na sociedade, nos meios de trabalho e apoio do governo às instituições que possuem alunos deficientes. Segundo as diretrizes da Política Nacional de Educação Especial na Perspectiva da Educação Inclusiva, “Os sistemas de ensino devem organizar as condições de acesso aos espaços, aos recursos pedagógicos e à comunicação que favoreçam a promoção da aprendizagem e a valorização das diferenças” (BRASIL, 2008, p. 13). A falta de materiais educativos apropriados às escolas que possuem tais alunos em salas regulares, entre outros motivos, dificulta a efetivação da inclusão, na prática. Ainda que tenham sido elaborados, cabe aos professores apropriarem-se destes materiais em sua metodo- logia de ensino, ajustando-os aos processos de aprendizagem dos conteúdos escolares em sala de aula. Pode-se supor outros motivos para esta dificuldade, tais como: falta de preparação de professores para a inclusão, necessidade de mais programas e trabalhos com a sociedade na in- clusão de pessoas com deficiência, transtornos globais do desenvolvimento e altas habilidades, ações que colaborem para sua inclusão no mercado de trabalho, e, de forma geral, condições para a implantação de políticas públicas de apoio a inclusão nas diferentes esferas da sociedade. p p p p p Paulino, Vaz e Bazon (2011, p. 678), ao estudarem recursos para inclusão de alunos com deficiência visual, afirmam que: [...] a intervenção educativa e a utilização de materiais didáticos adap- tados às necessidades perceptuais de estudantes com deficiência vi- sual ajudam no desenvolvimento de caminhos alternativos de desen- volvimento com o uso de recursos que favorecem a percepção tátil e a diferenciação de estruturas de forma a facilitar a compreensão do conteúdo tanto por alunos videntes, quanto com deficiência visual. Assim, dependendo de como são elaborados e utilizados metodologicamente pelos professores, os recursos didáticos adaptados favorecem a aprendizagem de todos, uma vez que se tornam capazes de contextualizar, de maneira didática, o conteúdo escolar, democratizando os conhecimentos na sociedade em que o aluno está incluído. Um termo que vem sendo utilizado de maneira crescente nos últimos anos é o da Tecnologia Assistiva. Criado em 1988 nos Estados Unidos e presente no Brasil, é assim defi- nido no documento oficial Tecnologia assistiva (BRASIL, 2009, p. Introdução 9): Tecnologia Assistiva é uma área do conhecimento, de caracte- rística interdisciplinar, que engloba produtos, recursos, meto- dologias, estratégias, práticas e serviços que objetivam promo- ver a funcionalidade, relacionada à atividade e participação de pessoas com deficiência, incapacidades ou mobilidade reduzida, visan- do sua autonomia, independência, qualidade de vida e inclusão social. A tecnologia assistiva engloba desde a criação de utensílios que ajudem no dia-a-dia da pessoa com deficiência, às adequações físicas de espaços, adequação de computadores ou ins- trumentos que auxiliem na comunicação aumentativa. Por exemplo, no ensino de Ciências, na área de Química, Benite et al. (2017), propõem o uso da tecnologia assistiva em experimento 357 Ciênc. Educ., Bauru, v. 25, n. 2, p. 353-374, 2019 Stella, L. F.; Massabni, V. G. sobre a extração do café envolvendo o conceito de temperatura. Neste experimento, demons- tra a utilização de um termômetro vocalizador capaz de emitir um som do valor indicado, para auxílio no aprendizado de pessoas com necessidades visuais.i sobre a extração do café envolvendo o conceito de temperatura. Neste experimento, demons- tra a utilização de um termômetro vocalizador capaz de emitir um som do valor indicado, para auxílio no aprendizado de pessoas com necessidades visuais.i Diversos estudos têm sido realizados a fim de encontrar metodologias de ensino di- ferenciadas para que a inclusão se efetive na prática docente em sala de aula. Porém, na área de ensino de Ciências, não se tem claro se os avanços no conhecimento acadêmico englobam estudos relacionados ao ensino de Ciências Biológicas, e cabe saber o que vem sendo publica- do em revistas e plataformas acadêmicas. A importância da discussão sobre o ensino de Ciências Biológicas e sua valorização se insere no campo das “[...] discussões sobre a natureza e importância da alfabetização em geral e da científica, em particular, atingem desde pesquisadores, trabalhando isoladamente, até instituições do porte e escopo da UNESCO [...]” (KRASILCHIK, 1992, p. 6). Pozo e Gómez Crespo (2009, p. 23) apresentam a ciência como saber histórico e mutável, a ser construído, assim como o aluno constrói seus conhecimentos quando as propostas de ensino priorizam seu processo construtivo em sala de aula. Relacionado à importância do ensino de Ciências Biológicas, destaca-se: Para que um país esteja em condições de satisfazer as necessidades fun- damentadas da sua população, o ensino das ciências e a tecnologia é um imperativo estratégico. 2 No original: “Para un país que quiere estar en condiciones de atender las necesidades fundamentales de su población, la enseñanza de la ciencia y la tecnología es un imperativo estratégico. Como parte de esa educación, los estudiantes deberán aprender a resolver problemas concretos y a atender las necesidades de la sociedad utilizando sus competencias y conocimientos científicos y tecnológicos”. Introdução Como parte dessa educação, os estudantes deverão aprender a resolver problemas concretos e a satisfazer as ne- cessidades da sociedade, utilizando as suas competências e conheci- mentos científicos e tecnológicos. (UNESCO, 1999, tradução nossa)2. Neste sentido, Cachapuz et al. (2005, p. 211) destacam os desafios das modificações metodológicas, as quais, nas pesquisas em ensino de Ciências, têm origem na investigação em torno dos problemas de ensino e aprendizagem das ciências. As pesquisas, que acarretam ou- tros arranjos metodológicos em aula, valorizam “[...] uma proposta efetiva dos estudantes na construção de conhecimentos e, marcadamente, de índole sócioconstrutivista”. No ensino de Ciências Biológicas são desenvolvidos conteúdos que abordam a natu- reza macroscópica e microscópica dos seres vivos e suas estruturas. A fim de auxiliar no pro- cesso de aprendizagem de ambas, tem-se como opção o uso de recursos didáticos que ampliam as estruturas e permitem seu tateamento, tais como modelos que buscam estimular a reflexão em torno da tridimensionalidade das estruturas, dos componentes, dos ciclos e afins, presen- tes no conteúdo das Ciências Biológicas. Para Ceccantini (2006) a aprendizagem de botânica recorre ao uso de imagens de base bidimensional, como desenhos de cortes ou fotos, porém é baixa a compreensão de estruturas espacialmente, pois os alunos tendem a descrever as células 358 Ciênc. Educ., Bauru, v. 25, n. 2, p. 353-374, 2019 Ensino de Ciências Biológicas: .. como círculos e não esferas. Segundo o autor, o alto preço e os erros dificultam o uso didático de modelos didáticos prontos pelos professores, os quais, com determinação e criatividade, podem confeccioná-los para o ensino. A utilização de recursos didáticos tridimensionais enri- quece a aula e estimula o aluno de maneira simples e acessível aos professores, que pode buscar apoio em investigações sobre o tema. como círculos e não esferas. Segundo o autor, o alto preço e os erros dificultam o uso didático de modelos didáticos prontos pelos professores, os quais, com determinação e criatividade, podem confeccioná-los para o ensino. A utilização de recursos didáticos tridimensionais enri- quece a aula e estimula o aluno de maneira simples e acessível aos professores, que pode buscar apoio em investigações sobre o tema. 3 Disponível em: http://www.scielo.org/. Acesso em: 23 abr. 2019. Objetivos Analisar a diversidade dos materiais propostos em estudos acadêmicos como recursos didáticos no ensino de Ciências Biológicas (ou Biologia) visando apoiar a aprendizagem de alu- nos com deficiência em revistas e plataformas de pesquisas acadêmicas, como Google e sites de bibliotecas de universidades na internet. Introdução Considerando o recurso didático importante ferramenta para o ensino e aprendiza- gem, logo, o presente estudo tem como preocupação agrupar e analisar investigações dedica- das a elaboração de materiais, pois estas buscam, na investigação, propor materiais sugerindo e atendendo critérios didáticos, lógicos, entre outros, com o rigor conceitual para confiabilidade no uso por professores. Buscam-se recursos didáticos já existentes que alcancem as neces- sidades de pessoas com necessidades especiais, procurando igualá-las nas oportunidades de aprendizado e a inclusão na prática. Ciênc. Educ., Bauru, v. 25, n. 2, p. 353-374, 2019 Material e Métodos Realizou-se uma revisão bibliográfica relativa a artigos do período de 2007 a 2016 sobre uso de materiais didáticos no ensino de Ciências Biológicas (ou Biologia) para alunos com deficiência, transtornos globais do desenvolvimento e altas habilidades, e o papel destas ferramentas na inclusão dos alunos, considerando o público almejado em cada publicação se- lecionada para análise. Foram consultadas as seguintes revistas em suas plataformas da internet ou por meio da base de dados Scielo3: - Caderno de Pesquisa; - Cadernos PDE (Programa de Desenvolvimento Educacional); - Ciência & Educação; - Educação e Pesquisa; - Educação e Sociedade; - Educação em Revista; - Revista Brasileira de Educação Especial; - Revista Brasileira de Educação; - Revista Brasileira de Estudos Pedagógicos - Revista Brasileira de Pesquisa em Educação em Ciências; 359 Stella, L. F.; Massabni, V. G. - Revista da SBEnBio: revista da Associação Brasileira de Ensino de Biologia;i - Revista da SBEnBio: revista da Associação Brasileira de Ensino de Biologia;i - Revista Eletrônica: debates em educação científica e tecnológica. - Revista Eletrônica: debates em educação científica e tecnológica. Foram realizadas consultas a 16 bases de dados, incluindo as revistas citadas, as ba- ses das bibliotecas virtuais de universidades públicas paulistas (USP, UNESP e Unicamp), o buscador do Google. Foi consultado também o Portal EduCapes da Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), um repositório de diversos mate- riais didáticos (imagens, vídeos, animações e jogos, entre outros), além de publicações como artigos. O Portal EduCapes4 foi consultado para buscar publicações de artigos na área de ensino de Ciências que se referissem aos recursos didáticos voltados ao ensino de Ciências Biológicas, preparados para uso com alunos com deficiência. Este portal permite que se con- sulte por palavras-chave as quais, ao serem digitadas tal como nas revistas, não atendiam ao filtro pretendido. Porém, ao se realizar este procedimento, trabalhos de diferentes temáticas não relacionadas à inclusão foram elencados pelo próprio buscador do Portal. Deste modo, optou-se por não incluí-lo no mapeamento. Além disto, como se trata de um repositório, as publicações resultantes estavam presentes nas revistas e eventos onde originalmente devem ter sido publicadas. Para encontrar os artigos foram escolhidas as palavras-chaves: Inclusão, Biologia, Deficiência, Recurso e Tecnologia Assistiva. Tais palavras eram incluídas nos buscadores dos sites das próprias revistas, colocadas isoladamente e em combinação, duas a duas, para averi- guação. Material e Métodos Após a busca conferiu-se, pela leitura, se os títulos e resumos abordavam a área de Biologia e a inclusão. Os que não se encaixavam neste critério ou se referiam a pesquisas reali- zadas em outros países ou ainda estavam em outro idioma que não a língua portuguesa foram excluídos da amostra. Em cada artigo encontrado, procurou-se analisar o recurso didático utilizado da se- guinte forma: identificação dos objetivos propostos pelos autores, deficiências indicadas (vi- sual, motora, auditiva ou cognitiva) e o conteúdo abordado do material didático proposto de maneira a obter-se tabela comparativa de materiais. 4 Disponível em: https://educapes.capes.gov.br/. Acesso em: 23 abr. 2019. Ciênc. Educ., Bauru, v. 25, n. 2, p. 353-374, 2019 Resultados e discussão Foi realizado levantamento de artigos sobre o ensino de Ciências Biológicas com pro- postas de materiais didáticos em 12 revistas consultadas em plataformas digitais, bibliotecas virtuais de três universidades do país e arquivo do Google, publicadas entre os anos de 2007 a 2016. As revistas que continham artigos na temática e atendiam a busca pelas palavras-chave indicadas foram as seguintes: 360 Ciênc. Educ., Bauru, v. 25, n. 2, p. 353-374, 2019 Ensino de Ciências Biológicas: ... - Revista da SBEnBio: artigos 3, 5, 6, 7, 8, 9, 10, 11 e 12; - Revista Brasileira de Pesquisa em Educação em Ciências: artigo 1;i - Revista Eletrônica: debates em educação científica e tecnológica: artigo 2; - Biblioteca Virtual da UNESP: artigo 13; - Biblioteca Virtual da UNESP: artigo 13; - buscador Google: seleção de apresentações em seminários e congressos disponíveis net: artigos 4, 14, 15, 16 e 18; g - Cadernos PDE: artigo 17. - Cadernos PDE: artigo 17. Foram encontrados 18 artigos acadêmicos relacionados aos recursos didáticos apro- priados a educação inclusiva voltados ao ensino de Ciências Biológicas listados no quadro 1. Quadro 1 – Relação de trabalhos acadêmicos encontrados sobre recursos didáticos apropriados a educação inclusiva em Biologia Nº Artigo Autor(es) e Título Referência Tipo de Necessidade Assunto Ano 1 PAULINO, A. L. S. ; VAZ, J. M. C.; BAZON, F. V. M. Materiais adaptados para ensino de biologia como recursos de inclusão de alunos com deficiência visual. In: ENCONTRO DA ASSOCIAÇÃO BRASILEIRA DE PESQUISADORES EM EDUCAÇÃO ESPECIAL, 7., 2011, Londrina. Visual Célula eucariótica animal, tradução e núcleo celular. 2011 2 LOPES, N. R.; ALMEIDA, L. A.; AMADO, M. V. Produção e análise de recursos didáticos para ensinar alunos com deficiência visual o conteúdo de mitose: uma pratica pedagógica no ensino de ciências biológicas. Debates em Educação Científica e Tecnológica, Vitória, v. 2, n. 2, dez. 2012. Visual Mitose. 2012 3 GOYA, P. R. L. et al. Materiais didáticos de ciências e biologia para alunos com necessidades educacionais especiais ou design e educação: projeto de um material didático para deficientes visual. Revista da SBEnBio, Niterói, n. 7, p. 6173- 6184, 2014. Visual / Intelectual Ecologia, teia alimentar, Leis de Mendel, Astronomia e fases da lua. 2014 4 ALVES JUNIOR, C. A. et al. Desenvolvimento de material didático para o ensino de ciências a estudantes deficientes visuais. In: COLÓQUIO LUSO-BRASILEIRO DE EDUCAÇÃO, 2., 2016, Joinville. Resultados e discussão Visual Kit laboratorial com descrições em braille. 2016 uadro 1 – Relação de trabalhos acadêmicos encontrados sobre recursos didáticos apropriados a ducação inclusiva em Biologia 361 Ciênc. Educ., Bauru, v. 25, n. 2, p. 353-374, 2019 Stella, L. F.; Massabni, V. G. Nº Artigo Autor(es) e Título Referência Tipo de Necessidade Assunto Ano 5 RIBAS, C. P. et al. Materiais alternativos para alunos cegos no ensino de ciências. In: ENCONTRO REGIONAL SUL DE ENSINO DE BIOLOGIA, 6., 2013, Santo Angelo. Visual Tabela periódica, estrutura de moléculas, espelhos côncavos, grandezas vetoriais, divisão celular e estruturas vegetais. 2013 6 SILVA, P. R.; RUST, N. M. Ensino de ciências: produção de material didático para alunos cegos e com baixa visão. Revista da SBEnBio, Niterói, n. 9, p. 2021- 2033, 2016. Visual Sistema respiratório. 2016 7 FERNANDES, A. F. F.; LAGE, D. A. Inclusão escolar no ensino de biologia: elaboração de materiais adaptados para deficientes visuais e auditivos. Revista da SBEnBio, Niterói, n. 9, p. 4920- 4931, 2016. Visual / Auditiva Árvores, artrópodes, genitália feminina e insetos. 2016 8 BASSO, S. P. S. et al. Material didático multissensorial: a fecundação para deficientes visuais. Revista da SBEnBio, Niterói, n. 5, 2012. Visual Fecundação. 2012 9 LIAÑO, G. A.; SANTOS, L. D.; VARANDA, L. L. Genética ao alcance das mãos: confecção e utilização de modelos táteis para a inclusão de alunos com deficiência visual no ensino regular. Revista da SBEnBio, Niterói, n. 9, 2016. Visual Genética. 2016 10 MENEZES, J. B. F.; NEVES, L.; NUNES, N. Educação inclusiva no ensino de biologia: experiência no estágio supervisionado. Revista da SBEnBio, Niterói, n. 9, 2016. Visual Bactérias e protozoários. 2016 11 PAULETTI, J. et al. Modelo didático tridimensional de epiderme foliar como estratégia para inclusão de alunos com deficiência visual no ensino de botânica. Revista da SBEnBio, Niterói, n. 7, 2014. Visual Epiderme foliar. 2014 continua Quadro 1 – Relação de trabalhos acadêmicos encontrados sobre recursos didáticos apropriados a educação inclusiva em Biologia uadro 1 – Relação de trabalhos acadêmicos encontrados sobre recursos didáticos apropriados a ducação inclusiva em Biologia 362 Ciênc. Educ., Bauru, v. 25, n. 2, p. 353-374, 2019 Ensino de Ciências Biológicas: ... Nº Artigo Autor(es) e Título Referência Tipo de Necessidade Assunto Ano 12 CARVALHO, P. M. S.; SILVA, F. A. Horta orgânica como ambiente de aprendizagem de educação ambiental para alunos com deficiência intelectual. Revista da SBEnBio, Niterói, n. 7, 2014. Intelectual Ecologia. Resultados e discussão Educação ambiental. Outros. 2014 13 FLORES, A. S. Recursos didáticos direcionados como complemento ao ensino de biologia para professores com deficiência visual: um estudo de caso. Trabalho de conclusão de curso (Bacharelado em ciências biológicas) – Faculdade de Engenharia de Ilha Solteira, Universidade Estadual Paulista, 2016. Visual Fotossíntese e células. 2016 14 PIRES, B. B. M.; JORGE, V. L. Confecção de modelos biológicos para alunos cegos no segundo segmento. In: SEMINÁRIO INTERNACIONAL DE INCLUSÃO ESCOLAR, 1., 2014, Rio de Janeiro. Visual Mitose, célula animal, célula vegetal e tradução RNAm. 2014 15 VIDAL, M. D.; CARGIN, A. B.; DALLABONA, K. G. Ensino de ciências e a deficiência visual: uma proposta de atividade para o estudo do corpo humano. CONGRESSO INTERNACIONAL ABED DE EDUCAÇÃO A DISTÂNCIA, 2016, Blumenau. Visual Órgãos do corpo humano. 2016 16 OLIVEIRA, T. G. C.; MARQUES, R. C. P. Utilização de modelos didáticos no ensino de biologia e o processo de inclusão na cidade de Apodi, RN. In: CONEDU, 3., 2016, Campina Grande. Visual / Intelectual Células. 2016 17 BEZ, J. J. V. O desenvolvimento da aprendizagem da célula em modelos didáticos táteis para alunos cegos e de baixa visão. Curitiba: Secretaria Estadual da Educação, 2013. (Cadernos PDE, 2). Visual Células, neurônio, óvulo, espermatozoide, hemácia e leucócito. 2013 18 GOULART, I. B.; MENEZES, M. F.; CARLOS, J. O jogo dos 7 erros como estratégia para o ensino das características celulares para portadores e não portadores de deficiência visual. In: ENCONTRO DE INICIAÇÃO CIENTÍFICA E TECNOLÓGICA DO IFSP, 2016, Araraquara. Visual Células. 2016 Fonte: elaborado pelas autoras. Quadro 1 – Relação de trabalhos acadêmicos encontrados sobre recursos didáticos apropriados a educação inclusiva em Biologia uadro 1 – Relação de trabalhos acadêmicos encontrados sobre recursos didáticos apropriados a ucação inclusiva em Biologia 363 Ciênc. Educ., Bauru, v. 25, n. 2, p. 353-374, 2019 Stella, L. F.; Massabni, V. G. No quadro 1 estão arrolados os 18 artigos encontrados e que são o corpus da pesqui- sa. Nota-se também (quadro 2) que de 2007 a 2010 não foram encontrados artigos publica- dos nas revistas investigadas. Por outro lado, em 2016, foram encontrados nove artigos. Esta constatação indica que a atenção ao tema na área de Ensino de Ciências Biológicas se deu após 2010. Resultados e discussão Este aspecto é positivo na medida em que revela maior preocupação dos pesquisadores com o tema, a ponto de desenvolverem na forma de pesquisa a proposição de novos materiais didáticos. É também relevante pois, embora sejam apenas 18 estudos, apresentam materiais com conteúdos específicos para atender ao currículo escolar na área de Biologia. Quadro 2 – Número de artigos por ano, entre 2007 e 2016 Ano Quantidade Ano Quantidade 2007 0 2012 2 2008 0 2013 2 2009 0 2014 4 2010 0 2015 0 2011 1 2016 9 Fonte: elaborado pelas autoras. Quadro 2 – Número de artigos por ano, entre 2007 e 2016 Ano Quantidade Ano Quantidade 2007 0 2012 2 2008 0 2013 2 2009 0 2014 4 2010 0 2015 0 2011 1 2016 9 Fonte: elaborado pelas autoras. Quadro 2 – Número de artigos por ano, entre 2007 e 2016 Ao analisar os títulos dos artigos e materiais didáticos propostos por cada um deles, percebeu-se que poderiam abranger mais conteúdos e propostas. E se, de fato, “[...] exis- tem diferentes propostas de recursos didáticos para o trabalho com inclusão no ensino de Ciências Biológicas estudados nos últimos anos, evidenciando assim maior interesse dos pro- fissionais da área pelo assunto” (SILVA; LANDIN, 2014, p. 34), o que pode levar, conforme Nepomuceno (2015, p. 54) a uma “[...] nova perspectiva nos estudos sobre a inclusão de pes- soas com deficiência”, nem todas as propostas existentes estão sendo publicadas em revistas dedicadas à pesquisa acadêmica. Nos conteúdos abordados são encontrados os assuntos: Genética (mitose/meiose, processo de tradução, organelas das células animais e vegetais, núcleo celular, lei de Mendel e moléculas), Reprodução (fecundação e órgãos reprodutivos), Astronomia (fases da lua), Ecologia (teia alimentar e artrópodes), Laboratório (kit laboratorial), Botânica (estruturas vegetais, fotossíntese e meios de produção agrícola) e corpo humano (sistema respiratório, neurônio, hemácia, leucócito e órgãos do corpo), sendo alguns assuntos estudados no Ensino Fundamental e outros, no Ensino Médio. O conteúdo de célula, presente em geral no Ensino Médio, é recorrente: foram en- contrados sete trabalhos, em 18, que tratavam especificamente de recurso didático para o ensino de célula (Figura 2). Outros artigos não tratavam especificamente de célula ou sua dife- renciação e caracterização, mas dependiam deste conteúdo para serem compreendidos (Figura 3). Ciênc. Educ., Bauru, v. 25, n. 2, p. 353-374, 2019 Ciênc. Educ., Bauru, v. 25, n. 2, p. 353-374, 2019 Resultados e discussão Como exemplificação, o trabalho oito (Figura 4), sobre fecundação e o trabalho 11 (Figura 5), sobre epiderme foliar, se dedicam a propor recursos para conteúdos que dependem da estrutura celular ou molecular para sua compreensão e também estão relacionados à compre- ensão de que os organismos vivos são, basicamente, constituídos de célula, as quais dependem 364 Ciênc. Educ., Bauru, v. 25, n. 2, p. 353-374, 2019 Ensino de Ciências Biológicas: ... de estruturas moleculares para manutenção da vida. Foram selecionados os exemplos a seguir, como ilustração: Figura 2 – Recursos didáticos para ensino do tema “Célula” Fontes: Paulino, Vaz e Bazon (2011, p. 676) e Flores (2016, p. 99). Figura 2 – Recursos didáticos para ensino do tema “Célula” Fontes: Paulino, Vaz e Bazon (2011, p. 676) e Flores (2016, p. 99). Estes modelos sobre células foram elaborados com materiais de baixo custo, cores fortes e texturas diferentes, ajustes didáticos que favorecem a educação inclusiva. Porém, o Trabalho 1 apresenta maior diferenciação e possibilidade de interação que os demais, uma vez que os estudantes devem mudar de local as peças componentes do modelo. Os outros trabalhos também são modelos ajustados a aprendizagem de Biologia de estudantes cegos e com baixa visão, pois permitem que passem as mãos e sintam diferentes texturas e relevos das estruturas. 365 Ciênc. Educ., Bauru, v. 25, n. 2, p. 353-374, 2019 Stella, L. F.; Massabni, V. G. Figura 3 – Modelo tátil para ensino do tema “Mitose” Fonte: Lopes, Almeida e Amado (2012). Figura 3 – Modelo tátil para ensino do tema “Mitose” Fonte: Lopes, Almeida e Amado (2012). O material didático da figura 3 foi produzido com barbantes, EVA, lixa e bijuterias. O estudante cego e de baixa visão, assim como os demais, deve tocar e sentir as diferentes textu- ras e relevos, ação que colabora na aprendizagem das fases da mitose. Figura 4 – Recurso didático para ensino do tema “Fecundação” Fonte: Basso et al. (2012). Os modelos da figura 4 foram produzidos com bexigas, biscuit, tintas e pano, tornan- do se capaz de a iliar a demonstração para o al no por meio do toq e também as diferenças Figura 4 – Recurso didático para ensino do tema “Fecundação” Fonte: Basso et al. (2012). Fonte: Basso et al. (2012). Fonte: Basso et al. (2012). Resultados e discussão Os modelos da figura 4 foram produzidos com bexigas, biscuit, tintas e pano, tornan- do-se capaz de auxiliar a demonstração, para o aluno, por meio do toque também, as diferenças Os modelos da figura 4 foram produzidos com bexigas, biscuit, tintas e pano, tornan- do-se capaz de auxiliar a demonstração, para o aluno, por meio do toque também, as diferenças 366 Ciênc. Educ., Bauru, v. 25, n. 2, p. 353-374, 2019 Ensino de Ciências Biológicas: ... entre as texturas e localização das estruturas do sistema reprodutor feminino e etapas da fe- cundação. entre as texturas e localização das estruturas do sistema reprodutor feminino e etapas da fe- cundação. Figura 5 – Recurso didático para ensino do tema “Epiderme Foliar” Figura 5 – Recurso didático para ensino do tema “Epiderme Foliar” Fontes: Pauletti et al. (2014). Fontes: Pauletti et al. (2014). Fontes: Pauletti et al. (2014). Por fim, este material da figura 5, sobre epiderme vegetal, foi elaborado com cerca, arames, conchas marinhas, isopor, algodão, massinha e ramos de flores artificiais onde, através do toque, o estudante cego ou de baixa visão é capaz de perceber pelo tato as diferentes es- truturas que compõem a epiderme foliar. Este e os demais modelos são visualmente atrativos, podendo também favorecer a aprendizagem de alunos videntes. De modo geral, é possível perceber que os artigos buscam trazer para o campo vi- sual ou tátil estruturas não visíveis a olho nu, evidenciando o aspecto tridimensional destas. Portanto, há preocupação dos autores em elaborar recursos didáticos que tenham como foco os conteúdos tidos como abstratos e possivelmente de compreensão mais difícil aos estudantes. i Somando os trabalhos que se referem especificamente à célula aos demais que se re- ferem a conteúdos microscópicos ou ultramicroscópicos (como as moléculas) nota-se um total de 12 artigos. Portanto, apenas 6 artigos entre os 18 (os de número 3, 4, 6, 7, 12 e 15) abordam conteúdos de natureza macroscópica e, entre estes, os artigos 3, 7 e 12 trazem assuntos rela- cionados ao meio ambiente e aos vegetais: Ecologia (artigo 3), órgãos das árvores (artigo 7) e horta (artigo 12). Resultados e discussão Percebe-se a falta de materiais relacionados a outros assuntos abordados na área de Ciências Biológicas e presentes no currículo escolar, tais como: Genética (processo de transcri- ção, sistema Rh e hereditariedade, por exemplo), Ecologia, ciclos da natureza, interação fauna e flora, problemas ambientais, por exemplo), Corpo Humano (sistema nervoso, sistema diges- tório, sistema circulatório, interação entre os sistemas, e hormônio, por exemplo), Botânica (respiração celular, xilema e floema, nutrição das plantas, adubação, por exemplo, conforme o que tradicionalmente é parte do currículo escolar na área de Biologia. Assim, nota-se que temas relacionados aos vegetais e ao meio ambiente estão entre os menos presentes, pois exis- tem menos recursos didáticos estudados nas plataformas acadêmicas e revistas utilizadas no estudo, merecendo investigações para o seu desenvolvimento. 367 Ciênc. Educ., Bauru, v. 25, n. 2, p. 353-374, 2019 Stella, L. F.; Massabni, V. G. Stella, L. F.; Massabni, V. G. Sugere-se a criação de novos modelos com orientações para o uso de situações- pro- blema pelo professor, de modo a apoiá- lo metodologicamente sem retirar sua autonomia na elaboração da própria prática. Para as questões ambientais, que fazem parte do conteúdo de Biologia, não foram encontrados recursos didáticos. Estes podem ser planejados, por exemplo, a partir de uma situação-problema, abordando a temática ambiental de maneira transversal baseada na realidade fornecida pelo próprio aluno (TEIXEIRA; SOUZA, 2015), contextuali- zando-o e facilitando a interdisciplinaridade. Considerando o proposto por cada autor em seu trabalho e analisando as necessi- dades especiais apresentadas, do total de 18 artigos, 17 tratam da educação para deficientes visuais, três para deficientes intelectuais, dois para deficientes auditivos e apenas um para defi- cientes motores como visto no gráfico 1. Gráfico 1 – Quantidade de publicações acadêmicas encontradas segundo a deficiência/necessidade especial do estudante 1 Ciênc. Educ., Bauru, v. 25, n. 2, p. 353-374, 2019 Buscou-se saber se o recurso proposto dava a possibilidade de o professor utilizá-lo para ir explicando, de forma a ir trocando posições de peças no modelo simulando uma situa- ção dinâmica, que favoreça a compreensão de processos biológicos. Ou ainda, o aluno poderia manipular o recurso, em um processo de análise para construir seus conhecimentos. Ao analisar o nível de interação aluno-material, utilizou-se como referência o quadro 3. Quadro 3 – Critérios utilizados para avaliar os níveis de interação e suas características na ob dos recursos didáticos para o ensino de Ciências Biológicas propostos nos artigos da revisão bibliográfica realizada Nível de interação Característica Pouca Aluno apenas olha ou escuta Média Aluno toca o material Grande Aluno toca com a possibilidade de realizar mudanças no material Fonte: elaborado pelas autoras. dro 3 – Critérios utilizados para avaliar os níveis de interação e suas características na observação ecursos didáticos para o ensino de Ciências Biológicas propostos nos artigos da revisão ográfica realizada No presente trabalho, para avaliar o potencial do material, optou-se por criar e esta- belecer critérios relacionados à possibilidade de interação do aluno com o recurso didático, justamente porque materiais que não requerem a interação pouco estimulam o aluno e pouco acrescentam aos recursos existentes como apoio ao ensino tradicional, que não valoriza o aluno ativo. Recursos planejados apenas para observação, como cartazes com esquemas ou figuras, além de não incentivarem a interação manual ou intelectual, são fáceis de serem encon- trados, sem se constituírem em um diferencial para o ensino da área de Biologia. Além disto, a possibilidade de o aluno realizar a manipulação e interagir com o material, permite que ele o explore, tire conclusões, tente analisar com sua própria capacidade, o que pode ser auxiliado pelo professor. Assim, torna-se uma atividade que pode colaborar com a ação mental, em especial se o estudante estiver no estágio operatório concreto do desenvolvimento cognitivo. Desta forma, favorece a construção de conhecimentos. Conforme Andrade e Massabni (2011), por meio da experiência física – realizada pelo aluno quando manipula e testa o objeto, ou realizada pelo professor, no caso de uma demons- tração – se retiram dados e se tecem conclusões sobre o mundo natural – que Piaget (1995) chama de abstração empírica. Critérios de análise, segundo as próprias autoras, sobre a necessidade especial que se buscou abordar com o recurso didático proposto. Em alguns artigos, por conta das características do material criado, mais de uma necessidade especial foi abordada. Critérios de análise, segundo as próprias autoras, sobre a necessidade especial que se buscou abordar com o recurso didático proposto. Em alguns artigos, por conta das características do material criado, mais de uma necessidade especial foi abordada. Critérios de análise, segundo as próprias autoras, sobre a necessidade especial que se buscou abordar com o recurso didático proposto. Em alguns artigos, por conta das características do material criado, mais de uma necessidade especial foi abordada. Fonte: elaborado pelas autoras. O artigo relacionado à necessidade motora é o de número 12, que também está vin- culado a todas as outras necessidades especiais, uma vez que trabalha com a implementação de horta em escolas. O trabalho com horta permite valorizar os sentidos dos alunos, se for basea- do no planejamento de atividades que requeiram a percepção de estímulos por meio dos cinco sentidos. Por exemplo, permite, ao professor, abordar com os estudantes fatores ambientais como luz e temperatura e alimentação por meio da degustação Se preparadas para envolver estímulos táteis, entre outros, ajusta-se a inclusão de alunos cegos e de baixa visão. Buscou-se inferir, ao ler os artigos, quais recursos propostos não eram apenas para ilustrar ou para serem observados pelo aluno, favorecendo metodologias de ensino diferencia- das. Nos artigos de número 2, 3, 5, 6, 7, 8, 9, 10, 11, 13, 14, 15, 16, 17 e 18 notou-se que era 368 Ciênc. Educ., Bauru, v. 25, n. 2, p. 353-374, 2019 Ensino de Ciências Biológicas: .. possível ou esperada a interação pois observou-se que, nos recursos propostos, o aluno poderia interagir, manuseando, construindo ou mesmo mexendo e modificando o modelo, alterando-o na prática. Buscou-se saber se o recurso proposto dava a possibilidade de o professor utilizá-lo para ir explicando, de forma a ir trocando posições de peças no modelo simulando uma situa- ção dinâmica, que favoreça a compreensão de processos biológicos. Ou ainda, o aluno poderia manipular o recurso, em um processo de análise para construir seus conhecimentos. possível ou esperada a interação pois observou-se que, nos recursos propostos, o aluno poderia interagir, manuseando, construindo ou mesmo mexendo e modificando o modelo, alterando-o na prática. No levantamento percebeu-se que em 83,33% do total de 18 artigos, os materiais apresentam médio e alto nível de possibilidade de interação, ou seja, os alunos podem, ou não, tocar e realizar modificações nos materiais, facilitando a aprendizagem de processos e a compreensão de estruturas conforme presente no gráfico 2. Por exemplo, o material do artigo 1 (figura 1) permite a interação uma vez que, para o aluno entender o processo de tradução celular, é preciso que haja movimentação das peças que compõem o material, ou seja, é neces- sário que haja mudança na estrutura do recurso criado. 369 Ciênc. Educ., Bauru, v. 25, n. 2, p. 353-374, 2019 Stella, L. F.; Massabni, V. G. Stella, L. F.; Massabni, V. G. São raros os modelos que permitem muita interação do estudante (artigos 1, 4 e 12), as quais podem facilitar a construção do conhecimento, especialmente quando se trata de ope- rações manuais que embasam construções mentais. Pressupostos teóricos da construção do conhecimento, como os presentes na teoria de Piaget e Inhelder (1982), sugerem que o objeto concreto presente materialmente é necessário nos estágios anteriores ao operatório formal graças à função simbólica presente no estágio nomeado Pré-operatório e ao desenvolvimento de noções de tempo, espaço, velocidade, ordem, entre outras relacionadas à abstração de dados da realidade durante sua passagem pelo estágio operatório-concreto. Neste sentido, por poderem facilitar a construção de conhecimento entre estudantes com dificuldades intelectuais que perpassam os estágios já descritos por Piaget de forma que necessite de mais tempo para aprendizagem, tais recursos interativos seriam os mais recomen- dados. Gráfico 2 – Nível de interação dos alunos com os materiais didáticos produzidos, segundo os critérios propostos neste estudo Fonte: elaborado pelas autoras. Fonte: elaborado pelas autoras. Verificou-se que a maioria dos recursos didáticos avaliados eram de média interação e foram utilizados materiais de diferentes cores, texturas e tamanhos para construção dos modelos criados pelos autores das publicações encontradas, indicando ajuste à realidade com- preensiva dos alunos, tanto os com deficiência, quanto os demais. Destaca-se a necessidade de haver descrições acompanhadas de explicações por parte do educador que alcancem as necessidades de cada deficiência como, por exemplo, incluir termos escritos em braille, figuras em Libras e/ou textos com letras grandes, assim como é realizado em determinados artigos. Ciênc. Educ., Bauru, v. 25, n. 2, p. 353-374, 2019 Considerações Finais O presente estudo teve como fundamento buscar uma maneira de colaborar com as escolas no alcance da meta presente no Plano Nacional da Educação e nas diversas leis e decretos vinculados à inclusão de pessoas com necessidades especiais na escola, porque são necessários avanços, no Brasil, para educar todas as crianças. Por sua vez, tais preocupações atingem áreas específicas do conhecimento como as Ciências Biológicas, sugerindo ajustes no ensino de Biologia para a inclusão. A elaboração de recursos didáticos e o desenvolvimento de metodologias de ensino que envolvem a interação do aluno com o recurso favorecem a inclu- são do aluno no ensino regular e também no ensino de alunos que estão nas escolas exclusivas. Para colaborar com ensino de Ciências Biológicas para alunos com necessidades espe- ciais, analisou-se a diversidade de materiais didáticos proveniente de pesquisas publicadas em revistas da área de Educação / Ensino de Ciências. Foram levantados artigos de 2017 a 2016, sendo que de 2007 a 2010 não foram encontrados artigos nas revistas investigadas. Um total de 18 artigos foi encontrado. Os resultados desta análise indicaram que: (1) os recursos eram eram de média interação e, dificultando uma aula dinâmica e metodologias diferenciadas; (2) são apenas 18 artigos em 16 bases de dados e a maioria abordava aspectos abstratos da área de Ciências Biológicas, como as estruturas celulares; (3) os recursos eram, na maioria, prepa- rados para alunos com necessidades visuais, demonstrando a carência de estudos relacionados às outras necessidades especiais; e, (4) há baixa diversidade de materiais sobre determinados conteúdos de Biologia. Materiais didáticos que necessitam apenas toque, como grande parte, pouco estimulam o aluno e pouco acrescentam aos recursos existentes como apoio ao ensino tradicional, que não valoriza o aluno ativo. Como ponto favorável, os materiais didáticos encontrados apresentaram baixo custo, tornando-se acessível a educadores e demonstram média interação com o aluno, indicando avanço no tipo de interação que se tem com o recurso. Sugere-se a elaboração de materiais que trabalhem mais a capacidade manual e intelectual dos alunos, incentivando criatividade e ha- bilidades em que os estudantes possam modificar, buscando atender a realidade de diferentes necessidades educativas. Outro ponto interessante é que, na maioria dos recursos didáticos propostos nas publicações, é utilizado material de baixo custo, dentre eles materiais recicláveis e de fácil manuseio e mon- tagem, aumentando a possibilidade de educadores construírem recursos com base nos artigos e os utilizarem em sala de aula. 370 Ciênc. Educ., Bauru, v. 25, n. 2, p. 353-374, 2019 Ensino de Ciências Biológicas: ... Considerações Finais Conclui-se que até o presente momento, poucos estudos abordam a criação de re- cursos didáticos para alunos com necessidades especiais no ensino de Ciências Biológicas e, dentre os existentes, a maioria dos trabalhos (12 em 18) se refere especificamente à célula e aos conteúdos microscópicos ou ultramicroscópicos (como as moléculas). Há a necessida- de da criação de recursos didáticos que abordem áreas que estes trabalhos não abordam, de forma alinhada a metodologias contextualizadas e com situações problemas, incluindo Meio Ambiente, por exemplo. i Por fim, estima-se que o uso de recursos didáticos em propostas metodológicas ela- boradas pelo professor é importante dentro da sala de aula, seja em escolas exclusivas ou em escolas regulares. Divulgar estas pesquisas e apropriar-se dos resultados nas escolas é tare- fa de cunho coletivo, de pesquisadores, revistas acadêmicas e sistemas de ensino para maior sensibilização, participação dos alunos e aprendizagem em Biologia e nas diversas áreas do conhecimento. Com o levantamento realizado neste artigo, espera-se colaborar com a temá- tica da inclusão em pesquisas na área do ensino de Ciências Biológicas e auxiliar o aluno com deficiência a aprender de maneira lúdica o mesmo conteúdo que outros alunos que estão em 371 Ciênc. 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https://openalex.org/W4298333657	https://zenodo.org/record/3485145/files/POPECON_article_36040.pdf	English	null	MAPPING THE NORTH CAUCASUS: DEMOGRAPHIC GRADIENTS AND INVESTMENT RISKS	Zenodo (CERN European Organization for Nuclear Research)	2,018	cc-by	4,112	Alexander N. Panin1 Faculty of Geography of the Lomonosov Moscow State University, GSP-1, Leninskie Gory, Main building Russian Federation, 119991, Moscow http://www.geogr.msu.ru Alexander N. Panin1 Faculty of Geography of the Lomonosov Moscow State University, GSP-1, Leninskie Gory, Main building Russian Federation, 119991, Moscow http://www.geogr.msu.ru Alexander N. Panin1 Faculty of Geography of the Lomonosov Moscow State University, GSP-1, Leninskie Gory, Main building Russian Federation, 119991, Moscow http://www.geogr.msu.ru 1 Alexander Nikolaevich Panin, Ph. D. (Geography), Senior Researcher, Laboratory of Com- plex Mapping of Faculty of Geography, the Lomonosov Moscow State University. E-mail: info@ geogr.msu.ru MAPPING THE NORTH CAUCASUS: DEMOGRAPHIC GRADIENTS AND INVESTMENT RISKS Abstract. The article examines approaches to the creation of geoinformation support in the field of socio-demographic and investment development of polyethnic territories on the example of the North Caucasus. With the help of the created geodatabase and maps, an assessment is made of the influence of ethnic mosaicism, the nature of the demographic and migration processes of municipalities on the investment development of the region. Key words: geoinformation monitoring, demographic processes, investment potential, conflicts. JEL CODES: J00, R11 JEL CODES: J00, R11 I. Geoinformation support of research and issues of information sources The Northern Caucasus is one of the most peculiar regions of Russia from the social and demographic standpoint. Here in a comparatively small area there are developed, stable peripherally and stably lagging regions / municipalities. Understanding the essence of these issues is impossible without system analysis based on the use of diverse spatial data and geoinformation technology. The paper uses the “open” geoinformation platform QGIS. It is based on the processes of updating data from diversified sources, spatial analysis, the production of final cartographic works.ifi Diversified sources of information are used in the paper: official Rosstat and regional statistical departments data, actual materials from household registers of municipalities, data from social networks, as well as a number of other statistical sources. The key idea of creating a geodatabase is to collect, systematize and process information at the municipal and settlement levels, which 133 Mapping the North Caucasus: Demographic Gradients and Investment Risks undoubtedly gives an opportunity to expand the research potential of the socio- demographic and investment landscape of the North Caucasus.fi undoubtedly gives an opportunity to expand the research potential of the socio- demographic and investment landscape of the North Caucasus.fi This approach is primarily due to the extremely low quality of official statistics. For example, the difference between de facto residing and de jure registered in some parts of the North Caucasus can range from 15 to 35%. The reason is the per capita financing of municipalities. Often, the authorities are directly interested in the fact that official sources of information do not record the true extent of the decline in the population [Belozerov et al., 2010; Belozerov et al., 2014; Kolosov et al., 2016; Shchitova, Chikhichin, 2014]. Another group of reasons is the change in accounting systems used by Rosstat itself. Thus, in 2001, the registration of ethnic aspects of the natural movement of the population (the form AO3 — born and died by nationality) ceased to exist. In 2007, Rosstat abolished statistics describing the ethnic composition of migrants (the form P12 MN1 — arrived migrants by ethnicity, the form P12 MN1 — departed migrants by ethnicity). It is important to note that the arrival / departure coupons of migrants and the records of births and deaths still contain a graph of ethnicity and are filled in many municipalities, but Rosstat does not process this data. I. Geoinformation support of research and issues of information sources Thus, modern research of demographic and migration processes requires a cautious approach to the choice of sources of information. This fact has particular importance for an objective analysis of the situation in multi-ethnic areas, where the nature of socio-demographic development directly affects the formation of conflict potential. II. Demographic and migration processes The North Caucasus is a region with a particular character of ethnodemographic and migration processes, which has an impact on the sustainability of Russia’s spatial development as a whole. We examine demographic and migration processes within the natural (slopes of the Caucasus mountains) and economic (shaped during the decades) borders of the Northern Caucasus. Here exist the areas of compact and mixed living of different ethnic groups, stable economic and ethno-cultural ties between them, and a variety of reproductive behavior. In terms of birth and death rates, the North Caucasus is quite clearly divided into a “relatively successful South” and “a dysfunctional North.” The range of the total fertility rate ranges from 25–26 to 10–11‰. In all the republics of the North Caucasus this indicator, despite a slight decrease, remains quite high. When moving to the northwest, the birth rate falls and reaches its minimum in the Krasnodar Krai. At the same time, one can not fail to notice the positive trend of slowly growing birth rates in the lowland regions, which is indicative not so much of the improvement of the social and economic situation here as the 134 Alexander N. Panin change in the ethno-demographic structure of the population. At the same time, it should be noted that in all the regions of the North Caucasus, as well as in the country as a whole, in the 1990s and early 2000s, the birth rate was noticeably lower than nowadays, and a kind of negative peak came to the very border of the centuries, which was a reflection of the widespread long-term demographic crisis caused by serious political and socio-economic upheavals (Fig. 1). Fig. 1. Birth rates in cities and regions of the North Caucasus (2010–2016). Fig. 1. Birth rates in cities and regions of the North Caucasus (2010–2016). Mortality of the population in the Krasnodar and Stavropol Territories is slightly lower, but close to average Russian indicators, while in the republics the rates of the crude death rate are minimal. Its values depend on at least two characteristics of the population. First, on the life expectancy, which is the highest in the North Caucasus republics: In Ingushetia — almost 81 years, in North Ossetia — almost 79 years, in Daghestan, Kabardino-Balkaria and Karachay-Cherkessia — 78 years. Secondly, mortality directly depends on the age structure of the population. II. Demographic and migration processes For example, the share of residents under the working age in the Stavropol Krai is 18%, and of pensioners — 22%, in Chechnia these figures are 35% and 9% respectively, in Ingushetia — 31% and 10%, in Daghestan — 27% and 11% [Kolosov et al., 2016]. From the regions of the North Caucasus, and indeed the country as a whole, the highest natural increase in the population is in the following republics: in Chechnia — 19.9 ‰, in Ingushetia — 17.7 ‰, in Daghestan — 13.3 ‰. In the Stavropol Krai, the natural growth rate is 0.9‰, in the Krasnodar Krai — 0.3 ‰, and in Adyghea there is a natural decline in population (Figure 2, 3). Mapping the North Caucasus: Demographic Gradients and Investment Risks 135 Mapping the North Caucasus: Demographic Gradients and Investment Risks Fig. 2. Death rates in cities and regions of the North Caucasus (2010–2016). Fig. 3. Population growth in the cities and regions of the North Caucasus (2010–2016). On the migration map of Russia, the North Caucasus serves as a transition zone for migrants from the South Caucasus states. Its regions found themselves on the path of a powerful ethnic migration stream, which for a long time Fig. 2. Death rates in cities and regions of the North Caucasus (2010–2016). Fig. 2. Death rates in cities and regions of the North Caucasus (2010–2016). Fig. 2. Death rates in cities and regions of the North Caucasus (2010–2016). Fig. 2. Death rates in cities and regions of the North Caucasus (2010–2016). Fig. 3. Population growth in the cities and regions of the North Caucasus (2010–2016). Fig. 3. Population growth in the cities and regions of the North Caucasus (2010–2016). Fig. 3. Population growth in the cities and regions of the North Caucasus (2010–2016). On the migration map of Russia, the North Caucasus serves as a transition zone for migrants from the South Caucasus states. Its regions found themselves on the path of a powerful ethnic migration stream, which for a long time was dominated by forced migrants, including ethnic Russians. 136 Alexander N. Panin Simultaneously, the plains are a kind of “corridor of migratory winds” between the unstable North Caucasian republics and the rest of the country. II. Demographic and migration processes Being at the forefront of the Caucasian issues, the Krasnodar Krai, and especially the Stavropol Region, turned out to be a Russian springboard in the Caucasus and at the same time a buffer that mitigates the severity of the ethnopolitical and socioeconomic crisis [Belozerov, Suprunchuk, 2012]. The direct location near the hot spots of Chechnia, Daghestan, the Ossetian-Ingush conflict zone, etc., as well as no less powerful centers of interethnic tension in the South Caucasian states (Georgian-South Ossetian, Azeri-Armenian, Georgian- Abkhazian) caused a massive influx of forced migrants into the Stavropol region, the Don and Kuban. Gradually, the stressful factors of migration lost their significance. The geopolitical lull, positive changes in the socio-economic and ethnopolitical situation have increased the influence of the economic factor on the course of migration processes. Fig. 4. Migration rate in cities and regions of the North Caucasus (2010-2016) Fig. 4. Migration rate in cities and regions of the North Caucasus (2010-2016) The complex modern nature of the economic development of most of the municipalities of the North Caucasus, the significant regional disparities in the distribution of investment and the national characteristics of demographic behavior (primarily a significant increase in the population due to natural growth in the national republics) determined the nature and extent of intra-regional migration. On the map (Figure 4), the “regional trends” of migration are clearly visible: “village-to-city”, “small city-to-big city”, “big city-to-regional capital”, “East- to-West”. It is also clear that the cities continue to grow at the expense of rural 137 Mapping the North Caucasus: Demographic Gradients and Investment Risks areas. The resource of “donors”, though, is almost exhausted. Plus, the suburbs of the regional capitals are actively growing: Mikhailovsk, Kaspiisk, and so on. This is because the cities are “clamped” in their administrative borders and almost do not have free land for housing construction. areas. The resource of “donors”, though, is almost exhausted. Plus, the suburbs of the regional capitals are actively growing: Mikhailovsk, Kaspiisk, and so on. This is because the cities are “clamped” in their administrative borders and almost do not have free land for housing construction. The key, and most tragic feature of migration processes in the North Caucasus is the outflow of North Caucasian youth to the ranks of ISIL. According to various estimates, from 2012 to 2015 the migration flow from the regions of the North Caucasus was about 8 thousand people. III. “Anchor” investment projects: risks and potential for their implementation The first few years of the State’s activation in the area of economic policy in the North Caucasus have convincingly demonstrated that not all standard mechanisms for attracting investments and development of territories work in this region. At present, priority is still given to large “anchor” projects, which are designed to immediately response most of the burning issues in the employment and filling of regional budgets. The multiplier effect on the economy of the regions of the North Caucasus is expected from such projects. i In the first year of the existence of the North Caucasus Federal District, two powerful development institutions were created to radically change the investment landscape of the entire region: OJSC North Caucasus Resorts (NCS) and OJSC North Caucasus Development Corporation (NCDC). Both companies are organized as a subsidiary of Vnesheconombank and JSC “Special Economic Zones” and have stable financial background. i Between 2012 and 2016 there were over 30 projects in the investment portfolios of two companies totaling 1.2 trillion rubles, including the “Tourist and recreational cluster” (Arkhyz, Veduchi, Elbrus-Bezengi, Mamison, Caspian coastal cluster, Lagonaki, Matlas, Tsori, SEZ in the Stavropol Territory) actively promoted by Aleksandr Khloponin, the Vice Prime Minister and the Authorized Representative of President Putin in the North-Caucasus Federal District. The logic of the strategic priorities for the development of the North Caucasus economy is distributed along the following lines: agro-industry, tourism, construction and the construction materials industry, small-scale energy, transport. Each of these directions is provided with a well-defined channel of state support, and a pool of potential investors has formed (the practicability of which is extremely difficult to judge. Thus, for example, none of the declared projects of the Interros Group has ever ventured to start investing in the Caucasus). i The first wave of problems faced by the authorities was due to the poor quality of drafting documents on the implementation of investment projects. Financial models, business plans and socio-economic grounds simply could not stand the criticism of experts involved in shaping the list of priority projects. Against this background, the level of lobbyism and pressure on the authorities behalf local elites, wishing to get state guarantees for the implementation of their own (often on ethnical grounds) businesses at all costs, was off scale. Hence the scandalous nature of the elaboration of the Strategy for the Socioeconomic Development of the North Caucasus until 2025. II. Demographic and migration processes Panin in social and economic development and quality of life of the population should take into account ethno-demographic features. in social and economic development and quality of life of the population should take into account ethno-demographic features. II. Demographic and migration processes (according to official data of the Federal Security Service, FSB — about 3 thousand people). At present, the migration vector is one-sided, most die in battle [Kolosov et al., 2016]. Examples of the return of people to their homes have been single cases, and practically, they are not voiced by official authorities. At the same time, there is a possibility of reverse migration, when well-trained and ideologically brainwashed people will be able to use the experience gained in the struggle for the redistribution of resources in the Caucasus. In addition, in the context of the economic crisis and the general decline in the standard of living, the proportion of the population that is loyal to the radical struggle for Islamic values in the North Caucasus is growing. In general, the overall scale of migration has decreased because of the depletion of migration resources of the countries of Transcaucasia and other regions of the post-Soviet space: all those who wanted and could leave there have already done so. At the same time, interregional migration growth in the plains regions will remain positive because of the high demographic potential in the republics. The population, and above all young people, will leave there, choosing more economically developed territories as their new residence. Thus, in general, the North Caucasus has a powerful and growing demographic potential. Practically all regions of the North Caucasus demonstrate population growth, while the rest of Russia is characterized by a negative population growth trend. However, it must be borne in mind that the mismatch of economic and demographic “well-being” is becoming a source of one of the key contradictions that increase the instability of the socio-economic and political development of these territories. The main source of increase in the population of the republics of the North Caucasus is natural increase. However, per capita incomes lag behind population growth, despite some revival in the economy. High fertility and life expectancy increase the dependency burden on the economically active population, in the structure of which the unemployed begin to predominate. The mismatch of demographic and socio-economic trends is becoming a source of political instability for the republics. The concentration of regions with high fertility and youth age structure of the population in one place requires an adequate response from federal authorities. Demographic policy should take into account regional specifics, and measures aimed at smoothing contrasts 138 Alexander N. III. “Anchor” investment projects: risks and potential for their implementation The realism of its activities was simply impossible to estimate and measure [Kolosov et al., 2016]. 139 Mapping the North Caucasus: Demographic Gradients and Investment Risks The second group of problems appeared where they were not expected - on the ground. The matter is that the implementation of projects with state support is possible only on a juridically “clean” plot of land. For example, in Dombai (a logical place for a cluster), such a site simply was not found. In many respects, the choice in favor of Arkhyz is related to the relatively easy procedure of obtaining the required territory. But even there there was a strong resistance from the local population, including shepherds, traditionally engaged in pasturing cattle. This problem has a special resonance at the present time both in the Caspian and other regions of the North Caucasus. The third and most important is the underestimate or explicit ignoring of the ethnodemographic, migration, economic and socio-political features of the North Caucasus. Few of the developers of such projects were aware of the social landscape of a cluster, a factory, a hotel, etc. If geology and engineering investigations were more or less quickly solved, other factors of the development of the territory were simply not taken into account. In this sense, mega-construction projects (like Olympic sites in Sochi or the APEC Summit) here pose colossal risks, and many of them resemble time bombs. Therefore, the absolute majority of projects are in a frozen or semi-frozen state (Fig. 5). Fig. 5. “Anchor” investment projects of the North Caucasus (2012–2016). Fig. 5. “Anchor” investment projects of the North Caucasus (2012–2016). 140 Alexander N. Panin The “anchors” are expected to provide jobs to local residents and stop the outflow of young people, but in fact it turns out on the contrary. The fact is that the mechanism for the appearance of large business is often different. Before entering the municipality, players try to negotiate vertically and horizontally with regional and local authorities. Often, this is a guarantee of stability and investment security. Accordingly, the holders of key competencies in government bodies should not change. Local elites, primarily the government, are actively involved in business activities. The authorities are gradually beginning to carry out not their direct responsibilities related to the management of the territory of the municipality, but is engaged in the development of a particular business. III. “Anchor” investment projects: risks and potential for their implementation Regretfully, this fact is typical for most of the country, but for municipalities where ethnic factors strongly affects economic development it is doubly risky. Since large business is closed, and small and medium businesses do not have sufficient resources, business activity in the municipality is not being formed. Small business is strongly disconnected and focuses mainly on providing services to the public. Most entrepreneurs at the municipal level are not satisfied with the current state of their businesses. Many of them associate a reduction in their incomes with increased competition and a decline in the population. For many, the problem is the underestimated, in their opinion, social status and insufficient attention to their problems behalf the local authorities. As a rule, castes do not mix. Examples of successful municipal businessmen coming to power are rather few. As a result, human, managerial and business lifts are clogged, and large projects have the opposite effect - there appears a social tension that leads to a migration outflow of the population. l Of course, the local population and their businesses should be built into the process of forming investment “cores”. It’s about local businesses within projects: shops, restaurants, rentals, etc. Without observing this condition, social tension will continue to grow. Residents are with good reason afraid that they will be pushed back by the big players. At the stage of planning investment projects, the interests of the local population must be clearly identified and formulated. For them, options for participation should be developed. And it is necessary to focus not only on those living in a conditional 30-kilometer zone, but also much further. For the population of such territories, other proposals are needed, for example, the organization of traffic flows in accordance with routes, the creation of local points of attraction, the production of ecological and traditional food products, souvenirs, etc. Therefore, it is necessary to take into account factors such as land, ethnic, religious, clan relations, which have a huge impact on the economy of the North Caucasus. And most importantly - it is necessary to take into account the specifics of each individual settlement. 141 apping the North Caucasus: Demographic Gradients and Investment Risks The municipal level of power is closest to the people, but it is also the most complex. 1. Belozerov A. A., Panin A. N., Prihod’ko R. A., 2010. Atlas ‘Educational complex of the Stavropol territory’: the resource edition – Stavropol: SGU Publishing House, 52 p. (in Russian). III. “Anchor” investment projects: risks and potential for their implementation Most municipalities are deeply subsidized territories with extremely low management efficiency, and, unfortunately, almost nothing has changed over the entire post-Soviet period: executives mostly exist within the framework of the Soviet management paradigm, many can not and do not want to change. Swapping heads of subjects can not change everything: the devil hides in details, and in this case — on the lower level of power. And in the North Caucasus this takes a very special form, since the boundaries of municipalities and even the subjects of the federation do not coincide with the boundaries of traditional resettlement, which means that the same investor does not have a ready answer to the main question: with whom to negotiate? These factors until recently were not taken into account in the investment agenda in the North Caucasus, but they are forming a high conflict potential. Effective businessmen react to them more sensitively than the authorities, and do not go to those territories where this potential exists — for example, there is not a single large cattle-breeding holding that would work in the eastern regions of Stavropol, although theoretically the opportunities for their appearance are very good. As before, small and medium-sized businesses in the North Caucasus are largely in the shadow or semi-shadow zone, and there are practically no convincing examples of them. One of the tools for this is the creation of industrial parks and production clusters, when a concentrated group of residents, including local producers, is formed on a small territory. In recent years we’ve seen a number of such attempts in the North Caucasus, but mostly they ended up with appearance of big players affiliated with the authorities and a significant part of the local asset was left behind. The big players, in turn, play cautiously — with an eye on the sanctions, the ruble rate and so on. We’ve got another paradox: businessmen of the North Caucasus invest in the economy of other regions of Russia and abroad, but there is no opposite investment vector. Therefore, there is no impressive economic success in the North Caucasus from the implementation of “anchor” investment projects. 3. Belozerov A. A., Panin A. N., Prikhodko R. A., Chikhichin V. V., Cherkasov A. A. 2014. Ethnic atlas of the Stavropol Krai. Stavropol: FOK-South Publishing House. 314 p. (in Russian). 2. Belozerov V. S., Suprunchuk I. P. 2012. Geography of terrorism: multiscale analysis of terrorist activities. Information-analytical atlas. Stavropol: SGU Publishing House. 48 p. (in Russian). 4. Kolosov V., Vendina O., Gritsenko A., Glezer O., Panin A., Sebentsov A., Zotova M. 2016. Consequences of economic and social transformation policies in the North Caucasus. CASCADE. DOI: http://www.cascade-caucasus.eu/wp-content/uploads/2016/04/ CASCADE-D4.1.pdf3 5. Kolosov V. A., Vendina O. I., Gritsenko A. A., Glezer O. B., Zotova M. V., Panin A. N., Sebentsov A. B. 2016. Results of the policy of post-Soviet economic and social reforms at the Northern Caucasus. Sustainable Development of Mountain Territories. 8(4). (in Russian). 6. Shchitova N. A., Chikhichin V. V. 2014. Comparative analysis of social and economic development of the North Caucasus regions. Science. Innovation. Technology. 1: 161-174. (in Russian). References 2. Belozerov V. S., Suprunchuk I. P. 2012. Geography of terrorism: multiscale analysis of terrorist activities. Information-analytical atlas. Stavropol: SGU Publishing House. 48 p. (in Russian). 142 Alexander N. Panin 5. Kolosov V. A., Vendina O. I., Gritsenko A. A., Glezer O. B., Zotova M. V., Panin A. N., Sebentsov A. B. 2016. Results of the policy of post-Soviet economic and social reforms at the Northern Caucasus. Sustainable Development of Mountain Territories. 8(4). (in Russian). 6. Shchitova N. A., Chikhichin V. V. 2014. Comparative analysis of social and economic development of the North Caucasus regions. Science. Innovation. Technology. 1: 161-174. (in Russian).
https://openalex.org/W3127022657	https://hal.inria.fr/hal-03686030/document	English	null	Learning of Art Style Using AI and Its Evaluation Based on Psychological Experiments	Lecture notes in computer science	2,020	cc-by	3,735	To cite this version: Cong Hung Mai, Ryohei Nakatsu, Naoko Tosa, Takashi Kusumi, Koji Koyamada. Learning of Art Style Using AI and Its Evaluation Based on Psychological Experiments. 19th International Conference on Entertainment Computing (ICEC), Nov 2020, Xi’an, China. pp.308-316, 10.1007/978-3-030-65736- 9_28. hal-03686030 Distributed under a Creative Commons Attribution 4.0 International License Mai Cong Hung1,5, Ryohei Nakatsu2, Naoko Tosa3, Takashi Kusumi4, Koji Koyamada2 Mai Cong Hung1,5, Ryohei Nakatsu2, Naoko Tosa3, Takashi Kusumi4, Koji Koyamada2 1Graduate School of Faculty of Science, Kyoto University, Japan 2Academic Center for Computing and Media Studies, Kyoto University, Japan 3Graduate School of Advanced Integrated Studies in Human Survivability, Kyoto University, Japan 4Graduate School of Education, Kyoto University, Japan 5RIKEN, Japan hungmcuet@gmail.com, nakatsu.ryohei@gmail.com, tosa.naoko.5c@kyoto-u.ac.jp, kusumi.takashi.7u@kyoto-u.ac.jp, koyamada.koji.3w@kyoto-u.ac.jp hungmcuet@gmail.com, nakatsu.ryohei@gmail.com, tosa.naoko.5c@kyoto-u.ac.jp, kusumi.takashi.7u@kyoto-u.ac.jp, koyamada.koji.3w@kyoto-u.ac.jp Abstract. GANs (Generative adversarial networks) is a new AI technology that has the capability of achieving transformation between two image sets. Using GANs we have carried out a comparison between several art sets with different art styles. We have prepared four image sets; a flower image set with Impressionism art style, one with the Western abstract art style, one with Chinese figurative art style, and one with the art style of Naoko Tosa, one of the authors. Using these four sets we have carried out a psychological experiment to evaluate the difference between these four sets. We have found that abstract drawings and figurative drawings are judged to be different, figurative drawings in West and East were judged to be similar, and Naoko Tosa’s artworks are similar to Western abstract artworks. Keywords: GANs, Cycle GAN, art history, transformation of art style, Impressionism, abstraction HAL Id: hal-03686030 https://inria.hal.science/hal-03686030v1 Submitted on 2 Jun 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 4.0 International License 1 Introduction Recently a new technology of deep learning in AI called GANs (Generative Adversarial Networks) has been proposed [1], and various attempts to create artworks by AI have been carried out. However, many of these methods merely let AI learn the style of a particular painter and output images with the learned style. Is there a different approach to the relationship between AI and art? For example, can AI approach basic questions such as what beauty is that exists at the basis of art, and what the difference between Oriental and Western perceptions of beauty is? In this paper, a new methodology for approaching the relationship between AI and art will be proposed, and the results of verification through psychological experiments will be shown. 3. Fluid art “Sound of Ikebana” The behavior of fluid is an important research subject in physics, and has been studied as "fluid dynamics." It is known that fluid creates extremely beautiful shapes under various conditions. As beauty is a fundamental element of art, it is natural to consider fluid dynamics as a basic methodology of art creation. Naoko Tosa, one of the authors, has been leading a project of creating “fluid art” by shooting the behavior of fluid with a high-speed camera. One of the techniques for creating fluid art is the creation of Ikebana-like shapes when sound vibration is applied to paint or other fluids and the phenomenon is shot with a high-speed camera. The detailed process is as follows. A speaker is placed upward, a thin rubber film is put on top of it, and a fluid such as paint is placed on top of it, and the speaker is vibrated with sound, then the paint jumps up and various shapes are created. Based on this method Naoko Tosa created a video art called "Sound of Ikebana" [3]. In April 2017, she exhibited Sound of Ikebana using more than 60 digital billboards at Times Square in New York. Figure 3 shows the event. Fig. 3. "Sound of Ikebana" at Times Square in New York. Fig. 3. "Sound of Ikebana" at Times Square in New York. It was interesting that the video art mentioned above let the authors consider what beauty is and also what Japanese beauty is. When Tosa, exhibited her media art around the world as Japan Cultural Envoy named by the Agency for Cultural Affairs in Japan, many foreign art-related people indicated, "In Tosa's media art, which expressed the beauty hidden in physical phenomena, there is the beauty which Westerners did not notice until now, and which might be the condensed consciousness and sensitivity unique to Japan.'' Inspired by this, the authors would like to compare Western and Oriental artworks using AI. Fig. 2. Conversion from a landscape photo to a Monet painting using Cycle GAN. ([3]) Fig. 2. Conversion from a landscape photo to a Monet painting using Cycle GAN. ([3]) Fig. 2. Conversion from a landscape photo to a Monet painting using Cycle GAN. ([3]) 2 Related Works Recently a new learning method called GANs (Generative Adversarial Networks), that can perform deep learning with a relatively small number of training data, has been proposed [1]. GANs are composed of two networks; a generator network and a discriminator network. By performing learning as a zero-sum game between these two networks, deep learning can converge even with a relatively small number of learning data. By modifying this basic configuration, various GANs have been proposed and interesting results have been obtained. Fig. 1 Basic concept of Cycle GAN. Fig. 1 Basic concept of Cycle GAN. Among them, Cycle GAN is a new method that enables mutual conversion between two image sets. Fig. 1 shows the basic concept of Cycle GAN [2]. In Cycle GAN, when two image sets (X, Y) are given, a transformation function G and an inverse transformation function F between them are considered. Also, two types of errors, Dx and Dy are considered; Dx is a difference between X and X' where X' is the transformation of X by applying G then F and Dy is an error caused by the difference between Y and Y’ where Y’ is a transformation of Y by applying F and then G. The training is carried out so that the sum of these two types of errors is minimized (Fig. 1). ) The feature of Cycle GAN is that, even if there was no one to one correspondence between image sets X and Y, the conversion between them is possible. By using this feature, for example, by learning a group of landscape photographs and a group of paintings of a specific painter, mutual conversion between these image groups becomes possible. Figure 2 shows how a Monet-style landscape painting is created from a landscape photograph [2]. 4. Framework of This Research As described in Chapter 2, Cycle GAN can be used to carry out the transformation between two image sets, even if there is no one-to-one correspondence between images belonging to each image set. So far, reference [3] merely states that landscape photographs could be converted into Monet-style paintings and vice versa. But the authors consider that Cycle GAN could be applied to research investigating the relationship between art and beauty. This paper is based on this basic thought. Considering that art is an essential feature extracted from real objects or natural phenomena, it is possible to use Cycle GAN to convert between real objects or natural phenomena and art that extracts their essence. In the long history of art, the painting originally tried to imitate nature. Western realism is the extension of this trend. As the times go down, however, the impressionism is born that tries to paint the light and its transitions perceived by human eyes, rather than trying to paint nature as it is. At this stage, the form of the objects depicted is still clear. Later, however, the history of Western painting was followed by Cubism and Surrealism, then followed by more recent abstract paintings. Nevertheless, it can be said that artists extract essential things they felt in their hearts from the surrounding nature and made them into abstract paintings. On the other hand, the history of Oriental painting is characterized by the fact that the painted objects have been clear since ancient times. Rather, it is characterized by the direction of minimalism that removes color like ink painting and by the way of drawing emphasizing the characteristics of the object like Ukiyo-e in Japan and remains at the level of figurative painting compared to the West. In such a situation, how is “Sound of Ikebana” described earlier positioned in the history of Oriental painting? It does not depict landscapes either human life and looks like abstract images and videos. Nevertheless, as mentioned earlier, many people overseas have said "The artwork has the feeling of Japanese beauty." Is it possible to use AI style learning and style conversion functions to find out how Sound of Ikebana is positioned compared to Western and Oriental figurative and abstract paintings? In this study, this important and interesting issue is approached using Cycle GAN. 4. Framework of This Research (1) Two types of image sets (image set A, and multiple image sets B1, B2, ...) are prepared. The image set A is to be converted into art. A multiple image sets B1, B2, ... consist of art images. (2) Using Cycle GAN, mutual conversion (Fig. 4) of an image set A and multiple image sets B1, B2, .. are achieved to obtain conversion functions (G1, F1), (G2, F2), …. Fig. 4. Mutual conversion between two types of image sets. Fig. 4. Mutual conversion between two types of image sets. (3) A psychological experiment is performed using the image sets G1 (A), G2 (A), ... that are obtained by performing the conversion G1, G2, … to the image set A. Depending on the purpose of the psychological experiment, questionnaires such as "Can you evaluate it as art?" "Do you feel beauty?" will be filled by the subjects. By doing this, it is possible to verify depending on different art styles what kind of information is extracted as essential information from real objects and made into artworks. 5. Learning of Various Art Style and Transformation of Art Style The following image sets were prepared. (Resolution of all images are 256x256) Image set A: 8069 flower images g g Image set B1: 1072 Monet paintings in the Monet2Photo dataset [2]. Image set B1: 1072 Monet paintings in the Monet2Photo dataset [ Image set B2: 123 Kandinsky abstract paintings from Wiki Art. Image set B1: 1072 Monet paintings in the Monet2Photo dataset Image set B2: 123 Kandinsky abstract paintings from Wiki Art. Image set B3: 238 Chinese hand-painted flower paintings called “Gongbi” from Stanford University project "Chinese Painting Generation Using Generative Adversarial Networks." Image set B4: 569 images selected from "Sound of Ikebana" g g The image set B1 includes paintings mainly for flowers drawn by the Impressionist Monet as a representative example of the Western figurative paintings. As a representative example of Western abstract paintings, Kandinsky paintings were prepared as image set B2. Image set B3 includes flower paintings of Chinese hand- painted painting, called “Gongbi [5],” as a representative example of Oriental figurative paintings. Image Set B4 is a set of still images taken from the media art "Sound of Ikebana" created by one of the authors, Naoko Tosa. The Sound of Ikebana is a video artwork created by shooting physical phenomena with a high-speed camera. As it is made from a physical phenomenon, it should not be said that it originally contains "Japanese beauty." In this experiment, the art style of the Sound of Ikebana was compared with Western and Oriental representative painting styles. p p p g y a b Fig. 5. Examples of mutual conversion between flower photos and Sound of Ikebana style images. (a: Flower to “Sound of Ikebana” style, b: “Sound of Ikebana” to flower-like) b a b a Fig. 5. Examples of mutual conversion between flower photos and Sound of Ikebana style images. (a: Flower to “Sound of Ikebana” style, b: “Sound of Ikebana” to flower-like) The four types of different image set B1, B2, B3, B4 and the image set A including were mutually converted using the Cycle GAN. Figure 5 shows examples of the result of the mutual conversion between the Sound of Ikebana and the photograph of the flower. The question here is why it is necessary to use AI for comparative evaluation of art styles. Why Monet art, Kandinsky art, Chinese Gongbi art, and Sound of Ikebana images were not directly compared and evaluated using psychological experiments. 5. Learning of Various Art Style and Transformation of Art Style There have been some studies that evaluated artworks through psychological The four types of different image set B1, B2, B3, B4 and the image set A including were mutually converted using the Cycle GAN. Figure 5 shows examples of the result of the mutual conversion between the Sound of Ikebana and the photograph of the flower. The question here is why it is necessary to use AI for comparative evaluation of art styles. Why Monet art, Kandinsky art, Chinese Gongbi art, and Sound of Ikebana images were not directly compared and evaluated using psychological experiments. There have been some studies that evaluated artworks through psychological experiments [4]. However, by using a copy of the original artwork, it can be relatively easy to identify the artist for each art. For example, knowing that a painting is Monet's art suggests that a subject has a prejudice of the work of Monet, a representative of the Impressionists in Western art history and that this will have a significant effect on evaluation experiments. To avoid this effect, there are research examples of using lesser-known works [4]. However, works of famous artists and art schools can be easily identified and would greatly affect evaluation experiments. On the other hand, using GAN allows for AI to learn an art style and to apply the art style on the input images with the art style from the input. Therefore, bias can be avoided in the evaluation experiment and this is the benefit of using AI to evaluate artworks. 6. Evaluation of Obtained Results Based on Psychological Experiment The experiment described in Chapter 5 yielded results of performing various style conversions on flower images. By having people evaluate the results of applying various style transformations to various flower images, is it possible to know what art is, what is the beauty behind it, and the culture of beauty? Is there any suggestion on how people receive Japanese beauty and the corresponding Western beauty? That is the goal of this research. Since this is a subjective evaluation, a method used in psychological experiments, which is to present a target image to a subject, to conduct a questionnaire survey, and statistically analyze the results, was used. 6.1 Psychological experiment Image group Goup1, Group2, Group3, and Group4 are prepared by selecting two images from each of image sets G1(A), G2(A), G3(A), and G4(A), which are obtained by converting image set A into image sets B1, B2, B3, and B4. The resolution of each image is 256x256. Twenty three Kyoto University students (12 male and 11 female, all are Japanese) were used as subjects. The gender ratio is almost half. Each of 8 images was printed out on A4 high-quality paper, and the eight images were presented to the subjects. The order of the presented images was set randomly for each subject. The subjects were asked to perform a seven-step subjective evaluation of the 6 items shown in Table 1. These items were selected to identify the difference between the Oriental and Western art styles. Table 1. Adjective pairs used for evaluation Individual - Ordinary Dynamic - Static Stable - Unstable Bold - Careful Artistic - Non-artistic Oriental - Western Table 1. Adjective pairs used for evaluation Individual - Ordinary Dynamic - Static Stable - Unstable Bold - Careful Artistic - Non-artistic Oriental - Western Table 1. Adjective pairs used for evaluation Table 1. Adjective pairs used for evaluation 6.2 Analysis The results of the subjective evaluations by the 23 subjects and for six items, which are Individual-Ordinary, Dynamic-Static, Stable-Unstable, Bold-Careful, Artistic-Non artistic, Oriental-Western, were averaged for each evaluation item, graphed, and t- tested. Figures 6 – 8 show the results of the averaged value and the standard error for each evaluation item. Also, the results of t-analysis (*:1%, :5%) are shown on these figures. Fig. 6. Subjective evaluation results for "individual-ordinary" (left) and “Dynamic-Static” (right). (Group1: Monet-style flower images, Group2: Kandinsky-style flower images, Group3: Chinese figurative art-style flower images, Grop4: Naoko Tosa art-style flower images.)- Fig. 6. Subjective evaluation results for "individual-ordinary" (left) and “Dynamic-Static” (right). (Group1: Monet-style flower images, Group2: Kandinsky-style flower images, Group3: Chinese figurative art-style flower images, Grop4: Naoko Tosa art-style flower images.)- Fig. 7 Subjective evaluation results for "Stable-Unstable" (left) and “Bold-Careful” (right). (Group1: Monet-style flower images, Group2: Kandinsky-style flower images, Group3: Chinese figurative art-style flower images, Grop4: Naoko Tosa art-style flower images.)- Fig. 8 Subjective evaluation results for "Artistic-Non-artistic" (left) and "Oriental-Western" (right). (Group1: Monet-style flower images, Group2: Kandinsky-style flower images, Group3: Chinese figurative art-style flower images, Grop4: Naoko Tosa art-style flower images.)- Fig. 8 Subjective evaluation results for "Artistic-Non-artistic" (left) and "Oriental-Western" (right). (Group1: Monet-style flower images, Group2: Kandinsky-style flower images, Group3: Chinese figurative art-style flower images, Grop4: Naoko Tosa art-style flower images.)- 6.3 Consideration (1) Group 2 vs Group 4 (1) Group 2 vs Group 4 Group 2 and Group 4 received similar evaluations. This indicates that there is a small significant difference between the style of Kandinsky and the style of Sound of Ikebana. Conversely, Group 4 is evaluated as having a significant difference from Group 1 and Group 3 for all items except “Oriental-Western”. This indicates that the Sound of Ikebana is considered to be abstract rather than figurative. If the transition from figurative painting to abstract painting is the history of Western painting, the Sound of Ikebana can be said to be positioned in the history of the transition from figurative painting to abstract painting in the Orient. (2) Group 1 vs Group 3 ( ) p p Similarly, Group 1 and Group 3 received similar evaluations. In particular, the hypothesis that there is a significant difference of 5% level between the "Dynamic- Static" and "Artistic-Non artistic" items have been rejected. Group 1 is an image set with a style of Western Impressionism, and Group 3 is an image set with the characteristics of Oriental figurative painting. Each of the original art could be judged that each has its characteristics, But in a more essential part, these styles may have something in common. g (3) Artistic or not ( ) It is interesting to note that Group 1 and Group 3 are around or below the median value of 4 for “Artistic-Non artistic.” Few people will rate Monet's original image as unartistic. Chinese hand-painted paintings have also been highly evaluated as elaborately depicting nature. However, Groups 2 and 4 are evaluated as being higher artistic than Groups 1 and 2. This is thought to be due to the young age of the subject. As the younger generation has more opportunities to watch abstract drawings and recent media art, they may have an aesthetic sense appreciating abstract paintings. Also, this means that our principle of using the converted photos with a specific art style instead of the original artworks worked well. By using the original art image for evaluation, it was relatively easy to identify who the artist was, or even the specific work itself, and it is guessed that this had a significant effect on the evaluation. (4) Oriental or Western As shown in Fig. 8, the answers to the question of Oriental or Western are all around the median of 4 except for Group 2. This indicates that the subject did not identify whether Oriental or Western for the artworks in these groups, and gave a response near the middle. Initially, the authors expected that the Sound of Ikebana would be evaluated as "Oriental" because of the overseas evaluation that the artwork contains Japanese beauty. But so far, such a result was not obtained. At the same time, Monet-style images and Chinese Gongbi-style images were evaluated as intermediate. This seems to indicate that at this time, the art style extracted by AI has not yet reached a level to identify Western or Oriental impression. 7. Conclusion In this paper, a new method of handling art with AI was described by using GANs to investigate where is the difference in art style, and what is the essence of the difference in aesthetic sense between Oriental and Western beauty. By using the method proposed in this paper, subjects evaluate the styles of Western and Oriental figurative and abstract paintings without bias created by identifying specific artists and/or artworks. As a result, it was shown that the figurative drawings of the Orient and the West are not very different. Also, it was shown that one of the authors' works "Sound of Ikebana," has no significant difference from the abstract drawings of the Western abstract drawings. However, it is not enough to clarify why the Sound of Ikebana is evaluated by Westerners as Oriental in the scope of this study. It is future work to clarify this. Also, it is necessary to use Western subjects to know the difference of sensitivity between Oriental and Western people. Oriental and Western people. 4. K. Freedman, “Judgement of Painting Abstraction, Complexity, and Recognition by Three Adult Educational Groups,” Visual Arts Research, Vol.14, pp68-78 (1988). 3. Naoko Tosa, Yunian Pang, Qin Yang, Ryohei Nakatsu, “Pursuit and Expression of Japanese Beauty Using Technology,” Arts journal, MDPI, Vol.8, No.1, 38, DOI (2019). 5. https://en.wikipedia.org/wiki/Gongbi Reference 1. Antonia Creswell, et al., “Generative Adversarial Networks: An Overview,” IEEE Signal Processing Magazine, Vol.35, No.1, pp.53-65 ( 2018). 2. Jun-Yan Zhu, Taesung Park, Phillip Isola, Alexei A. Efros, “Unpaired Image-to-Image Translation using Cycle-Consistent Adversarial Networks,” The IEEE International Conference on Computer Vision (ICCV), pp.223-2232 (2017). 3. Naoko Tosa, Yunian Pang, Qin Yang, Ryohei Nakatsu, “Pursuit and Expression of Japanese Beauty Using Technology,” Arts journal, MDPI, Vol.8, No.1, 38, DOI (2019).
https://openalex.org/W2940440479	https://www.graphyonline.com/archives/IJEES/2017/IJEES-141/article.pdf	English	null	The Effect of Living Wall Systems on the Heat Resistance Value of the Façade	International journal of earth & environmental sciences	2,017	cc-by	4,566	Introduction programs were used for that. The result of the performed showed, that the heat resistance value can be increased by 23.7 % by façade greening. As the paper [14] shows, the relative reduction of the energy demand caused by green roofs depends on the building itself. A well- insulated roof shows a lower reduction of the heat demand. The same is true for facades. The cities all over the world are growing. According to the projections of the UN in 2014, the amount of people who live in cities will grow up to 66 % in 2050 [1]. To provide housing for the future occupants, green areas will be reduced in the city and be replaced by buildings. This affects the life quality of urban inhabitants in a negative way. To set plants there is less area on the ground of the cities. A chance could be to set plants on buildings. Therefore the facades of the building could be used. In [15] the green wall was simulated with the UFORE simulation program [20]. This program could not give an output of the total reduction of the heat demand because of façade greening. But it was possible to find out, that the wind speed behind the greening is reduced. This reduces the heat demand. Additional to optical affects, the plants also influence the microclimate that surrounds them. They cool their surroundings on hot days, regulate the relative humidity, bind dust, reduce noise and provide habitat for animals [2-9]. Mahmudul Hasan showed in his master thesis [16] that green facades show a resistance value by 6.16 m².K.W-1. This is the sum of the heat resistances from the materials of a living wall system. The façade of the building and air gap behind the living wall are not considered. But the effect of the air gap, which is in contact with the exterior air, reduces the heat resistances dramatically. This is why the heat resistance value is that high in this thesis. The thesis also investigates the temperatures in the ventilation gap but focuses on summer temperatures and the cooling effect of green walls. The heat resistance value is not calculated in detail. Many product manufacturers of façade greening systems affirm that their systems also reduce the heating requirements of the building. So far there is no general proof for that. The published information is based on simulations. Corresponding Author: Prof. David Tudiwer, Vienna University of Technology, Institute for Building Construction and Technology, Karlsplatz 13, 1040 Wien, Austria; E-mail: david.tudiwer@tuwien.ac.at Introduction Usually only a few of several components which effect the thermal resistance of the façade are measured and the others are estimated. In [10] the gain of the thermal insulation was estimated based on the wind speed in the ventilation gap. Because of the measurements the authors of [10] found out, that the wind speed behind the green system was lower than 0.2 m/s. This is a wind speed which is usually at interior constructions. Considering this fact at the exterior surface resistance was equalized with the interior surface resistance. For the interior and the exterior surface resistance there exists a standardization which is explained in EN ISO 6946 [11]. Based on this standardization the interior surface resistance is higher than the outdoor surface resistance value by 0.09 m².K.W-1. The result of the authors of [10] is the improvement of the thermal resistance value because of the greening by 0.09 m².K.W-1. In [12] the total thermal resistance value of a green wall, is estimated to be 0.52 m².K.W-1 when the whole system (not only the reduction of the wind speed) is taken into account. This means that a façade is insulated better by the resistance of 0.52 m².K.W-1 when it gets greened. In other words, this publication suggests that the thermal resistance is increased by 0.52 m².K.W-1 through an applied facade greening. Note that this estimation integrates the overall improvement, not alone the improvement based on wind speed consideration. Green roofs are better known than green facades. In [17] five different green roof systems were investigated on a test cell in Greece. The dimensions of the cell are 8.4 m × 3.8 m × 3.6 m. On the top of the cell different roof greening systems were installed with the size of 0.6 m × 1.2 m. The heat flux was measured and simulations were performed. According to that simulations the energy savings for heating because of a green roof are 21.1 % - 28.4 % in comparison with a non-insulated roof. This contribution investigates the effects of façade greening systems on the heat flux through the facade in the winter (during the heating Citation: Tudiwer D, Korjenic A (2017) The Effect of Living Wall Systems on the Heat Resistance Value of the Façade. Int J Earth Environ Sci 2: 141. doi: https:// doi.org/10.15344/2456-351X/2017/141 Copyright: © 2017 Tudiwer et al. Abstract Received: September 20, 2017 Accepted: November 15, 2017 Published: November 17, 2017 This paper shows how façade greening effects the heat resistance value on an old building. The façades of the building are made out of bricks. Two greened façades with different living wall systems have been researched and measured for one heating season. At the same time two not greened parts of the façade were measured. So far there does not exist any general method, how to calculate the U-value or particular heat resistance values of the greened facades. The method which is developed within this research-paper makes the comparison between the greened part of the façade and the not greened part possible. It turned out that there is a difference of the thermal resistance between greened and not greened parts of the façades in winter. The improvement by the greening will be discussed within this paper. Keywords: Façade greening, Heat transmission coefficient, Heat demand, Heat transfer, U-value Façade greening, Heat transmission coefficient, Heat demand, Heat transfer, U-value Tudiwer et al, Int J Earth Environ Sci 2017, 2: 141 https://doi.org/10.15344/2456-351X/2017/141 Tudiwer et al, Int J Earth Environ Sci 2017, 2: 141 https://doi.org/10.15344/2456-351X/2017/141 Tudiwer et al, Int J Earth Environ Sci 2017, 2: 141 https://doi.org/10.15344/2456-351X/2017/141 Case Report International Journal of Earth & Environmental Sciences Open Access The Effect of Living Wall Systems on the Heat Resistance Value of the Façade D id T di d A K j i ç David Tudiwer* and Azra Korjenic Vienna University of Technology, Institute for Building Construction and Technology, Karlsplatz 13, 1040 Wien, Austria Publication History: Tudiwer et al, Int J Earth Environ Sci 2017, 2: 141 https://doi.org/10.15344/2456-351X/2017/141 Introduction 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. In [13] several simulations were performed to investigate the effect of green facades on heat resistance value. Different simulation Int J Earth Environ Sci ISSN: 2456-351X Int J Earth Environ Sci ISSN: 2456-351X IJEES, an open access journal Volume 2. 2017. 141 c A (2017) The Effect of Living Wall Systems on the Heat Resistance Value of the Façade. Int J Earth Environ Sci 2: 140. doi: https:// 2017/140 Citation: Tudiwer D, Korjenic A (2017) The Effect of Living Wall Systems on the Heat Resistance Value of the Façade. Int J Earth Environ Sci 2: 140. doi: https:// doi.org/10.15344/2456-351X/2017/140 Page 2 of 5 Page 2 of 5 Figure 2: Schematic representation of System B. Page 2 of 5 Figure 2: Schematic representation of System B. period). Also temperatures were measured on two different points of each façade (one part is greened, one is not greened) to calculate the heat resistance values (details see in chapter 2). The heat flux of the façade was measured on two different parts of the façade, one part was greened, the other one not. So it was possible to compare the greened part of the facade with the not greened part. Two different locations and two different greening systems were investigated. The two locations are in Vienna in urban surroundings. In this area the heating degree is 2768 Kd within one year [18]. This contribution is a demonstration of the first u-value in situ measurement that was taken on greened facades [19]. The results which are shown in this paper are calculated with simple formulas, based on the measurement and not simulated. Office building of the Municipal Department MA 48 The greening system installed on the facade of the MA48 building is based on aluminum troughs. This system will be referred to as "System Figure 1: Schematic representation of System A. Figure 3: Schematic representation of the installed sensors. A" from now on. The troughs are connected with each other, thereby forming a curtain façade (see Figure 1). The distance from the house façade is determined by the thickness of the metal rails. In this case it is 6 cm. The building is situated in urban surrounding. The measuring points of the façade are faced to south and to a big street with several lanes. This causes that the location is exposed to sun and wind. A" from now on. The troughs are connected with each other, thereby forming a curtain façade (see Figure 1). The distance from the house façade is determined by the thickness of the metal rails. In this case it is 6 cm. The building is situated in urban surrounding. The measuring points of the façade are faced to south and to a big street with several lanes. This causes that the location is exposed to sun and wind. 2. School Kandlgasse 39 (GRG 7) The detailed features of the green facades and the measurement setup are described below. The detailed features of the green facades and the measurement setup are described below. Within the following two paragraphs the investigated walls are introduced. 1. Office building of the Municipal Department 48 (MA 48) 2. School Kandlgasse 39 (GRG 7)h 2. School Kandlgasse 39 (GRG 7) Matherials and Mehtode The investigated greening systems were applied via curtain-wall technologies (including an air space). This is shown in Figure 1, Figure 2 and Figure 3. They are installed on rails, which are fixed on the façade of the building. The vegetation on the walls are herbs and grasses. Figure 2: Schematic representation of System B. Figure 2: Schematic representation of System B. Figure 3: Schematic representation of the installed sensors. The two locations which were investigated are: 1. Office building of the Municipal Department 48 (MA 48) School Kandlgasse 39 (GRG 7) This location is a grammar school. The greening system is a different system (here it is called System B) than the system that is referred to see above in (Figure 1). The plants grow horizontal out of aluminum modules. The roots of the plants are in direct contact with a capillary fleece, which distributes the water evenly. The substrate is inside the modules. They are mounted on metal rails, which are the connection to the façade (see Figure 2). The measuring points of the façade are faced to south into an inner court yard. This causes that System B is less exposed to wind. But as it is faced to south it is also exposed to the sun. Figure 1: Schematic representation of System A. Int J Earth Environ Sci ISSN: 2456-351X IJEES, an open access journal Volume 2. 2017. 141 Citation: Tudiwer D, Korjenic A (2017) The Effect of Living Wall Systems on the Heat Resistance Value of the Façade. Int J Earth Environ Sci 2: 140. doi: https:// doi.org/10.15344/2456-351X/2017/140 Citation: Tudiwer D, Korjenic A (2017) The Effect of Living Wall Systems on the Heat Resistance Value of the Façade. Int J Earth Environ Sci 2: 140. doi: https:// doi.org/10.15344/2456-351X/2017/140 Page 3 of 5 Page 3 of 5 Following filters where used to find the valid data: 1. Only the measurement period during the heating season was taken into account. The reason for that is, that u-values can be measured with a higher accuracy during high temperature differences between indoor and outdoor. 1. Only the measurement period during the heating season was taken into account. The reason for that is, that u-values can be measured with a higher accuracy during high temperature differences between indoor and outdoor. 2. 2. 2. Because of the storage mass of the façade the valid data need to have a high temperature difference in average between the air temperature outdoor and indoor, within the last 24 hours, the last 48 hours and the last 72 hours. This temperature was chosen to be above 10°C. There was also no moment within the last 24 hours, when the temperature difference decreased below 0°C (this happens very rarely during this measuring period, when the temperature outside is higher than inside). 2. Because of the storage mass of the façade the valid data need to have a high temperature difference in average between the air temperature outdoor and indoor, within the last 24 hours, the last 48 hours and the last 72 hours. This temperature was chosen to be above 10°C. There was also no moment within the last 24 hours, when the temperature difference decreased below 0°C (this happens very rarely during this measuring period, when the temperature outside is higher than inside). The sensors that were used for the measurements are described within the following lines. The Lin Picco A05 [21] is used to measure the air temperature and the air humidity. A picture of that sensor is shown in (Figure 4) left. The temperature of the surface of the façade was measured by PT 1000 shown in (Figure 4) right. The accuracy of this sensor is 0.1 K. It is glued on the façade and painted by façade color. 3. The measured heat flux was filtered to be above 0 W.m-2. The heat flux is measured on the surface of the construction and reacts very fast on air temperature conditions inside. When for example a window near the measuring station is opened, the heat flux decreases very fast. Even in winter it might happened that it decreases below 0 W.m-2 (because the construction temperature is above the air temperature inside). This data lead to wrong analyses. Monitoring Equipment This plastic cover is the protection for the sensor. Exterior the Lin Picco A05 is also protected by a protective shield (shown in Figure 5 middle). The effect of greened façades on the heat flux depends on the construction of the house. The impact of the improvement by greening systems can be considered to be more significant if applied on walls that show a high thermal transmittance in their original state. Because of that, the whole construction need to be investigated. One difference to the method that was used in [5] is, that here the surface resistances themselves were measured. This demands the measurement of air temperatures, heat flux and surface temperatures as shown in (Figure 3). The heat flux, the air temperature (outdoor and indoor) and the surface temperature on the façade interior and exterior was measured to get the needed information of the thermal insulation of the façade. It was measured at the same building, on the same façade on different parts (one part that is plain and the other one is greened). The heat flux measurement was carried out by a heat flux film which was fixed with adhesive tape on the wall. The adhesive tape closes the border of the heat flux film airtight. The heat flux film is measures the power of heat that leaves the room into the wall. The specific power is displayed by W.m-2. The accuracy of this device is 5 %. Following filters where used to find the valid data: Because of this reason they were filtered out of the data pool. The Lin Picco sensor has a protection cover made out of plastic. It is shown in Figure 5 left. s shown in Figure 5 left. Figure 4: Left Lin Picco A05: Used to measure air temperature and air humidity. Right PT 1000: Used to measure the temperature of the surface of the façade. Figure 5: Left: The Lin Picco sensor inside (protected by a plastic cover). Middle: Lin Picco sensor exterior protected by protective shield. Right: Heat flux film. Figure 4: Left Lin Picco A05: Used to measure air temperature and air humidity. Right PT 1000: Used to measure the temperature of the surface of the façade. 4. 4. Also the temperature fluctuation around the investigated façade should be very low within the last 24 hours. This way the conditions are more stationary. The difference between the air indoor and outdoor and the surfaces of the façade interior and exterior were below 2°C within the last 24 hours. This means that an increase of the indoor temperature of 3°C would only be taken into account, when the outdoor temperature also increases at least by 1°C. This way the interior and exterior temperature difference of the façade stays below 2°C. Otherwise the data is not considered because of too high fluctuation. This causes almost stationary conditions, comparable with the conditions in a laboratory. Figure 4: Left Lin Picco A05: Used to measure air temperature and air humidity. Right PT 1000: Used to measure the temperature of the surface of the façade. Figure 5: Left: The Lin Picco sensor inside (protected by a plastic cover). Middle: Lin Picco sensor exterior protected by protective shield. Right: Heat flux film. 5. MA48 – System A: The shown data are the average of 395 different measuring cycles. 6. GRG7 – System B: The shown data are the average of 1.389 different measuring cycles Results and Discussion In this chapter the measuring data are shown. The heat flux (Figure 6) illustrates the transmission losses through the façade. Given that the indoor temperatures can be considered as identical, the relationship between the heat flux values can be understood as the performance difference between greened and non-greened wall parts. In the building MA48 both parts of the façade (greened and not greened) are measured in the same room. In GRG7 two different rooms are necessary to measure both conditions (greened and not greened). Figure 5: Left: The Lin Picco sensor inside (protected by a plastic cover). Middle: Lin Picco sensor exterior protected by protective shield. Right: Heat flux film. Int J Earth Environ Sci ISSN: 2456-351X Int J Earth Environ Sci ISSN: 2456-351X IJEES, an open access journal Volume 2. 2017. 141 Citation: Tudiwer D, Korjenic A (2017) The Effect of Living Wall Systems on the Heat Resistance Value of the Façade. Int J Earth Environ Sci 2: 140. doi: https:// doi.org/10.15344/2456-351X/2017/140 Page 4 of 5 The formula to calculate the heat transfer coefficient (U-value). The outputs of Eq. 4 are shown in Figure 7. Figure 6: Heat flux of the measured walls Figure 7: The calculated U-values The formula to calculate the heat transfer coefficient (U-value). To consider the different temperatures in the room, or parts of the rooms and to get absolute values to compare between the greened and not greened parts of the facades, the resistances of the heat conductivity are calculated (formulas shown in Eq. 1, and Eq. 2). One of the resistances is defined by [11]. It is RSI = 0.13 m².K.W-1. The formula to calculate the heat transfer coefficient (U value). Figure 6: Heat flux of the measured walls Eq. 1 shows the way the resistances of the facades were calculated, by the measured data. The interior surface resistance is not changed because of the greening. The same is true for the resistance of the facade, but as the wall might be different (because in GRG7 two different floors are investigated). Eq. 2 shows the formulas to calculate the exterior surface resistances by the measured data. In [11] this value is definite with RSE = 0.04 m².K.W-1. But our measurements showed that the RSE is much higher than in the definition of [11]. It is about 0.11 m².K.W-1 at the MA48 and about 0.40 m².K.W-1 at GRG7. The exterior surface resistance value The exterior surface resistance value Conclusion The additional heat resistance of the greening system is considered in the RSE. The exterior air temperature is much lower than the temperature of the façade behind the greening system, because the greening system protects the façade and composes a buffer that is wormer than the exterior temperature. This causes a higher surface temperature of the façade behind the greening system. The average surface temperature of the blade façade in GRG7 is 2.80°C. Behind the greening system is 3.33°C even though the greening system shadows the sensor. In MA48 the surface temperature of the blade façade is 4.75°C in average and behind the greening the average surface temperature of the face is 8.27°C. The average temperatures at MA48 are higher because the measuring points are closer to the ground. They are at the level of the ground floor. In GRG7 they are at the level of the 3rd floor. This paper shows the increase of the heat resistance value (or the reduction of the U-value) because of façade greening. In the case of the investigated constructions the gain of thermal insulation is about 20 %. But this gain depends very highly on the construction of the façade itself. If there would be an insulation with XPS, the greening system might cause thermal bridges. It is obvious that the size of the ventilation gap is important for the results. Int J Earth Environ Sci ISSN: 2456-351X Units q = Heat flux = W.m-2 Rwall + RSE + RSI = m².K.W-1 U-value = W.m-2.K-1 Δ d l f (i i i ) °C q = Heat flux = W.m-2 Rwall + RSE + RSI = m².K.W-1 U-value = W.m-2.K-1 q = Heat flux = W.m-2 Rwall + RSE + RSI = m².K.W-1 U-value = W.m-2.K-1 Δtwall = delta surface temperature (interior – exterior) = °C Δtwall = delta surface temperature (interior – exterior) = °C ΔtSE = delta of air temperature (exterior) and surface temperature exterior = °C Rwall = Δtwall . q-1 The outputs of Eq. 4 are shown in Figure 7. The way to calculate the heat resistance value of the wall. The way to calculate the heat resistance value of the wall. The results show that there is a difference of the greened and the not greened part of the façade. System A increases the thermal resistance by 0.31 m².K.W-1. System B raises the thermal resistance by 0.67 m².K.W-1. RSE = ΔtSE . q-1 Results and Discussion The reason for that is, that both locations are urban and in a wind shadow zone. In [11] a wind speed of 4 m/s is assumed for the calculation. In GRG7 the RSE is higher because the façade is in an inner court yard. At MA48 the façade is faced to an open street with a wide free area. The outputs of Eq. 4 are shown in Figure 7. Figure 7: The calculated U-values Discussion In situ measurements of the U-value are not very accurate. Its results depend on different parameters. These parameters are for example the storage mass of the construction, the fluctuation of the temperature and the user behavior (the buildings are occupied and used) as ventilation, opened room doors or waste heat of computing and human. The way the data are filtered for the analysis in this paper is unique. There is no long year research to confirm that the methodic is without any mistake. The sum of the heat resistances (see Eq. 3) are the total heat resistance. Its inverse is the heat transfer coefficient U-value, see Eq. 4. Rtot = Rwall + RSI + RSE The formula to calculate the total heat resistance of the whole construction. Beside that only 1 dimensional heat flow is considered. References 1. World Urbanization Prospects, The 2014 Revision; Published by the United Nation 2014 1. World Urbanization Prospects, The 2014 Revision; Published by the United Nation 2014 2. Hoelschera MT, Nehlsa T, Jänickeb B, Wessolek G (2016) Quantifying cooling affects of facade greening: Shading, transpiration and insulation. Energy and Buildings 114: 283-290 3. Cameron RWF, Taylor JE, Emmett MR (2014) What’s cool in the world of green façades? How plant choice influences the cooling properties of green walls. Building and Environment 73: 198-207 4. Rakhshandehroo M, Yusof MJM, Najd MD (2015) Green façade (Vertical Greening): Benefits and Threats. Applied Mechanics and Materials 747: 12-15 5. Haggag M, Hassan A, Elmasry S (2014) Experimental study on reduced heat gain through green facades in ahigh heat load climate. Energy and Buildings 82: 668-674 6. Nori C, Olivieri F, Grifoni RC,Bedoya C (2013) Testing the performance of a green wall system on an experimental building in the summer PLEA 2013 - 29th Conference. Sustainable Architecture for a Renewable Future 7. Jaafar B, Said I, Rasidi MH (2011) Evaluating the Impact of Vertical Greenery System on Cooling Effect on High Rise Buildings and Surroundings: A Review. Senvar 8. Sheweka SM, Mohamed NM (2012) Green Facades as a New Sustainable Approach Towards Climate Change Energy Procedia 18: 507-520 9. U. Pitha et. al. Leitfaden Fassadenbegrünung (2013) https://www.wien. gv.at/umweltschutz/raum/pdf/fassadenbegruenung Leitfaden. 10. Perini K, Rosasco P (2013) Cost benefit analysis for green façades and living wall systems, Building and Environment 70: 110 -121 11. EN ISO 6946 Bauteile Wärmedurchlasswiderstand und Wärmedurchgangskoeffizient – BerechnungsverfahrenBerechnungsverfah ren für Wärmedurchlass – hinterlüftet Luftschichten (2015) 12. Perini K, Ottelé M, Haas EM, Raiteri R (2011) Greening the building envelope, façade greening and living wall systems. Open Journal of Ecology 1: 1-8 13. Pan XC, Xiao YX (2014) Simulation Analysis of Building Green Facade Eco-Effect. Applied Mechanics and Materials 548-549: 1701-1705 14. Gupta A, Hall MR, Hopfe CJ, Rezgui Y (2011) Building integrated vegetation as an energy conservation measure applied to non-domestic building typology in the UK, Proceedings of Building Simulation 14-16. 15. Bass, Green Roofs and Green Walls (2007) Potential Energy Savings in the Winter, Research Gate 16. Hasan MH (2013) Investigation of Energy Efficient approaches for the energy performance improvement of commercial buildings. Master thesis 17. Kotsiris G, Androutsopoulos A, Polychroni E, P.A. Nektarios PA (2012) Dynamic U-value estimation and energy simulation for green roofs. Energy and Buildings 45: 240-249 18. U-value = heat transfer coefficient = 1.Rtot -1 U-value = heat transfer coefficient = 1.Rtot -1 IJEES, an open access journal Volume 2. 2017. 141 Int J Earth Environ Sci ISSN: 2456-351X Int J Earth Environ Sci ISSN: 2456-351X Citation: Tudiwer D, Korjenic A (2017) The Effect of Living Wall Systems on the Heat Resistance Value of the Façade. Int J Earth Environ Sci 2: 140. doi: https:// doi.org/10.15344/2456-351X/2017/140 Page 5 of 5 20. https://www.nrs.fs.fed.us/tools/ufore/ Within the next steps the ventilation gap (size of the opening holes, distance between facades and greening construction) will be investigated more. The effect of the size of the opening hole and wideness of the gap on the U-value will be researched. 21. http://uk.farnell.com/ist-innovative-sensor-technology/linpicco-tm- basic-a05/sensor-humidity-module-0-5v/dp/9596119 Investigations of the thermal bridges because of fasteners. Simulations of the air streaming in the ventilation gap with computer programs. Competing Interests The authors declare that they have no competing interests. References Bundesministerium für Wissenschaft, Forschung und Wirtschaft Austria (2016) 19. Tudiwer D, Korjenic A (2017) The effect of living wall systems on the thermal resistance of the façade. Energy and Buildings 135: 10-19 IJEES, an open access journal Volume 2. 2017. 141 IJEES, an open access journal Volume 2. 2017. 141 Int J Earth Environ Sci ISSN: 2456-351X
https://openalex.org/W2146170170	https://rua.ua.es/dspace/bitstream/10045/2801/1/ETFA-07.pdf	English	null	Embedded web services for industrial TCP/IP services monitoring	null	2,007	cc-by	5,633	Abstract In this paper we present an approach based on the use of embedded network devices for the deployment of small network services suitable for IT management in industrial and manufacturing environments. The novelty of the proposal resides in the fact that allows to unfold IT services that can be deployed without the need for specialized IT staff, since the services in question have a zero maintenance design philosophy. They are presented under both conventional (client-server) and more open (SOA) standards, more specifically, as Web Services. To illustrate the specific the proposal, we have chosen as an example an embedded device able to monitor the correct operation of the applications and IT services located in the current manufacturing equipment and components. Our proposal consists of providing embedded IT management services in physical network devices — generally, small sized devices with simple services—, so that, in order to deploy those services, it is enough to select the specific device providing the service, and connecting it to the communications network. The device itself will obtain the minimum information required to activate the initial set up and, once this has been completed, execute the management tasks with minimal human intervention. In addition, since the service is provided from a physical device, it does not set in motion too many additional maintenance tasks relating to the infrastructures providing support for the new IT services. Embedded Web Services for Industrial TCP/IP Services Monitoring Virgilio Gilart-Iglesias University of Alicante Technology and Computer Science Department PO Box 99, 03080, Alicante, Spain vgilart@dtic.ua.es Diego Marcos-Jorquera University of Alicante Technology and Computer Science Department PO Box 99, 03080, Alicante, Spain dmarcos@dtic.ua.es Diego Marcos-Jorquera University of Alicante Technology and Computer Science Department PO Box 99, 03080, Alicante, Spain dmarcos@dtic.ua.es Francisco Maciá-Pérez University of Alicante Technology and Computer Science Department PO Box 99, 03080, Alicante, Spain pmacia@dtic.ua.es Francisco Maciá-Pérez University of Alicante Technology and Computer Science Department PO Box 99, 03080, Alicante, Spain pmacia@dtic.ua.es innovative technologies in manufacturing levels [4, 5, 6] with new production machinery incorporating IT elements [7, 8], ranging from the simplest, to the most complex. This situation is further aggravated by the fact that organisations run itself into a problem in training their employees or finding professionals with the requisite profile and skills. 2. Background The integration of manufacturing processes in the general organisation process map thus achieving continuity is one of the main goals of the industrial sector [9]. Due to physical and technological limitations, manufacturing processes have not reached the desired level of integration and automation, and in most cases they have to be considered as legacy systems. Until quite recently, integration proposals were centred on traditional automation models based on proprietary protocols situated at a resource level of the eBusiness model as systems external to business processes (Modbus, Profibus, AS-I, FIPIO, DeviceNET, Interbus or Ethernet industrial). These proposals were the first attempts to facilitate their integration with business components using ad-hoc adaptors [10]. The first open standards which attempted to address problems of IT management in a global manner were Simple Network Management Protocol (SNMP) and Common Management Information Protocol (CMIP) [15], proposed by the Internet Engineering Task Force (IETF); both protocols being principally oriented towards network monitoring and control. The main inconvenience of these administration models was their dependence on the platform. Based on these, and seeking an integration with heterogeneous systems, two principal lines of work arose: an attempt to achieve integration of the systems using the same network management protocol, as is the case of [16] and [17] with the use of Common Object Request Broker Architecture (CORBA); and more ambitiously, to propose a network management protocol which would be independent of the infrastructures. Some of the more extensive proposals include: CORBA/JIDM, specification of the work group Joint Inter-Domain Management (JIDM) [18] of the Object Management Group (OMG) [19]; CIM/WBEM, proposal of the Distributed Management Task Force (DMTF) [20] using techniques oriented towards Computer Integrated Manufacturing (CIM) objects and interoperation using HTTP and XML with Web Based Enterprise Management (WBEM); JMX specification defined by the Java Community Process (JCP) [21] which defines a series of Application Programming Interfaces (API) oriented to Java for network management; and WS- Management specification carried out by various companies in the sector (Sun, Intel, MS, AMD) for the integration of service management systems and resources based on Web Services. With the development of internet and electronic advances, solutions have been proposed based on service paradigms, distributed systems and embedded devices with the computational and communications capacity suitable for environments with hostile physical conditions such as those occurring in industrial atmospheres. 1. Introduction Internet capacity for providing clients with a choice of the most up to date and available consumer goods and at competitive prices is currently requiring the industrial sector to progress from traditional mass production manufacturing paradigms towards models which facilitate mass customization [1]. Obviously, from a functional point of view the services offered by these devices are totally compatible with the traditional network services and therefore their integration and interoperability are ensured. More precisely the services implemented are compatible with standard Web Services and with other more traditional client-server protocols within the scope of systems management such as: telnet, TFTP, HTTP or SNMP. With these new production models, the customer is no longer considered as a mere entity, external to the manufacturing process itself, but rather, becomes a part of it as an active component, determining the specific characteristics of the desired product. By way of illustration and with the aim of arguing the motivating of the proposal, in this work we suggest a specific management service: a service for the network service monitoring (NSM) build in the current manufacturing equipment as well as the physical device associated with that service (eNSM device). The goal of this service is to reduce stoppage times in the event of failures and discontinuity in the production process. In order to ensure that these manufacturing models are viable, the manufacturing processes must be fully integrated in the organisation global process map [2]. Although new technologies may help considerably in this aim, it is also true that they require a change of scenario for which not all organisations are completely ready. [3]. The basic function of the service will be to indicate to the eNSM device which equipment and which service or services of that equipments we wish to check in order to assure that they are operating correctly (generally by using an IP address, a port and the expected result) If One of the main problems faced by these organisations is the emergence of new management tasks deriving from the introduction of complex and there is any incident the device will register it and report in that respect. significant results and are progressing towards Service- Oriented Architectures (SOA) and embedded devices in industrial machinery as valid technologies [7, 14]. 1. Introduction In the following sections we provide a review of the current state of the art of the technologies involved, describing the NSM service, the hardware and software structure of the device in which it is embedded, the application protocol specified and its implementation as Web Service. Finally, the conclusions on the research and our current lines of work are exposed. The scientific community is clearly interested in the use of IT paradigms which have established the present WEB bases as a technological framework which supports the execution of processes associated with production elements. However, as information technologies continue to inundate production plants increasingly further new associated tasks arise, namely the management of the new services and infrastructures used. These tasks are gaining greater importance as the organisation becomes increasingly reliant on IT, requiring the same levels of robustness and security as in the industrial sector. 2. Background As a result of the considerable number of tasks associated with network management as well as its diversity and complexity, the work of maintaining these systems involves a high cost for organisations both in terms of resources and also in terms of time and personnel; added to this are the difficulties inherent in engaging staff with the required skills for addressing this new scenario. The monitoring procedure consists of establishing connections with the services and applications to be monitored by means of its own protocols based on TCP/IP, analysing the responses to standard requests in search of errors, either in the operation of the protocol itself or in the data received. In order to relieve these problems, the current trend in IT management is to use outsourcing as a strategy to recoup investments, ensure the continuous availability of infrastructures and services, and to achieve sufficient levels of quality to enable organisations to keep abreast of a changing environment. However, outsourcing is not usually a valid strategy for handling production environments due to problems raised by security privacy and immediacy. The eNSM device represents a basic part of the system. Figure 1 shows a diagram of the main elements and actors involved in the service, together with the existing relation between them. We may synthesise these as: eNSM Device, Manufacturing Components, Discovery Service, NSM Center, NSM Clients and a set of Software Agents. In areas where automated handling of information and those where several devices are involved, such as industrial processes or domotics, there has been a trend in the development of autonomous management towards architectures designed for services for embedded systems [12, 14]. This final framework includes monitoring systems developed by third parties but residing with the client, who is responsible for their control and management. Along these lines we find proposals such as NAGIOS [27], MON [28], MUNIN/MONIT [29, 30] or nPULSE [31] generic monitoring systems for network services for Linux, with Web interface, highly configurable and based on open code which monitors the availability of network services and applications. The disadvantage of these proposals lies in the complexity of their installation and configuration in environments without qualified system administrators, in addition to the complex systems and infrastructures required for their implementation. g Figure 1. Organization of functional elements of the NSM service. Figure 1. Organization of functional elements of the NSM service. 2. Background Schneider was one of the first manufacturers of automation and industrial control devices to have incorporated these ideas in its automation apparatus in order to communicate with management applications. This trend is reflected in concepts such as transparent factory [11]. Other manufacturers such as ABB, go somewhat further by raising communication to higher levels of organisation using the Simple Object Access Protocol (SOAP), and incorporating intelligence, self- management and proactivity into its embedded devices [12]. Along the same lines, in [5] the use of Web Services is proposed as a normalised means of accessing functionalities of the devices so that they can be integrated with Enterprise Resource Planning systems (ERP). In [13] is proposed to use these same techniques to raise the level of abstraction of production elements to a business level so that the integration of resources, processes and in general business logic are produced naturally and in a transparent manner within the current business models. The use of multi-agent systems for computer network management provides a series of characteristics which favour automation and self reliance in maintenance processes [22, 23]. The creation of projects such as AgentLink III, the first Coordinated Action on based on Agents financed by the 6th European Commission Framework Programme [24], is a clear indicator of the considerable degree of interest in research into software agents. Within the framework of European research projects there are some important initiatives which bear out our interest in this line of research, and which have produced In [25], a proposal is made for a group of basic operations for a Web Service to be standardised within the management networks as a counterpart to standardisation of the SNMP information model under XML development in other works [26]. This device is small in size, transparent to existing IT infrastructures and with minimum maintenance required from the system administrators. The administrator informs the device which of the equipment’s services or applications need checking. The eNSM device has sufficient knowledge of each service to carry out this task. This knowledge is included in monitoring software agents displaced to the device. In this way if the device receives a request for monitoring a new service, it will be sufficient to request the adequate monitoring of the agent in order to carry out its work. 2. Background The eNSM Device, as may be seen, is the cornerstone of the service. It is designed to act as a proxy between the broad area network and the local area network for which it provides support. Instructions from the WAN consist of orders defined in the NSMP protocol which they use SOAP as transport protocol (it is possible to use other options: HTTP, SNMP or telnet). Communications between the device and the manufacturing components of the services and applications to be monitored shall be made directly using native protocols for each of these services or applications. 3. Description of the Monitoring Service The NSM Centre is also responsible for managing the repository of monitoring agents with the know-how of each monitoring service. The NSM Center usually acts as a control panel for all the possible eNSM devices distributed through Internet. This control is implemented through the planning agent who carries out, executes and verifies all the previously established tasks on the eNSM devices. The NSM Centre is also responsible for managing the repository of monitoring agents with the know-how of each monitoring service. The NSM Client provides the user, through the NSM agent, access to the NSM Center (to manage work plans or query logs files) and to the eNSM Devices (to manage particular services of specifics manufacturing components directly). The NSM Client provides the user, through the NSM agent, access to the NSM Center (to manage work plans or query logs files) and to the eNSM Devices (to manage particular services of specifics manufacturing components directly). repository in the NSM Centre. Figure 2. Sequence diagram of the NSM main functionality. The Software Agents consists of a multi-agent system which is actually responsible for executing all the tasks involved in the system. The Register Agents reside in an eNSM Device and undertake to check the monitoring service in a Discovery Service. The Management Agents reside in an NSM Client and are responsible for providing an appropriate interface for the administrators so that they can access the NSM Centre or an eNSM Device from any node connected to Internet. The Planning Agents reside in the NSM Centres and undertake the planning management of eNSM Devices. The SOA Agents and the CS Agents reside in an eNSM Device and represent the SOA interface or client-server, respectively, which compiles the service requests. The NSM Agents are assigned to an eNSM Device and are responsible for coordinating work requests contained in its Work Plan, selecting the appropriate Monitoring Agents for carrying out the work. The Monitoring Agents are stored in the agent repositories managed by the NSM Centres, and they contain the service logic for each type of application or service requiring monitoring, and they are transferred to an eNSM Device, where they carry out the tasks for which they have been programmed. Finally, the Employer Agents are assigned to an eNSM Device and are responsible for locating the Monitoring Agents required by the device to carry out its task. Figure 2. 3. Description of the Monitoring Service Sequence diagram of the NSM main functionality. 4. Monitoring Service Protocol The NSM service protocol (NSMP) gathers all monitoring service functionality through a set of instructions. The protocol has defined like a request- response text based protocol. This permits that it may become easily adapted to different models, such as client-server (over basic protocols like HTTP, SMTP or telnet) and SOA (over protocols like SOAP). The sequence diagram in figure 2 shows the basic operation of the service and the multi-agent system. The diagram comprises three blocks and is executed constantly in parallel mode. In the first block the device interface agents (SOA Agent and CS Agent) are on standby for requests either from a Planning Agent, or directly from a Management Agent. When they receive a request they attend to it and execute it through the eNSM service kernel. When they receive monitoring requests these are programmed in the Work Plan register of the NSM Agent. The syntactic elements for an instruction are: 3. Description of the Monitoring Service The Embedded Network Service Monitoring (eNSM) service is a Web Service which enables TCP/IP application active monitoring, using for this purpose Internet standard protocols and service-oriented architectures, which render the service regardless of the administrator’s location or the platform used. The main task of the monitoring service is to check that the services and applications contained in the manufacturing components are operating correctly. A network device (known as an eNSM Device) has been designed for this purpose and in which this functionality has been embedded. The Embedded Network Service Monitoring (eNSM) service is a Web Service which enables TCP/IP application active monitoring, using for this purpose Internet standard protocols and service-oriented architectures, which render the service regardless of the administrator’s location or the platform used. The Manufacturing Components are the object of the management and comprise all those devices connected to the network and acting as network services containers, which will be monitored. The main task of the monitoring service is to check that the services and applications contained in the manufacturing components are operating correctly. A network device (known as an eNSM Device) has been designed for this purpose and in which this functionality has been embedded. The Discovery Service comprises a standard UDDI registration service. It is responsible for maintaining the pages describing the NSM services in WSDL format, as well as facilitating that information to the clients wishing to access the service. The third block concerns the contracting of the Monitoring Agents. In blocks 1 and 2, in the event that the monitoring of a service is requested in which the device does not have a Monitoring Agent able to deal with it; both the NSM Agent and the SOA and CS Agents who have detected this lack may make a request to the Employer Agent programming it into its Work Plan. The Employer Agent then undertakes to obtain the Monitoring Agents required by the device. This agent is responsible for negotiating and validating the whole process. The Monitoring Agents are located in the agent repository in the NSM Centre. The NSM Center usually acts as a control panel for all the possible eNSM devices distributed through Internet. This control is implemented through the planning agent who carries out, executes and verifies all the previously established tasks on the eNSM devices. The syntactic elements for an instruction are: CMD defines the service actions and corresponds to the name of the remote procedure which implements the functionality of the NSMP command. ACTION is a special parameter which discriminate the functionality of the request. The second block of the diagram corresponds to the execution of the programmed tasks. In this case the NSM Agent is constantly checking the Work Plan on the basis of which it programmes the different Monitoring Agents to carry out the tasks requested. ARGS represents the necessary information for executing the <action>. Table 1. Main instructions of the NSM protocol The main instructions (showed in table 1) that can be embedded in a NSM requests are: SET, GET, PUT, MONITOR and ALERT. Service-oriented architecture, in particular, Web Services, it has utilizing SOAP as mechanism of messages interchange. This case is a little more complex and it requires some more attention. SET command manages the configuration of the internal system variables which determines their function mode. PUT and GET commands, combined with the SCHDL action, program and obtain, respectively, the agents work planning. GET command, with STATUS action, allows getting specific information from the device state. In SOAP request Document style and RPC style are supported. In the case of Document style each NSM instruction is embedded in the body of a SOAP message which contains the NSMP document, which implements the functionality of the command and the arguments required for its execution. The syntax of NMSP is defined in his correspondent DTD. MONITOR command provides access to the principal service of the device. The instruction syntax to activate a monitoring service is: In the SOAP messages of RPC style, will exist a single operation (EXECUTE) whose single argument is a text string that will contain the instruction NSMP, of very similar form to as it makes in client-server cases. MONITOR ON <host> <port> <time> <service> [args]* As well, in order to disable the monitoring of a service, the instruction format will be: 5. Device Design <host> is the IP address or the name of the device to monitoring In the same way as it has been described previously, the monitoring service is part of a broadest system in which the eNSM device is the cornerstone. The eNSM device combines hardware and software in order to deploy all its functionality, achieving the different requirements that have been established before. <port> identifies the service or application whose status wants to verify. <service> identifies the monitoring agent that must take care of making the analysis of the service. <service> identifies the monitoring agent that must take care of making the analysis of the service. ALERT Command is designed so that the eNSM device could to notify of the error to the NSM center. ALERT Command is designed so that the eNSM device could to notify of the error to the NSM center. For this reason, a device general architecture has been defined (figure 3). This device architecture is layer-based organized and has a widely accepted structure for embedded devices. In the lower layer the device hardware has been defined, based on a computational system including microprocessor, volatile memory (for the execution), non volatile memory (for the stored system) and communication module for its connection to the network. Because is about a request-respond protocol, for each NSMP request there will be a corresponding NSMP response. Basically, this request will be OK, if the order is correctly execute or conversely ERROR if it is not. In some cases the answer can contain more specific information about the realized operation, as GET SCHDL command, which returns the list of tasks programmed. In addition of these basic components, it is pertinent to highlight the absence of mechanic elements (as hard disks) and the inclusion of auxiliary elements (as watchdog mechanism or Power over Ethernet supply). These instructions must become embedded inside the specific request/response protocol that it be used as transport layer. In the case of choosing a telnet service, instructions and the result of his execution will be literal. In the case of HTTP, the instructions will have to go inserted in a HTTP request body. In strategies to support On the hardware layer is placed the embedded operative system. In order to satisfy industrial environment specific requirements —where the device will be included and will provide support— real time operative systems has been chosen. Figure 3. eNSM device architecture. The first two important elements in this layer comprise different network services, commonly used in the management field, under the client-server model and services oriented architectures (SOA). These modules give basic services so the device can communicate, at the application level, with other external components. In order to make suitable this communication to the syntax of monitoring service instructions, the NSMP protocol is implemented on these modules (in both SOA and CS versions). One of the other important services placed in this layer is the core which contains all the procedures offered by the NSM services. In the same layer is located the middleware platform in order to provide support to the service software agents. These agents are placed in the last layer (application layer) and really, they take charge of provide the service: acting as interface with other services and applications, registering the service in a discovery service, coordinating the monitoring service or, simply, monitoring selected services. Figure 4. Architecture of the eMSM device prototype. Among other I/O interfaces, the device has a Fast Ethernet network interface which allows suitable external communications ratios. In addition, in order to ensure the correct system operation there are several auxiliary elements such as: a watchdog which monitors the CPU and prevents it from blocking; and a PLL frequency divider required to set up the frequency of the system clock with an adjustable clock signal (CLK) to optimise consumption or performance according to needs. 5. Device Design The following layer contained the middleware platform to provide services to the applications. picture of an eNSM device prototype connected to the service network can be observed ). The various memory modulates provided by this device undertake specific tasks according to their intrinsic features: the execution programmes and the dates handled by the device SRAM memory it resides in the (1,25MB); the ROM memory (16KB) holds the system start up application and, finally, the flash memory, with 4MB, stores information which though non-volatile, is susceptible to change, such ace the Sep up of the eNSM device or the system applications which may be updated. These capacities are sufficient for the memory requirements of the software developed for implementing the protocol. Figure 4. Architecture of the eMSM device prototype. 7. Conclusions In this paper we have presented a system for the provision of IT services directed to manage applications and embedded services in machinery and components of production in the industry. The proposal aims to provide a reference frame in which we could design and implement this kind of service in a systematic way. One of the most relevant aspects of this system consists of providing these embedded management services in network devices. The devices must be adapted to the characteristics of the production and IT environments: small size, simple, low power consumption, adjusted costs, autonomous, designed with safety criteria and robustness, and compatible with the traditional network services through the standard protocols such as: SOAP, SMTP or HTTP. With the aim to validate the proposal, a concrete service has been designed and implemented in order to monitoring the IT embedded services in the current components of manufacture, together with functional prototype of the network device. The communication service middleware is upheld by standard protocols and technologies included in the Evolution OS. In the CS based NSMP implementation, a C module has been developed to provide a protocol syntactic analyser. In the SOA based NSMP implementation, development has been done using the cSOAP library, which is appropriate for these devices [32]. However, some changes have been done in the original cSOAP library due to device limitations (restriction of memory use, proprietary libraries, etc.). These limitations have forced us to replace cSOAP XML parser, LibXML2 (more than 1 MB in size), by another adapted XML parser with limited but enough functionalities to achieve our objective. Due to cSOAP limitations, only RPC style that use the same protocol analyser used in the client-server version has been developed. Besides, in order to registry and to publish the services, an UDDI embedded version has been implemented based in UDDI version 2.0 which simply permits publishing the WSDL document associated with the monitoring service. Figure 6 shows a fragment of the WSDL page with the definition of the RPC procedure for the EXECUTE command. We are currently working with other embedded network services and integrating them all in a model based on Semantic Web services, so that in future they will not only be compatible with existing services, but also with new services or setups which were not considered in its initial design. 6. Device Implementation In this section the implementation of a prototype of eNSM device is presented taking into account the general architecture exposed in the previous section and specifying the different structural blocks according to the available technologies. In figure 4 the resulting architecture has been given shape graphically. As real time operating system, the device incorporates the version 3 of the Lantronix OS, Evolution OS™. Through an confidentiality agreement with Lantronix, we have had access to the different modules of the system. Given the space restrictions, this has been crucial to develop a made-to-measure version of this OS. As main elements to point out, in this version, a TCP/IP stack has been included together with several client- server application protocols (HTTP, TFTP, SNMP and telnet). The hardware platform chosen for the prototype development is a Lantronix Xport® AR™ device which has a 16 bit DSTni-EX™ processor with 120MHz frequency reaching 30MIPS respectively (in figure 6 a In the service layer, the implementation process has been conditioned by the characteristics of XPort device. Although, with the current hardware miniaturization level it has been enlarged their computational capacities, the devices continue presenting important limitations in their resources. library, the monitoring service intrinsic functionalities are achieved. Figure 5. An eNSM Device prototype. Figure 6. WSDL document fragment for MONITOR command (RPC style). In order to implement service agents, a division in the implementation process between static and mobile agents has been realized. In the first case, an ad-hoc implementation for the XPort device has been development in C language, using operative system as agents’ container. In the second case, in order to establish an execution framework for the monitoring agents, a Python embedded engine (ePython version 2.5) has been adapted to the XPort features. These monitoring agents are implemented as Python text scripts. Figure 5. An eNSM Device prototype. In this layer, three services blocks are implemented: the middleware that provides the communication mechanisms of the monitoring service, the NSM service kernel with the implementation of NSM instructions and the middleware platform that provides the execution of software agents. References [1] C. Younghwan, K. Kwangsoo, K. Cheolhan. “A design chain collaboration framework using reference models,” International Journal of Advanced Manufacturing Technology. 26 (1) pp. 183-190. July, 2005. [15] RFC Project: http://www.rfc.net (URL). [2] L. Avella y D. Vázquez. Is Agile Manufacturing a New Production Paradigm? Universia Business Review, ISSN 1698-5117, Nº 6, pp. 94-107, 2005. [16] Jeong, M.S., Kim, K.H., Kwon, J.H., Park, J.T.”CORBS/CMIP: Gateway Service Scheme for CORBA/TMN Integration. Knom Review, Vol.2, No. 1, pp. 55-62, 1999. [3] P. Harmon, M. Rosen, M. Guttman. Developing E- business Systems and Architectures: A Manager’s Guide. Morgan Kaufmann Publishers, San Francisco, USA. 2001. [17] Aschemann, G., Mohr, T., Ruppert, M. “Integration of SNMP into a CORBA- and Web-Based Management Environment”in Proc. Kommunikation in Verteilten Systemen, , Heidelberg, 1999, pp. 210—221. [4] D.C. McFarlane and S. Bussmann. “Developments in Holonic Production Planning and Control.” Intenational Journal of Production Planning and Control, Vol. 11, No. 6, pp. 522 -- 536, 2000. [18] Work Group JIDM: http://www.opengroup.org (URL). [19] OMG: http://www.omg.org (URL) [20] DMTF: http://www.dmtf.org (URL). [5] A.P. Kalogeras, J.V. Gialelis, C.E. Alexakos, M.J. Georgoudakis, S.A. Koubias. “Vertical integration of enterprise industrial systems utilizing web services”, IEEE Transaction on industrial informatics. VOL II. nº 2, pp. 120-128. 2006. [21] JCP: http://www.jcp.org (URL). [22] T.C.Du, E.Y. Li, and A.P. Chang, “Mobile Agents in Distributed Network Management”, in Communications at the ACM, 46(7), 2003, pp127-132. [23] J. Guo,Y. Liao, and B. Parviz. “An Agent-Based Network management system”, presented at the 2005 Internet and Multimedia Applications. [6] J.L.M. Lastra, M. Delamer. “Semantic web services in factory automation: fundamental insights and research roadmap”, IEEE Transaction on industrial informatics. VOL II. nº 1, pp. 1-11. 2006. [24] European co-ordination action for agent-based computing: http://www.rfc.net (URL). [25] J. Sloten, A. Pras, and M. Van Sinderen, “On the standardisation of web service management operations”, presented at the 2004 X EUNICE Summer School and IFIP WG 6.3 Workshop. [7] F. Jammes, H. Smit. “Service-Oriented paradigms in industrial automation”, IEEE Transaction on industrial informatics. VOL I. nº 1, pp. 62-70. 2005. [8] S-M. Lee et al. “A component-based distributed control system for assembly automation”, Proceedings of 2nd International Conference on Industrial Informatics. INDIN 2004. [26] T. Klie, and F. Straub, “Integrating SNMP agents with XML-based management systems,” IEEE Communications Magazine vol. 42 Issue 7, July 2004, pp 76-83. [9] A.P. Kalogeras, J.V. Gialelis, C.E. Alexakos, M.J. Georgoudakis, S.A. Koubias. 7. Conclusions The final objective of our research is focussed on assuring continuity in the manufacturing processes through technologies that should be as transparent as possible to the users. The NSM service kernel has been implemented as a functions library write in C language and offered as API for the others eNSM device modules. By means of this Acknowledgements [13] V. Gilart-Iglesias, F. Maciá-Pérez, J.A. Gil-Martínez- Abarca and A. Capella-D’alton. “Industrial Machines as a Service: A model based on embedded devices and Web Services”. Proceedings of 4th International IEEE Conference on Industrial Informatics (INDIN'06). Singapore, 2006. This work was supported by the Generalitat Valenciana under Grant GV/2007175 and Office of the Vice President for International Relations & Cooperation of the University of Alicante. [14] F. Jammes, H. Smit, J.L. Martinez-Lastra, I.M. Delamer. “Orchestration of Service-Oriented Manufacturing Processes,” Proc. of the 10th IEEE International Conference on Emerging Technologies and Factory Automation, ETFA 2005, Catania, Italy, September 19- 22, 2005. References “Manufacturing Collaborative Process Integration Utilizing State of the Art Technologies” Proceedings of the IEEE International Symposium on Industrial Electronics, ISIE 2005. 2005. [27] NAGIOS: http://nagios.org (URL). [28] MON: http://www.kernel.org/software/mon/ (URL). [29] MUNIN: http://munin.projects.linpro.no/ (URL). [30] MONIT: http://www.tildeslash.com/monit/ (URL). [31] nPULSE: http://www.horsburgh.com/h_npulse.html (URL). [10] R.P. Moreno. Ingeniería de la automatización industrial. Ra-Ma, Madrid, Spain, 2004. [32] V. Miori, L. Tarrini, and R. Bianchi, “LIGHT: XML- Innovative Generation for home Networking Technologies”, Ercim News, Number 62, July2005. [11] Transparent Factory. Manual de usuario y planificación. [Online] Available: http://www.modicon.com, 2001. [12] U. Toop, P. Muller, J. Konnertz, A. Pick, "Web based Service for Embedded Devices," LNCS vol. 2593, 2002, pp. 141-153.
https://openalex.org/W2963617681	https://e-space.mmu.ac.uk/625603/1/THNKEN~2.PDF	English	null	Parasite-induced colour alteration of intermediate hosts increases ingestion by suitable final host species	Behaviour	2,019	cc-by	8,664	Please cite the Published Version Please cite the Published Version DOI: https://doi.org/10.1163/1568539X-00003568 Publisher: Brill Version: Published Version Downloaded from: https://e-space.mmu.ac.uk/625603/ Usage rights: Creative Commons: Attribution 4.0 Additional Information: This is an Open Access article published in Behaviour, published by Brill, copyright The Author(s). Enquiries: If you have questions about this document, contact openresearch@mmu.ac.uk. Please in- clude the URL of the record in e-space. If you believe that your, or a third party’s rights have been compromised through this document please see our Take Down policy (available from https://www.mmu.ac.uk/library/using-the-library/policies-and-guidelines) Parasite-induced colour alteration of intermediate hosts increases ingestion by suitable ﬁnal host species Timo Thünken a,b,∗, Sebastian A. Baldauf a, Nicole Bersau a, Joachim G. Frommen b and Theo C.M. Bakker a a Institute for Evolutionary Biology and Ecology, University of Bonn, An der Immenburg 1, 53121 Bonn, Germany b Division of Behavioural Ecology, Institute of Ecology and Evolution, University of Bern, Wohlenstrasse 50a, 3032 Hinterkappelen, Switzerland Corresponding author’s e-mail address: tthuenken@evolution.uni-bonn.de Timo Thünken a,b,∗, Sebastian A. Baldauf a, Nicole Bersau a, Joachim G. Frommen b and Theo C.M. Bakker a a Institute for Evolutionary Biology and Ecology, University of Bonn, An der Immenburg 1, 53121 Bonn, Germany b Division of Behavioural Ecology, Institute of Ecology and Evolution, University of Bern, Wohlenstrasse 50a, 3032 Hinterkappelen, Switzerland Corresponding author’s e-mail address: tthuenken@evolution.uni-bonn.de Received 26 February 2019; initial decision 25 March 2019; revised 14 June 2019; accepted 19 June 2019 Abstract Parasites with complex life cycles often alter the phenotypic appearance of their intermediate hosts in order to facilitate ingestion by the ﬁnal host. However, such manipulation can be costly as it might increase ingestion by less suitable or dead-end hosts as well. Species-speciﬁc parasitic manipulation is a way to enhance the transmission to suitable ﬁnal hosts. Here, we experimen- tally show that the altered body colouration of the intermediate host Gammarus pulex caused by its acanthocephalan parasite Pomphorhynchus laevis differently affects predation by different ﬁsh species (barbel, perch, ruffe, brown trout and two populations of three-spined stickleback) depend- ing on their suitability to act as ﬁnal host. Species that were responsive to colour manipulation in a predation experiment were more susceptible to infection with P. laevis than unresponsive species. Furthermore, three-spined stickleback from different populations responded to parasite manipu- lation in opposite directions. Such increased ingestion of the intermediate host by preferred and suitable hosts suggests ﬁne-tuned adaptive parasitic manipulation and sheds light on the ongoing evolutionary arms race between hosts and manipulative parasites. DOI: https://doi.org/10.1163/1568539X-00003568 If you have questions about this document, contact openresearch@mmu.ac.uk. Please in- clude the URL of the record in e-space. If you believe that your, or a third party’s rights have been compromised through this document please see our Take Down policy (available from https://www.mmu.ac.uk/library/using-the-library/policies-and-guidelines) brill.com/beh Behaviour (2019) DOI:10.1163/1568539X-00003568 © The authors, 2019 DOI 10.1163/1568539X-00003568 This is an open access article distributed under the terms of the prevailing CC-BY license at the time of publication. Heruntergeladen von Brill.com07/21/2019 02:10:46PM via Universitatsbibliothek Bern © The authors, 2019 Behaviour (2019) DOI:10.1163/1568539X-00003568 Behaviour (2019) DOI:10.1163/1568539X-00003568 2 Keywords visual manipulation, trophic transmission, Gasterosteus aculeatus, evolutionary arms race, ﬁshes, dead-end hosts. © The authors, 2019 DOI 10.1163/1568539X-00003568 This is an open access article distributed under the terms of the prevailing CC-BY license at the time of publication. Heruntergeladen von Brill.com07/21/2019 02:10:46PM via Universitatsbibliothek Bern Heruntergeladen von Brill.com07/21/2019 02:10:46PM via Universitatsbibliothek Bern 1. Introduction / Behaviour (2019) Acanthocephalans represent a well-described example of manipulative parasites infecting arthropods as intermediate hosts and vertebrates as ﬁ- nal hosts (Bethel & Holmes, 1973, 1977; Crompton & Nickol, 1985; Stone & Moore, 2014; Bakker et al., 2017). In these systems, inter- and intra- speciﬁc variation in host responses have been described repeatedly (e.g., Thomas et al., 2011; Poulin, 2013; Thünken et al., 2018). The acantho- cephalan Pomphorhynchus laevis uses Gammarus species as intermediate hosts — in which they develop into the infectious cystacanths — and differ- ent ﬁsh species as ﬁnal hosts. Gammarids are common in many freshwater ecosystems and represent an important food source for many ﬁshes and birds (Wootton, 1990; MacNeil et al., 1999). Consequently, a broad array of ﬁsh species is exposed to P. laevis infected gammarids under natural conditions. Based on ﬁeld studies from different rivers, Kennedy (2006) listed a mini- mum of 16 ﬁsh species that can serve as ﬁnal hosts for P. laevis. Interestingly, these species seem to differ in suitability, e.g., due to variation in ﬁsh immune defence or gastrointestinal morphology (Lagrue et al., 2011). Barbel (Barbus barbus) and chub (Leuciscus cephalus) are highly suitable hosts of P. laevis (Kennedy, 2006). However, other species can serve as host for the parasite as well. Thus, the usage of a species as ﬁnal host differs between populations depending on ecological factors like the composition of ﬁsh communities (Kennedy, 2006; Thomas et al., 2010). Pomphorhynchus spec. manipulate their amphipod intermediate hosts in several ways (Crompton & Nickol, 1985; McCahon et al., 1991; Kennedy, 2006). For example, uninfected Gammarus pulex avoid the odour of ﬁsh predators, whereas individuals infected with P. laevis prefer the scent of the parasite’s ﬁnal host (Baldauf et al., 2007; Perrot-Minnot et al., 2007). Be- sides behavioural manipulation, P. laevis also alters the visual appearance of its intermediate host (Bakker et al., 1997). The cystacanth of P. lae- vis is visible through the gammarid’s cuticle and appears as a conspicuous orange dot in the dorsal coelom. Such colouration increases the conspicu- ous of intermediate hosts to ﬁsh predators (Bakker et al., 1997; Kennedy, 2006). Bakker et al. (1997) provided experimental evidence that the altered colouration of G. pulex caused by P. laevis increases predation by ﬁshes and, thus, facilitates transmission to ﬁnal hosts. Uninfected gammarids that were painted with an orange spot mimicking a P. 1. Introduction Parasites with complex life cycles often depend on the ingestion of their intermediate host by a ﬁnal host (trophic transmission), where they reach sexual maturity and reproduce (Lafferty, 1999; Lafferty & Kuris, 2002; Moore, 2002). Therefore, parasites’ interests strongly conﬂict with those of the intermediate host, leading to high selective pressures on parasites to manipulate their intermediate host in a way that increases the proba- bility of trophic transmission (parasitic manipulation). Numerous studies show that parasites alter — often dramatically — the conspicuousness or (anti-predator) behaviour of their intermediate hosts (e.g., Lefevre et al., 2009; Poulin, 2010; Moore, 2013). For example, sporocysts of the dige- nean parasite Leucochloridium macrostomum turn the antennae of the snail Succinea putris into colourful blinker lamps (Wesołowska & Wesołowski, 2014), while nematode-parasitized tropical arboreal ants, (Cephalotes atra- tus) resemble ripe fruits in the rain forest (Yanoviak et al., 2008). Both manipulations eventually lead to an increased ingestion of the parasite by birds. However, not all potential predators of the intermediate host are equally suited as ﬁnal host for the parasite. Thus, parasite-induced phenotypic al- teration of the intermediate host might not only increase predation by the favoured ﬁnal host, but also by less suitable or even non-host species (dead- end hosts, Mouritsen & Poulin, 2003; Poulin et al., 2005; Thomas et al., 2005; Seppälä et al., 2008). Indeed, some studies showed increased preda- tion of infected individuals by non-hosts compared to uninfected individuals (Milinski, 1985; Poulin et al., 2005; Kaldonski et al., 2008), while others found evidence for infection-dependent predation by non-hosts (Seppälä et al., 2006). Consequently, the net beneﬁt for the parasite resulting from the manipulation could be lower than expected, which might lead to a systematic overestimation of the adaptive signiﬁcance of parasitic manipulation (Sep- pälä & Jokela, 2008; Cézilly et al., 2010). However, parasitic transmission will be increased when suitable ﬁnal hosts preferentially prey upon manipulated intermediate hosts (host-speciﬁc manipulation) (Levri, 1998; Seppälä et al., 2006). Testing this host-speciﬁc manipulation hypothesis requires to link suitability to act as ﬁnal host of different predators with their responsiveness to parasite-manipulated inter- mediate hosts. Such studies are scarce thus far (but see Seppälä et al., 2008). Heruntergeladen von Brill.com07/21/2019 02:10:46PM via Universitatsbibliothek Bern 3 T. Thünken et al. Heruntergeladen von Brill.com07/21/2019 02:10:46PM via Universitatsbibliothek Bern 1. Introduction laevis infection were more fre- quently eaten by predatory three-spined stickleback (Gasterosteus aculeatus) than control-treated gammarids (Bakker et al., 1997). This ﬁnding strongly Heruntergeladen von Brill.com07/21/2019 02:10:46PM via Universitatsbibliothek Bern Behaviour (2019) DOI:10.1163/1568539X-00003568 4 supports the hypothesis that parasite-induced alteration of body colouration increases trophic transmission. Interestingly, a subsequent study applying a similar experimental approach found no evidence for increased predation of parasite-mimics by brown trout (Salmo trutta) (Kaldonski et al., 2009). As different ﬁsh species appear to vary in responsiveness to P. laevis-induced colour alteration and in suitability to act as ﬁnal host, the described host- parasite system is ideal to test the host-speciﬁc manipulation hypothesis. In the present study, we investigate variation in the predatory response of different potential ﬁsh hosts to P. laevis-induced colour alteration of the intermediate host G. pulex. We experimentally manipulated gammarids’ colouration by painting an orange spot on the cuticle of uninfected individu- als, mimicking a P. laevis infection (cf., Bakker et al., 1997; Kaldonski et al., 2009). Examined ﬁshes include barbel, the preferred ﬁnal host of P. laevis as well as perch (Perca ﬂuviatilis), ruffe (Gymnocephalus cernuus), brown trout and three-spined stickleback. Additionally, in order to investigate intra- speciﬁc variation, a stationary and a migratory stickleback population was examined. Finally, we aimed to link ﬁshes’ behavioural responsiveness to their susceptibility to P. laevis infections. Therefore, we tested the suscep- tibility of each of the ﬁsh species to infection with P. laevis parasites under standardised laboratory conditions. Following the host-speciﬁc manipulation hypothesis, we predict that ﬁshes’ responsiveness is positively related to their susceptibility to parasitic infection and, thus, suitability to act as ﬁnal hosts. Heruntergeladen von Brill.com07/21/2019 02:10:46PM via Universitatsbibliothek Bern 2.1. Experimental animals 2. Material and methods To examine the effect of colour alteration on the predation risk of the in- termediate host independent from other infection related factors, such as parasite-induced changes of behaviour, we experimentally manipulated the colouration of uninfected G. pulex. To estimate ﬁshes’ suitability to act as ﬁnal host, we fed them with P. laevis-infected G. pulex and subsequently examined the presence of adult P. laevis in the intestine. 2.1. Experimental animals 2.1.1. Intermediate host Gammarus pulex 2.1.1. Intermediate host Gammarus pulex Several thousand uninfected G. pulex were sampled with a hand net in Febru- ary 2010 from the Katzenlochbach brook (50°41′59.03′′N, 7°4′54.27′′E) near Several thousand uninfected G. pulex were sampled with a hand net in Febru- ary 2010 from the Katzenlochbach brook (50°41′59.03′′N, 7°4′54.27′′E) near Heruntergeladen von Brill.com07/21/2019 02:10:46PM via Universitatsbibliothek Bern Heruntergeladen von Brill.com07/21/2019 02:10:46PM via Universitatsbibliothek Bern 5 T. Thünken et al. / Behaviour (2019) Bonn, Germany, by kick sampling (Heynes 1954). Subsequently, the gam- marids were transported to the laboratory where they were kept under stan- dardised winter conditions (light/dark regime: 8.5/15.5 h, temperature 12 ± 2°C). Dead leaves served as shelter and nutrition. Only uninfected G. pulex with a body size larger than 5 mm were used in the colour-manipulation experiments. Heruntergeladen von Brill.com07/21/2019 02:10:46PM via Universitatsbibliothek Bern 2.1.2. Predatory ﬁshes 20% of the female parasites carried eggs indicating that sticklebacks are suitable ﬁnal hosts for P. laevis. Perch, ruffe, and three- spined stickleback (Rhine population) were wild-caught ﬁsh captured in the river Rhine close to Grieth, Germany (51°47′15.17′′N, 6°19′7.32′′E). Differ- ent populations of the same species might face variation in environmental conditions and thus may differ in responsiveness to parasitic manipulation. 2.2. Variation in ﬁshes’ response to parasite-induced colouration 2.1.2. Predatory ﬁshes In the present study, we used ﬁve different ﬁsh species, all of which are described to prey on small invertebrates like Gammarus under natural con- ditions (Kottelat & Freyhof, 2007). P. laevis is present in every natural ﬁsh population examined. The suitability of three-spined stickleback as hosts for P. laevis is ambiguous thus far. In the Appendix, we therefore provide un- published results of a study in which three-spined stickleback from a Swiss population were artiﬁcially exposed to P. laevis (Mazzi & Bakker, 2003). In short, 76% of the P. laevis-exposed ﬁsh were infected with a median number of 2 parasites per ﬁsh. 20% of the female parasites carried eggs indicating that sticklebacks are suitable ﬁnal hosts for P. laevis. Perch, ruffe, and three- spined stickleback (Rhine population) were wild-caught ﬁsh captured in the river Rhine close to Grieth, Germany (51°47′15.17′′N, 6°19′7.32′′E). Differ- ent populations of the same species might face variation in environmental conditions and thus may differ in responsiveness to parasitic manipulation. To shed light on this variation we investigated a second stickleback popula- tion that was collected from a small brook close to Euskirchen, Germany (Kuchenheim population; 50°40′8.05′′N, 6°49′35.82′′E). Brown trout and barbel were purchased from ﬁsh farms (brown trout: Stolberg-Schevenhütte, Germany; barbel: Gersfeld, Germany) at the age of one year. As hatchery- reared and wild trout show similar feeding habits (Johnsen & Ugedal, 1990), they are suitable for the experiment. Further, to habituate experimental ﬁsh to gammarids as food they were fed daily with defrosted gammarids (Hyalella azteca) and with live uninfected G. pulex for several months. Fish were kept under similar conditions as G. pulex in tanks of variable size (adapted to ﬁsh size), except for trout that were kept in a large outdoor tank with continuous water ﬂow. In the present study, we used ﬁve different ﬁsh species, all of which are described to prey on small invertebrates like Gammarus under natural con- ditions (Kottelat & Freyhof, 2007). P. laevis is present in every natural ﬁsh population examined. The suitability of three-spined stickleback as hosts for P. laevis is ambiguous thus far. In the Appendix, we therefore provide un- published results of a study in which three-spined stickleback from a Swiss population were artiﬁcially exposed to P. laevis (Mazzi & Bakker, 2003). In short, 76% of the P. laevis-exposed ﬁsh were infected with a median number of 2 parasites per ﬁsh. 2.2.1. Colour manipulation We aimed at creating highly standardised prey mimics that imitate the natural reﬂectance of P. laevis. Therefore, the reﬂectance of the parasite cystacanth Heruntergeladen von Brill.com07/21/2019 02:10:46PM via Universitatsbibliothek Bern Heruntergeladen von Brill.com07/21/2019 02:10:46PM via Universitatsbibliothek Bern Behaviour (2019) DOI:10.1163/1568539X-00003568 6 was measured through the cuticle of 10 naturally infected gammarids origi- nating from a brook near Müllekoven, Germany (50°46′49.9′′N, 7°6′36.1′′E) with a spectrophotometer (Avantes USB 2000) connected to a deuterium- halogen light source (Avantes DH-2000). Reﬂection measurements were taken perpendicular to the body surface with a 1 mm probe under standard- ised conditions relative to a Spectralon white standard. For each individual a mean reﬂectance value deriving from 20 single measurements was taken. Subsequently, the mean reﬂectance value of the ten individuals was calcu- lated, which served as a reference for the reﬂection values of the colour used for imitating infection on uninfected mimics. Similarly, the reﬂection reference for uninfected control mimics was calculated. Here, the mean re- ﬂectance of the cuticle of uninfected G. pulex (N = 20) served as a reference. The imitation of the cystacanth’s colour was achieved by mixing acrylic model paint colours (Revell enamel colour; yellow (order number 32 112), brown (32 180), orange (32 130) and black (32 107)), until spectrophoto- metrical readings conﬁrmed that the colour matched with natural reﬂection values (see Figure A1 in the Appendix). This was achieved with a ratio of 18:4:0.25:0.2 of the respective colours. For the control, a clear, transparent colour was used (Revell Email enamel colour clear (32 101)). Twenty-four hours before the start of the experiments, uninfected gam- marids were haphazardly caught from their habitat tank. They were anes- thetised with carbonated water, dried on a cellulose cloth, and their length was measured. A colour spot of approx. 1.5 mm diameter mimicking a P. laevis cystacanth was applied with a toothpick on a position where natural infections would be visible (colour manipulation treatment). In the con- trol treatment, gammarids received a transparent spot in order to represent uninfected prey. The procedures took no longer than three minutes per in- dividual. Gammarids of both treatment groups were transferred to small holding tanks containing tap water. Only gammarids that recovered and showed similar swimming behaviour as untreated gammarids were used in the experiment the next day. 2.2.1. Colour manipulation Gammarids of the two treatment groups did not differ in body size (Wilcoxon rank-sum test: Nmimics = 2220, Ncontrol = 2220, W = 2499698, p = 0.39). Heruntergeladen von Brill.com07/21/2019 02:10:46PM via Universitatsbibliothek Bern 2.2.2. Experimental setup and procedure p p p Experiments were conducted in tanks (50 × 30 × 30 cm) that were laminated with a brown, self-adhesive foil, which matched in its reﬂection with spec- trometric readings of the substrate from the natural gammarid habitat (own Heruntergeladen von Brill.com07/21/2019 02:10:46PM via Universitatsbibliothek Bern 7 T. Thünken et al. / Behaviour (2019) measurements). The foil was ﬁxed to the bottom of the tanks and the lower 10 cm of the sidewalls to simulate sheltered and unsheltered areas as present in the natural habitat. The test tank was surrounded by opaque polystyrene plates in order avoid visual disturbance from outside. A ﬂuorescent tube with daylight spectrum without UV (Sylvania Luxline plus Daylight de Luxe F36 W1860) was installed 75 cm above the test tank. Twenty-four hours before experimental trials started, one haphazardly chosen individual of each ﬁsh species was isolated in a tank that was of similar size and appearance as the test tank to acclimate to the experimental conditions. Each test tank was ﬁlled with aged tap water up to a height of 20 cm. Then, 20 colour-manipulated gammarids and 20 gammarids from the control treatment group were placed in each tank. Subsequently, the test ﬁsh was placed in a cylinder in the mid- dle of each test tank (diameter 20 cm). In total, 15 barbel, 19 perch, 20 ruffe, 17 brown trout and 20 three-spined stickleback from each population were tested. After 30 minutes of acclimatization the cylinder was removed. The ﬁsh was allowed to swim freely and to prey upon the gammarids. With the exception of barbel, each ﬁsh species was allowed to prey upon the gam- marids for one hour after release from the cylinder. Pilot studies revealed that barbel needed 2 hours after release from the cylinder to show preda- tory behaviour. Thus, this species was tested for three hours. After the trial, ﬁsh were removed from the test tanks and all surviving colour-manipulated gammarids and control gammarids were counted. Heruntergeladen von Brill.com07/21/2019 02:10:46PM via Universitatsbibliothek Bern 2.3. Variation in susceptibility to the P. laevis parasite in ﬁshes Ten individuals of each ﬁsh species (except three-spined stickleback) were captured randomly to examine their susceptibility to P. laevis. For the Rhine stickleback population seven ﬁsh were used, for the Kuchenheim stickleback population 11. All ﬁsh were food-deprived for one day. After determining the standard length (SL) and body mass (M), each ﬁsh was placed individually in a tank where it was allowed to ingest ten G. pulex infected with mature P. laevis originating from the brook in Müllekoven. A gammarid was deter- mined as infectious if it showed a clearly visible orange spot through the cuticle. The ﬁsh stayed in the tank until it had ingested all infected gam- marids. After the infection procedure, ﬁsh were transferred to a community pool with other infected conspeciﬁcs. Here, ﬁsh were daily fed with a mix- ture of defrosted uninfected gammarids and chironomid larvae. Fish were kept under standardised winter conditions (light/dark 8.5/15.5 h, tempera- ture 12 ± 2°C). Four months (±5 days) after exposure, ﬁsh were killed by Heruntergeladen von Brill.com07/21/2019 02:10:46PM via Universitatsbibliothek Bern Heruntergeladen von Brill.com07/21/2019 02:10:46PM via Universitatsbibliothek Bern Behaviour (2019) DOI:10.1163/1568539X-00003568 8 a blow to the head, followed by immediate decapitation. Subsequently, the intestine as well as abdominal cavity and viscera were searched for living P. laevis or their remains. The total number of parasites found in the intestine and abdominal cavity of each ﬁsh was recorded. Heruntergeladen von Brill.com07/21/2019 02:10:46PM via Universitatsbibliothek Bern 2.4. Statistical analysis In the analysis, we refer to the different ﬁshes as ﬁsh populations (one trout, perch, ruffe and barbel population each and two three-spined stickleback populations). In order to analyse whether ﬁsh populations differ in respon- siveness to colour-manipulated gammarids Manly’s alpha was calculated (Manly, 1974; see also Klecka & Boukal, 2012). Manly’s alpha takes into account that during a trial some prey items are eaten, and thus accounts for changes in the proportions of available prey classes. We excluded all ﬁsh that consumed more than 50% of the gammarids in a trial, as these would have been forced to feed against their potential preferences (see Bakker et al., 1997). Therefore, 5 perch, 5 ruffe and 10 trout were not included in this analysis. Including these ﬁsh would not qualitatively change the results (data not shown). Three-spined stickleback and barbel never consumed more than 20 prey items in a given trial. Differences between populations were examined by an ANOVA. Responsiveness of each ﬁsh population was in- vestigated by one-sample t-tests. Signiﬁcant deviation from 0.5 thus reports responsiveness (preference or avoidance of colour-manipulated gammarids, respectively) to colour-manipulated gammarids. The total number of ingested gammarids by ﬁsh of the different popu- lations was analysed by ﬁtting a GLM with quasi-Poisson distribution to account for overdispersion of the data. Population and ﬁsh size were used as explanatory variables. Additionally, we investigated whether ﬁsh respon- siveness was related to the total number of ingested gammarids. To this end, the mean number of ingested gammarids per species was calculated. A linear model was ﬁtted with mean number of ingested mimics as depen- dent variable, which was explained by responsiveness (population responded to colour manipulation yes/no). A population was considered as responsive to manipulation when ﬁsh discriminated between coloured and uncoloured gammarids (see one-sample t-tests in the paragraph above). To analyse differences in susceptibility to P. laevis infection between ﬁshes of the different populations, a GLM was ﬁtted with quasi-Poisson dis- tribution to account for overdispersion of the data. Total number of parasites Heruntergeladen von Brill.com07/21/2019 02:10:46PM via Universitatsbibliothek Bern T. Thünken et al. / Behaviour (2019) 9 found in the intestine and abdominal cavity served as dependent variable and ﬁsh population as explanatory variable. Body size was added as covari- ate. 2.5. Ethics Permissions were obtained to artiﬁcially infect and dissect ﬁshes with P. laevis (LANUV NRW 8.87-51.04.20.09.352). Fish were killed according to §4 of the German animal welfare act. 2.4. Statistical analysis The total number of parasites (intestine plus body cavity) signiﬁcantly correlated with the number of parasites found in the intestine, where the para- site reaches sexual maturity (Spearman rank correlation: N = 58, S = 8718, p < 0.001). In order to link ﬁshes’ susceptibility to P. laevis infection to their respon- siveness to colour manipulation, a GLMM with binomial distribution was ﬁtted with parasites presence (yes/no) in the individual ﬁsh as dependent variable. As we had no behavioural data of these individual ﬁsh, we entered whether an individual belongs to a species that responded to colour manipu- lation or to an unresponsive species as explanatory variable. Fish population was entered as random factor to control for the multiple uses of ﬁsh originat- ing from the same population. In all models, likelihood ratio tests (“LRT”) assessed whether the removal of a variable caused a signiﬁcant decrease in model ﬁt. Non-signiﬁcant vari- ables were removed, while signiﬁcant ones remained in the model. In case of binomial test, χ2-values are reported. For models assuming quasi-Poisson distribution we report F-values. All given test probabilities are two-tailed throughout. All analyses were performed using R 3.02 statistical package (R Core Team, 2013). Heruntergeladen von Brill.com07/21/2019 02:10:46PM via Universitatsbibliothek Bern 3.1. Variation in response to parasite-induced colouration The proportion of ingested colour-manipulated and control gammarids was different between ﬁsh populations (ANOVA: df = 5, F = 7.075, p < 0.001, Figure 1). Barbel (N = 15) and three-spined stickleback (NKuchenheim = 20, NRhine = 20) were responsive to the colour manipulation and their prey choice deviated from random (Figure 1). Colour-manipulated gammarids were more often eaten than control gammarids by barbel (one-sample t-test, df = 13, t = 5.076, p < 0.001, Figure 1) and by the Kuchenheim stick- The proportion of ingested colour-manipulated and control gammarids was different between ﬁsh populations (ANOVA: df = 5, F = 7.075, p < 0.001, Figure 1). Barbel (N = 15) and three-spined stickleback (NKuchenheim = 20, NRhine = 20) were responsive to the colour manipulation and their prey choice deviated from random (Figure 1). Colour-manipulated gammarids were more often eaten than control gammarids by barbel (one-sample t-test, df = 13, t = 5.076, p < 0.001, Figure 1) and by the Kuchenheim stick- Heruntergeladen von Brill.com07/21/2019 02:10:46PM via Universitatsbibliothek Bern Behaviour (2019) DOI:10.1163/1568539X-00003568 10 Figure 1. Manly’s alpha (means ± SE) representing selective predation of the different ﬁshes on colour-manipulated gammarids. In each trial, 20 colour-manipulated gammarids and 20 control gammarids were offered. Values greater than 0.5 indicate higher consumption of colour-manipulated gammarids. Asterisks indicate whether consumption signiﬁcantly devi- ated from random predation (0.5). ns indicates p > 0.5; (∗)0.05 < p < 0.1; ∗∗p < 0.01; ∗∗∗p < 0.001. Figure 1. Manly’s alpha (means ± SE) representing selective predation of the different ﬁshes on colour-manipulated gammarids. In each trial, 20 colour-manipulated gammarids and 20 control gammarids were offered. Values greater than 0.5 indicate higher consumption of colour-manipulated gammarids. Asterisks indicate whether consumption signiﬁcantly devi- ated from random predation (0.5). ns indicates p > 0.5; (∗)0.05 < p < 0.1; ∗∗p < 0.01; ∗∗∗p < 0.001. lebacks (one-sample t-test, df = 18, t = 1.995, p = 0.062, Figure 1). By contrast, Rhine sticklebacks avoided the consumption of colour-manipulated gammarids (one-sample t-test, df = 18, t = −3.605, p = 0.002, Figure 1). Perch (N = 14), ruffe (N = 15) and brown trout (N = 7) did not show any signiﬁcant discrimination between experimental groups (all p values > 0.53, Figure 1). Heruntergeladen von Brill.com07/21/2019 02:10:46PM via Universitatsbibliothek Bern 3.2. Variation in susceptibility to P. laevis parasites in ﬁshes The number of parasites found in the intestine and body cavity (ruffe: no parasites; perch: 1 parasite in 1 ﬁsh; trout: 3 parasites in 2 ﬁsh; Kuchenheim stickleback: 4 parasites in 3 ﬁsh; Rhine stickleback: 8 parasites in 3 ﬁsh; barbel: 20 parasites in 7 ﬁsh) differed between ﬁsh populations (LRT: df = 5, F = 5.862, p < 0.001), and was independent from ﬁsh body size (LRT: df = 1, F = 0.174, p = 0.678). Fishes that were responsive to colour manip- ulation had a higher probability to be infected by P. laevis than unresponsive ones (LRT: χ2 = 5.945, p = 0.015). 3.1. Variation in response to parasite-induced colouration The total number of ingested gammarids varied between populations (LRT: df = 5, F = 16.924, p < 0.001, Figure A2 in the Appendix) and was positively correlated to ﬁsh body size (LRT: df = 1, F = 27.370, p < 0.001, Figure A2 in the Appendix). The mean number of ingested gammarids per population was signiﬁcantly related to the respective responsiveness for vi- Heruntergeladen von Brill.com07/21/2019 02:10:46PM via Universitatsbibliothek Bern n Brill.com07/21/2019 02:10:46PM via Universitatsbibliothek Bern 11 T. Thünken et al. / Behaviour (2019) sual manipulation, with responsive ﬁshes eating a lower total amount of gammarids (LRT: F = −14.084, p = 0.019, Figure A2 in the Appendix). ual manipulation, with responsive ﬁshes eating a lower total amount of gammarids (LRT: F = −14.084, p = 0.019, Figure A2 in the Appendix). Heruntergeladen von Brill.com07/21/2019 02:10:46PM via Universitatsbibliothek Bern 4. Discussion The present study demonstrates variation in the responsiveness of different predatory ﬁsh species to artiﬁcially coloured G. pulex mimicking an infec- tion by P. laevis. This variation was positively related to the susceptibility of the respective ﬁsh species to P. laevis infection. Although infection with a parasite does not guarantee that the parasite will reproduce (Pattersson et al., 2011, 2013), our results are in accordance with other studies on the suitability of different ﬁshes to act as ﬁnal host (i.e., allowing parasitic reproduction). The preferred host of P. laevis under natural conditions, the barbel (Britton & Pegg, 2011), preferentially consumed colour-manipulated gammarids and showed highest susceptibility to artiﬁcial infections. This result indicates that the colour alteration of the intermediate host caused by P. laevis increases ingestion by the optimal host and, thus, that colour alteration increases trans- mission of the parasite to a suitable ﬁnal host. Therefore, our study supports the adaptive manipulation hypothesis (Bakker et al., 1997). In that scope, we were also able to conﬁrm the results of Bakker et al. (1997), who reported that three-spined stickleback respond to the colour manipulation of P. lae- vis. The present study shows that three-spined stickleback are suitable hosts for P. laevis (see below). Furthermore, we found support for the results of Kaldonski et al. (2009), who reported that parasite-induced colour alteration plays no role for parasite transmission to brown trout. Brown trout, perch and ruffe showed very low infection rates in our experiment. Also, under natural conditions they do not seem to be the main hosts of P. laevis (e.g., Kennedy Heruntergeladen von Brill.com07/21/2019 02:10:46PM via Universitatsbibliothek Bern Behaviour (2019) DOI:10.1163/1568539X-00003568 12 et al., 1978; Kennedy, 2006). These species did not response to colour ma- nipulation. Therefore, our study provides a solution to the conﬂicting results of the different studies. It implies that colour alteration of the intermediate hosts represents a strategy of parasites to increase transmission to suitable ﬁnal hosts without increasing transmission to less suitable hosts. Speciﬁc manipulation by the parasite could be achieved, for example, by exploiting physiological differences in the visual system of ﬁshes (Lythgoe, 1979). This could lead to different responses to speciﬁc colours such as to the orange spot of P. laevis cystacanths. Heruntergeladen von Brill.com07/21/2019 02:10:46PM via Universitatsbibliothek Bern 4. Discussion Future experiments could address whether (natural) variation in carotenoid-based colouration of the parasite depend on the ﬁnal host species present in the system, the production costs of the colouration and potential ﬁne-tuned species-speciﬁc feeding prefer- ences of ﬁnal hosts. An alternative explanation for the link between ﬁshes’ responsiveness and susceptibility is a possible secondary adaptation of the parasites to the intestinal tract of predatory ﬁshes that are attracted by the colour of infected gammarids and thus increasingly prey on them. Future studies, based on phylogenetic comparisons, could address these questions. Intriguingly, ﬁsh from the two three-spined stickleback populations re- sponded to parasite manipulation, but in opposite directions. This result sug- gests intra-speciﬁc variation between populations in the evolutionary arms race between hosts and parasites. The difference in the behavioural response might depend on variation of the costs for the stickleback host that are as- sociated with different parasite prevalence in host populations. Although an acanthocephalan infection does not seem to seriously harm most ﬁsh hosts (Kennedy, 2006), P. laevis, which deeply penetrates the tissue of the diges- tive tract, can cause considerable damage, particularly in small ﬁshes like three-spined stickleback (Schmidt et al., 1974; Mazzi & Bakker, 2003). In- creased activity of the immune system of the host following an infection and associated changes in the metabolism (Dezfuli et al., 2002) can indirectly affect ﬁtness-related traits like the intensity of ornamentation, which plays a central role in sexual selection (Bakker & Milinski, 1993). This should select for counter-adaptation by the host, like strategies to actively avoid in- fected prey as it has been shown in three-spined stickleback from a stationary Swiss population (Mazzi & Bakker, 2003). Host responses to parasitic ma- nipulation are assumed to differ between populations due to differences in selective pressure exerted by parasites. For instance, the relative abundance of intermediate hosts as well as the composition of the ﬁsh community may T. Thünken et al. / Behaviour (2019) 13 vary between different habitats inﬂuencing the prevalence of infection of ﬁ- nal hosts (Kennedy, 2006). Three-spined stickleback populations from the Rhine and from Kuchenheim differ in both aspects. The Rhine harbours a much higher ﬁsh diversity than the small Kuchenheim brook. This should also affect P. laevis prevalence across as well as within species. Heruntergeladen von Brill.com07/21/2019 02:10:46PM via Universitatsbibliothek Bern 4. Discussion The results of the present study encourage future work on the interplay between varia- tion in parasite prevalence and ﬁsh diversity (Thomas et al., 2011). Kaldonski et al. (2009) and Cezilly et al. (2010) concluded that parasite colour alteration of intermediate hosts generally plays a limited role for in- creased transmission to ﬁnal ﬁsh hosts, based on the ﬁnding that brown trout did not respond to the manipulation. The authors argued that the contradict- ing results of Bakker et al. (1997) resulted (1) from methodological ﬂaws while mimicking the parasite-infection and (2) from the usage of an unsuit- able ﬁnal host, i.e., three-spined stickleback. In the light of the present study, this criticism does not hold. Barbel, which is the most important host of P. laevis, were responsive to the colour alteration of G. pulex which clearly supports the hypothesis by Bakker et al. (1997), stating that colour alter- ation caused by the parasite increases transmission to ﬁnal hosts. Based on data obtained from only three individuals, Hine & Kennedy (1974) prema- turely labelled three-spined stickleback as non-suitable host of P. laevis. In their study, two out of three individuals carried a single parasite each, which thus excluded the presence of gravid female parasites, a criterion for the suitability of the host. In contrast, the data reported in the Appendix indi- cate that sticklebacks are physiologically compatible to serve as ﬁnal host and, thus, are suitable hosts for P. laevis for reproduction in nature. Indeed, three-spined stickleback may be an important host for P. laevis under chang- ing natural environments. Even under harsh conditions, when ﬁsh diversity decreases temporally (e.g., due to anthropogenic impacts) the robust three- spined stickleback is often able to maintain stable populations (Wootton, 1976), ensuring at least basic opportunities for parasites’ reproduction and survival. Field data on natural infections, particularly from smaller rivers where three-spined stickleback are often the dominant species, are required to clarify their importance as ﬁnal host for P. laevis under natural conditions. Overall, larger ﬁshes were less infected by P. laevis but consumed a higher absolute number of gammarids compared to the smaller, more in- fected ﬁshes. The consequences for parasitic transmission in the wild are unclear and may depend on several factors like the proportion of host and Behaviour (2019) DOI:10.1163/1568539X-00003568 14 non-host species and the general predation risk (see Seppälä & Jokela, 2008). 4. Discussion Gammarids are an important component in the food web of aquatic systems (MacNeil et al., 1999), where they represent an important food source for many ﬁshes. Parasites (particularly those with complex life cycles) have the potential to alter and maintain biodiversity (Lafferty et al., 2008) by adding complexity (Dunne et al., 2013). Our results suggest that manipulative par- asites may contribute to population dynamics (see Heggelin et al., 2007) as increased conspicuousness of intermediate hosts induced by parasitic manip- ulation might increase consumption by predator species. Besides changing colouration, P. laevis and other acanthocephalans are known to alter a range of behaviours of their intermediate gammarid host like geo- and phototatic behaviour or activity (e.g., Bethel & Holmes, 1973, 1977; Thünken et al., 2010, 2018; Bakker et al., 2017), or predator evasion (Baldauf et al., 2007; Perrot-Minot et al., 2007). In three-spined stickleback changes in colouration and behaviour of infected G. pulex alone lead to an increased risk of predation for the intermediate host (Mazzi & Bakker, 2003). Furthermore, intermediate and ﬁnal hosts may respond in diverse manners to deal with the risk of parasitic infection (Kennedy, 2006). Thus, in order to evaluate the overall ﬁtness consequences of parasitic infections integrative approaches are required combining laboratory experiments with ﬁeld data. As we were interested in parasitic manipulation, we experimentally examined a speciﬁc trait of the intermediate host that is altered by the parasite (colouration) and analysed its impact on parasitic transmission independent from other host traits potentially affected by the infection. Summarising, the present study shows that the altered colouration of the intermediate host G. pulex caused by its acanthocephalan parasite P. laevis differently affects predation by ﬁshes, depending on their suitability to act as ﬁnal host. This could be a result of parasitic adaptation to use predators preferring coloured gammarids as ﬁnal hosts. However, our results could also hint towards speciﬁc parasitic manipulation of the intermediate host to reach a suitable ﬁnal host. Thus, this study highlights the importance of parasite-induced colour alteration of the intermediate host for the evolution of successful transmission of the parasite to its ﬁnal host. Heruntergeladen von Brill.com07/21/2019 02:10:46PM via Universitatsbibliothek Bern Acknowledgements T.T. and S.A.B. contributed equally to this study. We are grateful to In- golf Rick for advice concerning colour measurement and discussion, and to Heruntergeladen von Brill.com07/21/2019 02:10:46PM via Universitatsbibliothek Bern Heruntergeladen von Brill.com07/21/2019 02:10:46PM via Universitatsbibliothek Bern T. Thünken et al. / Behaviour (2019) 15 Rudi Held, Ivar Steinmann and Thomas Lepich for their help with catching and transporting ﬁsh. Saskia Hesse, Kathrin Langen, Tobias Ottenheym and Manuel Thelen helped in ﬁsh maintenance. The manuscript beneﬁtted by thoughtful comments of anonymous referees. T.T. was supported by SNF- Grant 31003A 144191 to J.G.F. Authors’ contributions: T.T., S.A.B., J.G.F. and T.C.M.B. designed the study. N.B. conducted the main experiments and were supported by T.T. and S.A.B. T.C.M.B. collected infection data of the Swiss stickleback population. Data were analysed by T.T., S.A.B., N.B., and T.C.M.B. T.T. and S.A.B. wrote the manuscript and were supported by J.G.F. and T.C.M.B. All authors approved the ﬁnal version of the manuscript. Behaviour (2019) DOI:10.1163/1568539X-00003568 Behaviour (2019) DOI:10.1163/1568539X-00003568 16 Hegglin, D., Bontadina, F., Contesse, P., Gloor, S. & Deplazes, P. (2007). Plasticity of pre- dation behaviour as a putative driving force for parasite life-cycle dynamics: the case of urban foxes and Echinococcus multilocularis tapeworm. — Funct. Ecol. 21: 552-560. Hine, P.M. & Kennedy, C.R. (1974). Observations on the distribution, speciﬁcity and pathogenicity of the acanthocephalan Pomphorhynchus laevis (Müller). — J. Fish Biol. 6: 521-535. Johnsen, B.O. & Ugedal, O. (1990). Feeding by hatchery and pondreared brown trout, Salmo trutta L., ﬁngerlings released in a lake and in a small stream. — Aquacult. Res. 21: 253- 258. Kaldonski, N., Perrot-Minnot, M.J., Motreuil, S. & Cézilly, F. (2008). Infection with acan- thocephalans increases the vulnerability of Gammarus pulex (Crustacea, Amphipoda) to non-host invertebrate predators. — Parasitology 135: 627-632. Kaldonski, N., Perrot-Minnot, M.J., Dodet, R., Martinaud, G. & Cézilly, F. (2009). Carotenoid-based colour of acanthocephalan cystacanths plays no role in host manipu- lation. — Proc. Roy. Soc. Lond. B: Bio. Sci. 276: 169-176. Kennedy, C.R., Broughton, P.F. & Hine, P.M. (1978). Status of brown and rainbow-trout, Salmo trutta and S. Gairdneri as hosts of the acanthocephalan, Pomphorhynchus laevis. — J. Fish Biol. 13: 265-275. Kennedy, C.R. (2006). Ecology of the Acanthocephala. — Cambridge University Press, Cam- bridge. Klecka, J. & Boukal, D.S. (2012). Who eats whom in a pool? A comparative study of prey selectivity by predatory aquatic insects. — PLoS ONE 7: e37741. Kottelat, M. & Freyhof, J. (2007). Handbook of European freshwater ﬁshes. — Kottelat, Cornol. afferty, K.D. (1999). 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Nat. 171: 536-544. Heruntergeladen von Brill.com07/21/2019 02:10:46PM via Universitatsbibliothek Bern Suitability of three-spined stickleback as a ﬁnal host for Pomphorhynchus laevis Suitability of three-spined stickleback as a ﬁnal host for Pomphorhynchus laevis According to the classiﬁcation of Hine & Kennedy (1974), three-spined stickleback are unsuitable as ﬁnal hosts for Pomphorhynchus laevis (also referred by Kaldonski et al., 2009). However, this classiﬁcation was based on only three individuals and thus must be considered with caution because parasite prevalence is often below 100%. Therefore, for the present study, we re-examined 60 P. laevis that were dissected out of artiﬁcially infected three- spined stickleback originating from the same population as the ﬁsh used in the study of Bakker et al. (1997). Details of the infection procedure are de- scribed in Mazzi & Bakker (2003). 76% of the exposed ﬁsh were infected with a median number of 2 parasites per ﬁsh. Worms were preserved in 70% ethanol. Digital photographs including a size standard were taken from the preserved individuals to determine body length. The sex of each parasite and presence of eggs was noted to analyse whether the parasite reaches sexual maturity in sticklebacks. The sex of one individual could not be determined, reducing the total sample size to 59 individuals. The body size of ethanol- preserved (shrunken) P. laevis (mean ± SD = 10.09 mm ± 1.54) largely Heruntergeladen von Brill.com07/21/2019 02:10:46PM via Universitatsbibliothek Bern T. Thünken et al. / Behaviour (2019) 19 conforms to the classiﬁcation of Hine & Kennedy (1974) for body size of P. laevis found in suitable hosts (12–15 mm). The body size of males (N = 25, mean ± SD = 9.91 mm ± 1.59 mm) and females (N = 34, mean ± SD = 10.21 mm ± 1.55 mm; for one female the body size could not be determined) was comparable (t-test: t = 0.74, df = 57, p = 0.46). Seven out of 35 fe- males (i.e., 20%) carried eggs. Fecund females were larger in body size than females without eggs (with eggs: mean ± SD = 11.59 mm ± 1.81 mm, with- out eggs: mean ± SD = 9.92 mm ± 1.35 mm, Student’s t-test: t = 2.60, df = 32, p = 0.014). Hine & Kennedy (1974) state that hosts should be classiﬁed as suitable when ripe parasite females are frequently present. Accordingly, we consider three-spined stickleback as a suitable host for P. laevis repro- duction. Figure A1. Mean reﬂectance spectra of natural Gammarus pulex (black lines) and experi- mentally treated gammarids (grey lines). Heruntergeladen von Brill.com07/21/2019 02:10:46PM via Universitatsbibliothek Bern Heruntergeladen von Brill.com07/21/2019 02:10:46PM via Universitatsbibliothek Bern Suitability of three-spined stickleback as a ﬁnal host for Pomphorhynchus laevis The left panel shows the reﬂectance of the cuticle of naturally uninfected gammarids and control treated gammarids that were painted with a trans- parent spot. The right panel shows the reﬂectance of P. laevis cystacanths measured through the gammarid’s cuticle and the colour-manipulated gammarid painted with an orange spot. Figure A1. Mean reﬂectance spectra of natural Gammarus pulex (black lines) and experi- mentally treated gammarids (grey lines). The left panel shows the reﬂectance of the cuticle of naturally uninfected gammarids and control treated gammarids that were painted with a trans- parent spot. The right panel shows the reﬂectance of P. laevis cystacanths measured through the gammarid’s cuticle and the colour-manipulated gammarid painted with an orange spot. Figure A1. Mean reﬂectance spectra of natural Gammarus pulex (black lines) and experi- mentally treated gammarids (grey lines). The left panel shows the reﬂectance of the cuticle of naturally uninfected gammarids and control treated gammarids that were painted with a trans- parent spot. The right panel shows the reﬂectance of P. laevis cystacanths measured through the gammarid’s cuticle and the colour-manipulated gammarid painted with an orange spot. Heruntergeladen von Brill.com07/21/2019 02:10:46PM via Universitatsbibliothek Bern Heruntergeladen von Brill.com07/21/2019 02:10:46PM via Universitatsbibliothek Bern Behaviour (2019) DOI:10.1163/1568539X-00003568 20 Figure A2. Relationship between body size of experimental ﬁsh and the total number of eaten gammarids by ﬁsh in the predation experiment (both gammarid treatment groups are pooled). Data for barbel, perch, ruffe, trout and the 2 three-spined stickleback populations (Rhine and Kuchenheim population) are shown. Fishes that responded to colour manipulation (barbel and the two stickleback populations) consumed on average less gammarids than unresponsive ﬁshes (perch, ruffe and trout). Lines are least-square regression lines. Figure A2. Relationship between body size of experimental ﬁsh and the total number of eaten gammarids by ﬁsh in the predation experiment (both gammarid treatment groups are pooled). Data for barbel, perch, ruffe, trout and the 2 three-spined stickleback populations (Rhine and Kuchenheim population) are shown. Fishes that responded to colour manipulation (barbel and the two stickleback populations) consumed on average less gammarids than unresponsive ﬁshes (perch, ruffe and trout). Lines are least-square regression lines.
https://openalex.org/W4300866585	https://zenodo.org/records/6977024/files/2%20Dr.%20Emine%20ET%20OLTULU.pdf	English	null	The Effects Of Corporate Reputation Perception On Organizational Performance: A Study In Private Hospitals	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	6,494	ABSTRACT In today’s world, reputation which is an important element for both public institutions and private sector companies is an abstract concept that means prestige and to be respected. Corporate reputations are seen as an abstract value place in the minds of partners, employees, customers, suppliers and in short all shareholders of a business. Reputation is a significant factor in enabling customer satisfaction and increasing market share. Therefore, reputation comes in the first place for companies. The purpose of this study is to measure the effects of corporate reputation on organizational performance. In this study, reputation and corporate reputation terms are researched and performance and organizational performance concepts are studied. In the last section, the relationship between corporate reputation and organizational performance is analyzed also in this section which is the application part of this research, in light of the findings based on literature review, a questionnaire was conducted with 50 managers working at managerial positions at 8 different private hospitals to find out more about the effects of corporate reputation perception on organizational performance. Collected data was analyzed through SPSS software program. According to analysis results, answers regarding the effects of corporate reputation perception on organizational performance were evaluated. Keywords: Corporate reputation perception, organizational performance The Effects Of Corporate Reputation Perception On Organizational Performance: A Study In Private Hospitals Dr. Emine ET OLTULU Orcid No: 0000-0002-1180-4157 ATLAS Journal International Refereed Journal On Social Sciences ATLAS Journal International Refereed Journal On Social Sciences Arrival Date : 10.01.2022 Published Date : 28.05.2022 2022, Vol:8, Issue:48 pp: 2738-2747 DOI: https://doi.org/10.5281/zenodo.6977024 2.1.1. Reputation Concept Reputation involves the perspectives of both organization employees and customers who are projected as external partners. Reputation is an Arabic word and it means being respected, the case of being valuable and credible, credibility and prestige. There are numerous definitions for reputation in the literature. Some of these are as follows: “Reputation is an abstract entity which shows the way how an organization is perceived by its employees and customers (Bedük, 2012: 108). Reputation is the collective evaluation related to the ability of providing invaluable results to partner organizations’ representative groups (Fombrun vd., 2000: 243). Reputation is the positive and negative impressions of the public about the organization. Reputation expresses the public’s positive or negative value judgements about the individuals or the organization (Bedük, 2012: 108-109). Reputation is the cumulative reflection of past work experiences and observed individual identifications (Markwick and Fill, 1997: 398). In other words, reputation is defined as a collective system regarding subjective beliefs among the members of a social group (Bromley, 2001: 316). beliefs among the members of a social group (Bromley, 2001: 316). Reputation matters. It influences the attitudes and behaviours not only of existing and potential investors, customers, employees, and suppliers, but also those of regulators and the voice of local communities, pressure groups and media. Stakeholders not only have the ability to influence corporate reputation, they are its arbiters too (Neely vd., 2002: 1). As of today, reputation has become an important asset that the organizations cannot give up on. It provides organizations with such advantages of overcoming crisis easily and acquiring new customers. Organizations gain reputation at the end of a long and difficult process. Alongside with this difficulty, they might lose it in a short time too. In this sense, sustaining reputation is as important as acquiring it. 1. INTRODUCTION Organizations must create a good reputation in order to reach their goals and to attain reputation in the eyes of the stakeholders and society, and this obtained reputation needs being managed and sustained in a correct way. In the environments where the competitiveness increased as an important result of globalization, companies are looking for possible way to make difference and help them gain value in their products and services. At this point, corporate reputation is seen as a significant change maker tool and it is given importance. Corporate reputation is of great importance in terms of providing a competitive advantage for businesses. The purpose of this study is to measure the effects of corporate reputation on organizational performance. In this study, reputation and corporate reputation terms are researched and then performance and organizational performance concepts are studied. In the last section, the relationship between corporate reputation and organizational performance is analyzed also in the last section which is the application part of this research, in light of the findings based on literature review, a questionnaire was conducted with 50 managers working at managerial positions at 8 different private hospitals to find out more about the effects of corporate reputation perception on organizational performance. Collected data was analyzed through SPSS software program. According to analysis results, answers regarding the effects of corporate reputation perception on organizational performance were evaluated. ATLAS INTERNATIONAL REFEREED JOURNAL ON SOCIAL SCIENCES 2.2.1. Performance Concept Either they manufacture goods or provide services; organizations are settled on realizing a number of tasks and goals. The degree of organizational purposes such as efficiency, productivity and profitability etc. in resource utilization has an important role in deciding to what extent the organization behave according to pre-set performance criteria or standards (Şimşek and Çelik, 2013: 131). Nowadays as a result of rapidly increasing competitiveness, business places need to analyze themselves in the process of product and service production and compare themselves with other businesses. In this comparison process, institutions keep the track of key indicators such as quality, cost, and delivery speed which represents organizational performance, they compare their overall performance in these areas with other businesses, and they endeavor to be multiplexed to the desired level of overall organizational performance criteria (Demir and Taşkın, 2012: 2). For managers, it is important to ensure that the employees perform at the desired level. To make sure employees are working at the level expected of them and to evaluate the work of employees in the organization seriously, managers realize a systematic performance assessment process (Robbins vd., 2013: 176). ATLAS INTERNATIONAL REFEREED JOURNAL ON SOCIAL SCIENCES appreciated by society. The importance of reputation can only be understood when it is lost (Kadıbeşegil, 2006: 55). The reputation of an institution or organization is among the most important values for the institution. Good reputation does not only help an organization gain an economic profit, but also overcome a crisis easily with the help of committed employees and customer. appreciated by society. The importance of reputation can only be understood when it is lost (Kadıbeşegil, 2006: 55). The reputation of an institution or organization is among the most important values for the institution. Good reputation does not only help an organization gain an economic profit, but also overcome a crisis easily with the help of committed employees and customer. 2.2.2. Definition of Performance Performance can be defined as the results obtained by an employee through carried out works or duties in a certain limit of time (Şimşek and Öğe, 2014: 295). According to Akal, “performance, in general meaning, is a qualitative or quantitative term which is obtained as a result of purposeful and planned activities. This result can be explained absolutely or relatively such as an athlete’s individual ranking or ranking in general order in high jump or production ratio of a planned production. For determining performance, the result of the performed activities must be considered. Value is a measurement tool used for explaining an asset or fact coming out as a result of an activity in an understandable, explicable, and reduced in specifications and increased in objectivity” (Akal, 2011: 17). 2.1.2. Corporate Reputation Corporate reputation, as a result of recently realized rapid changes in the business environment, has become a concept which is slightly increasing importance every day and even assumed to have a determining role in organizational sustainability. In a general expression, corporate reputation concept is the whole of public opinion, judiciary and thought about an organization, and the belief against an organization and the credibility. In other words, corporate reputation can be expressed as a set of all types of perceptions for organization (Eroğlu and Başak, 2012: 1). Corporate reputation is the total of assumptions and opinions of organization environment regarding the business place. Corporate reputation concept expresses emotional reactions such as good or bad, and weak or strong by organization employees, customers, investors and the general public towards the organization itself (Fombrun, 1996: 37). While it takes a long time to gain, it might be lost all of sudden. The components of corporate reputation are divided by Charles Fombrun into six as follows: “product and service (to provide high quality products and services, to give value for money, to support innovation and their product), vision and leadership (to capture market opportunities, to have a clear vision for the future, to have excellent leadership skills), working environment (to provide a nice place to work, to have good employees), emotional appeal (reliable, honest, and arousing good feeling), corporate social responsibility (to be sensitive to the environment, to avoid damaging the environment, to promote the good things), and financial performance (strong profitability, low risk, strong growth potential, to perform better than its competitors)” (Bilbil, 2013: 163). Corporate reputation is the worth in total market share of the trust created by the company. It is an expression of the company's intangible values. It is a prerequisite for being an institution liked and 2738 Year: 2022 Vol:8 Issue: 48 Year: 2022 Vol:8 Issue: 48 2.2.4. Organizational Performance Assessment Organizational performance assessment is defined as quantitative indicators used to determine the effectiveness and efficiency of business operations, while performance assessment system is defined as the set of indicators used to measure an organization’s activities’ efficiency and its effectiveness (Neely vd., 1995: 80). The companies have so far developed various performance measurement systems for assessment of their organizational performance. In initial times, the companies only used financial measures but recent years shows that they use and develop non-financial performance measurement models. The financial performance measurements are financial methods based on accounting data and they are generally conducted based on the principles of efficiency and profitability. Cost analysis, volume- profit analysis, budgets, standard costs are examples of. Financial objectives of the organizations are usually operating income; the profitability of capital, and economic value added which are measurable/digitized goals (Akgemci, 2008: 53). Non-financial performance measures are classified as the following (Koçel, 2014: 449-450): a) Indicators related to customers (customer satisfaction, the number of new customers, customer profitability, et al.), a) Indicators related to customers (customer satisfaction, the number of new customers, customer profitability, et al.), b) Indicators related to corporate learning and innovation (employee skills, employee satisfaction, indicators related to information technology, the motivation of employees to the awards, the number and amount of turnover and absenteeism rates), b) Indicators related to corporate learning and innovation (employee skills, employee satisfaction, indicators related to information technology, the motivation of employees to the awards, the number and amount of turnover and absenteeism rates), c)Performance indicators related to internal processes (quality, new products, production error rate etc.) The basic criteria that can be used to measure organizational performance in general can be summarized as follows (Quoted by Şimşek and Öğe, 2014: 297): The basic criteria that can be used to measure organizational performance in general can be summarized as follows (Quoted by Şimşek and Öğe, 2014: 297):  Quality  Productivity  Profitability  Cost  Innovation Customer satisfaction  Innovation  Customer satisfaction  Employee satisfaction p y It is underlined that organizational performance assessment serves many purposes in the field of human resources. One of them is that it helps human resources managers on the issues of promotion, transfer and dismissal. These assessments also indicated the needs of training and development. They also demonstrate employees’ ability and skills for the development of solution-generating programs. 2.2.3. Organizational Performance Organizational performance is the expression of the degree of fulfillment of business objectives or tasks obtained after a certain period of output/outcome (Akal, 2011: 15-16). Organizational performance, which is the achievement indicator of an organization’s activities, is the sum of each employee’s individual performance (Vural and Coşkun, 2007: 163). With the advent of globalization and rapid change in the world, it is observed that organizations’ understanding of competition is changing or they have to be in a position to change it. Today, organizations have to keep up with quality, innovation, flexibility, speed and distinctness features of competition dimensions. In this sense, it is important to use tangible and intangible assets owned by the organization in such a way that the organization obtains a competitive advantage by having these resources (Akgemci, 2008: 478). Organizations founded with the goal of manufacturing goods and services necessary for human needs are in contact with their environments in terms of input and output. Organizations can perform high and sustain for longer periods of time when this interaction continue in a good way. In this context, 2739 Year: 2022 Vol:8 Issue: 48 ATLAS INTERNATIONAL REFEREED JOURNAL ON SOCIAL SCIENCES performance values of those who work in the organization and their competencies are placed among the significant indicators of organizational effectiveness and efficiency (Göksel, 2013: 2). 2.2.4. Organizational Performance Assessment Finally, it provides feedback indicating how employees see their organizations performance (Welch, 2013: 565). At the same time, a decision about humans is a special condition which requires individual rules for each person. These decisions have the greatest impact on the performance of the organization (Drucker, 2012: 30). 3.1.3. Scales In the first part, 6 items regarding the demographics (age range, education level, length of service in the profession, the number of hospital staff, the hospital's bed capacity, and position in the organization) were asked. 3.1.1.Research Objective The purpose of this study is to research the relationship between corporate reputation and organizational performance. For this goal, private hospital managers operating in Konya province were contacted in order to measure the effects of corporate reputation on organizational performance and a questionnaire was conducted with 50 managers who are working at 8 different private hospitals. 3.1.2. Sample The research sample is consisted for private hospital managers in Konya province. The reason for that is only 8 of the contacted hospitals responded back. A questionnaire was carried out as a data collection tool and 50 questionnaires were administered. Research questionnaires are consisted of 3 parts. 2.3. Corporate Reputation and Organizational Performance Relationship The relationship between corporate reputation and organizational performance in business life has been a topic of interest and controversy for more than half a century. Moreover; company’s reputation 2740 2740 Year: 2022 Vol:8 Issue: 48 ATLAS INTERNATIONAL REFEREED JOURNAL ON SOCIAL SCIENCES affects the company performance in economy ways. Together with the technology employed, they determine transaction and transformation costs that make up total costs (North, 1991: 120). affects the company performance in economy ways. Together with the technology employed, they determine transaction and transformation costs that make up total costs (North, 1991: 120). The quality of manufactured goods and provided services affects corporate reputation and it also influences or determines employee performance. Having a good reputation in the eyes of the society is a sign of high quality and stronger performance. Therefore, employees are successful to the extent that they satisfy the customers. Positive customer feedback and high customer satisfaction indicate higher performance and the gained reputation as a result of this (Quoted by Izgar, 2012: 168). In today organizations, protection and development of corporate reputation is crucial in learning, developing and growing. It also becomes prerequisite for Continuous learning, high job performance and organizational activity (Barutçugil, 2004: 334). Corporate reputation provides benefits to many aspects of the businesses. For instance, when investors increase the company's market share, customers pay a high price to buy the products of a reputable company (Fombrun, 1996: 39). Employees might prefer to work with organizations with higher reputation –when all other conditions are the same- due to the cost advantages. Suppliers tend to prefer companies with a positive reputation regarding the dangers of the contract. When such kinds of organizations lead in decreasing operating and monitoring costs, it gives the opportunity to suppliers to concern less (Robert and Dowling, 2002: 1079). These advantages of corporate reputations also help organizational performance to increase. ATLAS INTERNATIONAL REFEREED JOURNAL ON SOCIAL SCIENCES ATLAS INTERNATIONAL REFEREED JOURNAL ON SOCIAL SCIENCES 3.2.Findings 3.2.1. Research Hypothesizes 3.1.4. Corporate Reputation Scale In the second part, corporate reputation scale consisting of 22 items and adapted to Turkey by Altıntaş in 2005 developed by Charles J. Fombrun was used. The questionnaire was taken from Irfan Yurt’s Master’s thesis (Yurt, 2012: 92). Collected data was evaluated by 5-point Likert scale. Statements in the scale are as follows: 1= strongly disagree, 2= Disagree, 3= Undecided, 4= Agree, and 5= Strongly Agree. 5-point Likert scale is consisted of 22 items and 7 sub-dimensions. These sub-dimensions are as follows: Having general information about institution, Management Quality, Product and Services, Leadership, Difference, Reliability, Social Responsibility. 2741 Year: 2022 Vol:8 Issue: 48 3.1.5. Organizational Performance Scale In the third part, organizational performance scale developed by Latinen and consisting of 10 items was used. The questionnaire was taken from Umut Avcı’ s Doctorate thesis (Avcı, 2005:163). Collected data was evaluated by 5-point Likert scale. Statements in the scale are as follows: 1= Strongly disagree, 2= Disagree, 3= Undecided, 4= Agree, and 5= Strongly Agree. 5-point Likert scale is consisted of 10 items and 2 sub-dimensions. There is a meaningful and positive correlation between two dimensions of organizational performance (financial performance dimension and non-financial performance dimension). Study Method: This study provided frequencies based on data. After the provision of this data, data was tested with Normal Distribution Compliance Kolmogorov-Smirnov Normality Test in order to decide on which test to use. As a result of data normality and homogeneous variance, Parametric Tests applicability was decided to conduct. To investigate the differences between the averages, one- way variance analysis (ANOVA) was used. For correlations Pearson Correlation coefficient was used while for reliability analysis Cronbach’s Alpha Coefficient was used. ATLAS INTERNATIONAL REFEREED JOURNAL ON SOCIAL SCIENCES Pearson Correlation test was used for the correlation between social responsibility dimension of corporate reputation and financial performance dimension of organizational performance, and a Having general information about institution Manage ment Quality Product and Services Leaderssh ip Differen ce Reliabilit y Social Responsibili ty Financial Performance Dimension 0,281 0,372 0,396 0,311 0,449 0,401 0,481 p-value 0,048 0,008 0,004 0,028 0,011 0,004 0,000 Non-Financial Performance Dimension -0,037() 0,605 0,681 0,650 0,659 0,677 0,797 P- value 0,797 0,000 0,000 0,000 0,000 0,000 0,000 Pearson Correlation test was used for the correlation between social responsibility dimension of corporate reputation and financial performance dimension of organizational performance, and a positive 48,1% correlation was found as a result of this test. As p value is 0,000<0,05, it is possible to suggest that there is a meaningful correlation between social responsibility dimension of corporate reputation and financial performance dimension of organizational performance. Pearson Correlation test was used for the correlation between social responsibility dimension of corporate reputation and financial performance dimension of organizational performance, and a positive 48,1% correlation was found as a result of this test. As p value is 0,000<0,05, it is possible to suggest that there is a meaningful correlation between social responsibility dimension of corporate reputation and financial performance dimension of organizational performance. Pearson Correlation test was used for the correlation between having general information about institution dimension of corporate reputation and non-financial performance dimension of organizational performance, and a negative %3,7% correlation was found as a result of this test. Asp value is 0,797>0,05, it is possible to suggest that there is a meaningful correlation between having general information about institution dimension of corporate reputation and non-financial performance dimension of organizational performance. All correlations among the other dimensions are meaningful and positive. All correlations among the other dimensions are meaningful and positive. Hypothesis 4: Is there a difference between age variance groups according to Corporate Reputation and Organizational Performance scale. s 4: Is there a difference between age variance groups according to Corporate Reputatio izational Performance scale. g N Average Std. 3.2.1. Research Hypothesizes Hypothesis 1: There is a positive and meaningful correlation between corporate reputation and organizational performance. Organizational Performance Corporate Reputation Pearson Correlation Coefficient 0,706 p value 0,000 : There is a positive and meaningful correlation between corporate reputation and l performance. Pearson Correlation test was used for the correlation between corporate reputation and organizational performance, and it was found that there was a positive 70,6% correlation. As p value is 0,000<0,05, it is possible to suggest that there is a meaningful correlation between Corporate Reputation and Organizational Performance. Hypothesis 2: Corporate reputation perspectives of research participants are positive. Hypothesis 2: Corporate reputation perspectives of research participants are positive. One-sample t-test N Average Std Deviation Corporate Reputation 50 4,1773 0,56087 Test Value 4 T Degrees of Freedom P value Average Difference 95% Confidence İnterval For The Difference Lower Upper Corporate Reputation 2,235 49 0,015 0,17727 0,0179 0,3367 Corporate reputation perspectives of research participants are positive (p 0 015 < 0 05) orporate reputation perspectives of research participants are positive (p=0,015 < 0,05). Hypothesis 3: There is a positive and meaningful correlation between 7 dimensions of corporate reputation and 2 dimensions of organizational performance. Year: 2022 Vol:8 Issue: 48 ATLAS INTERNATIONAL REFEREED JOURNAL ON SOCIAL SCIENCES Pearson Correlation test was used for the correlation between social responsibility dimension of corporate reputation and financial performance dimension of organizational performance, and a positive 48,1% correlation was found as a result of this test. As p value is 0,000<0,05, it is possible to suggest that there is a meaningful correlation between social responsibility dimension of corporate reputation and financial performance dimension of organizational performance. Having general information about institution Manage ment Quality Product and Services Leaderssh ip Differen ce Reliabilit y Social Responsibili ty Financial Performance Dimension 0,281 0,372 0,396 0,311 0,449 0,401 0,481 p-value 0,048 0,008 0,004 0,028 0,011 0,004 0,000 Non-Financial Performance Dimension -0,037() 0,605 0,681 0,650 0,659 0,677 0,797 P- value 0,797 0,000 0,000 0,000 0,000 0,000 0,000 ATLAS INTERNATIONAL REFEREED JOURNAL ON SOCIAL SCIENCES ATLAS INTERNATIONAL REFEREED JOURNAL ON SOCIAL SCIENCES Again in the same table, as p value is 0,275>0.05 it was found that hospital employee numbers variable has no effect (or in other ATLAS INTERNATIONAL REFEREED JOURNAL ON SOCIAL SCIENCES Reputation University 32 91,468 2,327 Master and above 12 91,833 3,007 Organizational Performance High School 6 41,666 1,145 0,826 0,444 University 32 39,781 0,779 Master and above 12 41,583 1,856 According to findings, as the p value is 0,877>0.05 in the table above, it was found that education level variable has no effect (or in other words, there is no difference among education level groups with regards to corporate reputation) on corporate reputation. Again in the same table, as p value is 0,826>0.05 it was found that education level variable has no effect (or in other words, there is no difference among education levels with regards to organizational performance) on organizational performance. Hypothesis 6: Is there a difference between hospital employee numbers according to Corporate Reputation and Organizational Performance scale. Hypothesis 6: Is there a difference between hospital employee numbers according to Corporate Reputation and Organizational Performance scale. Hospital Employee Numbers N Average Std. Deviation F-test Value P-Value Corporate Reputation 51-100 3 87,500 5,238 1,047 0,381 101-150 12 90,041 3,980 151-250 20 95,200 2,345 250 and above 15 90,000 3,544 Organizational Performance 51-100 3 41,333 2,185 0,275 0,843 101-150 12 40,083 1,469 151-250 20 39,900 0,940 250 and above 15 41,266 1,491 According to findings, as the p value is 0,381>0.05 in the table above, it was found that hospital employee numbers have no effect (or in other words, there is no difference among hospital employee numbers with regards to corporate reputation) on corporate reputation. Again in the same table, as p value is 0,275>0.05 it was found that hospital employee numbers variable has no effect (or in other words, there is no difference among hospital employee numbers with regards to organizational performance) on organizational performance. 2744 Year: 2022 Vol:8 Issue: 48 Hypothesis 7: Is there a difference between hospital bed capacity according to Corporate Reputation and Organizational Performance scale. Hospital Bed Capacity N Average Std. ATLAS INTERNATIONAL REFEREED JOURNAL ON SOCIAL SCIENCES Deviation F-test Value P-Value Corporate Reputation 51-100 3 87,500 5,238 1,047 0,381 101-150 12 90,041 3,980 151-250 20 95,200 2,345 250 and above 15 90,000 3,544 Organizational Performance 51-100 3 41,333 2,185 0,275 0,843 101-150 12 40,083 1,469 151-250 20 39,900 0,940 250 and above 15 41,266 1,491 According to findings, as the p value is 0,381>0.05 in the table above, it was found that hospit employee numbers have no effect (or in other words, there is no difference among hospital employe numbers with regards to corporate reputation) on corporate reputation. Again in the same table, as value is 0,275>0.05 it was found that hospital employee numbers variable has no effect (or in oth words, there is no difference among hospital employee numbers with regards to organization performance) on organizational performance. H th i 7 I th diff b t h it l b d it di t C t R t ti ATLAS INTERNATIONAL REFEREED JOURNAL ON SOCIAL SCIENCES Reputation University 32 91,468 2,327 Master and above 12 91,833 3,007 Organizational Performance High School 6 41,666 1,145 0,826 0,444 University 32 39,781 0,779 Master and above 12 41,583 1,856 According to findings, as the p value is 0,877>0.05 in the table above, it was found that education level variable has no effect (or in other words, there is no difference among education level groups with regards to corporate reputation) on corporate reputation. Again in the same table, as p value is 0,826>0.05 it was found that education level variable has no effect (or in other words, there is no difference among education levels with regards to organizational performance) on organizational performance. Hypothesis 6: Is there a difference between hospital employee numbers according to Corporate Reputation and Organizational Performance scale. Hospital Employee Numbers N Average Std. Deviation F-test Value P-Value Corporate Reputation 51-100 3 87,500 5,238 1,047 0,381 101-150 12 90,041 3,980 151-250 20 95,200 2,345 250 and above 15 90,000 3,544 Organizational Performance 51-100 3 41,333 2,185 0,275 0,843 101-150 12 40,083 1,469 151-250 20 39,900 0,940 250 and above 15 41,266 1,491 According to findings, as the p value is 0,381>0.05 in the table above, it was found that hospital employee numbers have no effect (or in other words, there is no difference among hospital employee numbers with regards to corporate reputation) on corporate reputation. ATLAS INTERNATIONAL REFEREED JOURNAL ON SOCIAL SCIENCES Deviation F-test Value P-Value Corporate Reputation 18-29 4 87,500 14,062 0,835 0,482 30-43 24 90,041 2,508 44-55 20 95,200 1,645 56 and above 2 90,000 10,000 Organizational Performance 18-29 4 39,000 3,109 0,925 0,436 30-43 24 39,541 1,058 44-55 20 41,800 0,907 56 and above 2 40,500 3,500 According to findings, as the p value is 0,482>0.05 in the table above, it was found that age variable has no effect (or in other words, there is no difference among age groups with regards to corporate reputation) on corporate reputation. Again in the same table, as p value is 0,436>0.05it was found that age variable has no effect (or in other words, there is no difference among age groups with regards to organizational performance) on organizational performance. 274 Year: 2022 Vol:8 Issue: 48 Hypothesis 5: Is there a difference between education level groups according to Corporate Reputation and Organizational Performance scale. Education Level N Average Std. Deviation F-test Value P-Value Corporate High School 6 94,333 5,327 0,132 0,877 5: Is there a difference between education level groups according to Corporate nd Organizational Performance scale. 2743 ATLAS INTERNATIONAL REFEREED JOURNAL ON SOCIAL SCIENCES Reputation University 32 91,468 2,327 Master and above 12 91,833 3,007 Organizational Performance High School 6 41,666 1,145 0,826 0,444 University 32 39,781 0,779 Master and above 12 41,583 1,856 According to findings, as the p value is 0,877>0.05 in the table above, it was found that educatio level variable has no effect (or in other words, there is no difference among education level group with regards to corporate reputation) on corporate reputation. Again in the same table, as p value 0,826>0.05 it was found that education level variable has no effect (or in other words, there is n difference among education levels with regards to organizational performance) on organization performance. Hypothesis 6: Is there a difference between hospital employee numbers according to Corpora Reputation and Organizational Performance scale. Hospital Employee Numbers N Average Std. ATLAS INTERNATIONAL REFEREED JOURNAL ON SOCIAL SCIENCES ATLAS INTERNATIONAL REFEREED JOURNAL ON SOCIAL SCIENCES 90-120 7 35,285 1,614 150 and above 7 43,000 1,690 According to findings, as the p value is 0,043<0.05 in the table above, it was found that hospital bed capacity has an effect (or in other words, there is a difference among hospital bed capacity with regards to corporate reputation) on corporate reputation. Tukey was used in order to find from which group this effect is stemmed, and it was found that this is rooted from 30 and below and 30-60 groups. Again in the same table, as p value is 0,014>0.05 it was found that hospital bed capacity variable has an effect (or in other words, there is a difference among hospital bed capacity with regards to organizational performance) on organizational performance. Tukey was used in order to find from which group this effect is stemmed, and it was found that this is rooted from 90-120 group and other three groups. 90-120 groups have the least organizational performance score. Hypothesis 8: Is there a difference between hospital working duration according to Corporate Reputation and Organizational Performance scale. Hypothesis 8: Is there a difference between hospital working duration according to Corporate Reputation and Organizational Performance scale. Hospital Working Duration N Average Std. Deviation F-test Value P Value Corporate Reputation Less than 5 years 5 90,400 6,675 0,643 0,638 5-8 years 16 89,125 3,771 8-14 years 16 95,875 1,823 14-20 11 90,818 3,895 More than 20years 2 92,000 12,000 Organizational Performance Less than 5 years 5 39,000 2,323 1,010 0,413 5-8 years 16 38,875 1,244 8-14 years 16 41,437 1,056 14-20 11 41,363 1,383 More than 20 years 2 43,500 6,500 According to findings, as the p value is 0,638>0.05 in the table above, it was found that hospital working duration has no effect (or in other words, there is no difference among hospital working durations with regards to corporate reputation) on corporate reputation. Again in the same table, as p value is 0,413>0.05 it was found that hospital working duration variable has no effect (or in other words, there is no difference among hospital working durations with regards to organizational performance) on organizational performance. ATLAS INTERNATIONAL REFEREED JOURNAL ON SOCIAL SCIENCES Deviation F-test Value P-Value Corporate Reputation 30 and below 8 82,250 5,759 2,695 0,043 30-60 23 95,956 1,767 60-90 5 97,200 4,465 90-120 7 87,000 3,970 150 and above 7 90,714 6,014 Organizational Performance 30 and below 8 39,750 2,059 3,500 0,014 30-60 23 40,956 0,758 60-90 5 42,800 2,332 Hypothesis 7: Is there a difference between hospital bed capacity according to Corpora and Organizational Performance scale. there a difference between hospital bed capacity according to Corporate Reputation nal Performance scale. ATLAS INTERNATIONAL REFEREED JOURNAL ON SOCIAL SCIENCES 90-120 7 35,285 1,614 150 and above 7 43,000 1,690 4. CONCLUSION AND RECOMMENDATIONS Being reputable is morally a necessity for both individuals and institutions. Feeling important in the society from the perspectives of individuals and organizations, and having some advantages in the environment they live in is only possible with the help of reputation. When strong organizations are observed, they manage to be afloat with their mutual relations by using various credits even when they experience economic problems and at crisis times. Reputation, which is one of the most significant assets of organizations, is a value that is earned through long time but can be lost at a short span of time. Therefore, it is important to protect this credibility and not to lose the reputation earned. Having a positive credibility in the eyes of the society for an organization is the indicator for high quality and better performance. Customer satisfaction levels of organizations are at the same time an 2745 Year: 2022 Vol:8 Issue: 48 Year: 2022 Vol:8 Issue: 48 ATLAS INTERNATIONAL REFEREED JOURNAL ON SOCIAL SCIENCES important indicator of their achievements. So, organizations are successful as long as they satisfy their customers, partners and the community. important indicator of their achievements. So, organizations are successful as long as they satisfy their customers, partners and the community. This study was conducted with the purpose of researching the effects of corporate reputations on organizational performance involving 50 managers working at managerial positions in 8 private hospitals in Konya province. According to research findings, it was concluded that there is a meaningful correlation between Corporate Reputation and Organizational Performance and corporate reputation perspectives of research participants are positive. It is possible to suggest that there is a meaningful correlation between social responsibility dimension of corporate reputation and financial performance dimension of organizational performance. On the other hand, it was found out that there is not meaningful correlation between having general information about institution dimension of corporate reputation and non-financial performance dimension of organizational performance. It was detected that age variable and education level has no effect on organizational performance, and that hospital employee numbers have no effect on organizational performance. It was also concluded that hospital bed capacity has not effect on corporate reputation and organizational performance. Consequently, it is important to note that working duration at hospital has no effect on corporate reputation. SOURCE Akal, Z. (2011). İşletmelerde Performans Ölçüm ve Denetimi Çok Yönlü Performans Göstergeleri, 7. Basım, Milli Prodüktivite Merkezi Yayınları no:473, Ankara, Akal, Z. (2011). İşletmelerde Performans Ölçüm ve Denetimi Çok Yönlü Performans Göstergeleri, 7. Basım, Milli Prodüktivite Merkezi Yayınları no:473, Ankara, Akgemci, T. (2008). Stratejik Yönetim, 2.baskı, Nisan, Gazi Kitabevi, Ankara. Avcı, U. 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https://openalex.org/W4389311035	https://www.frontiersin.org/articles/10.3389/fgstr.2023.1241901/pdf?isPublishedV2=False	English	null	Current and future opportunities for the management of primary biliary cholangitis	Frontiers in gastroenterology	2,023	cc-by	8,458	TYPE Review PUBLISHED 04 December 2023 DOI 10.3389/fgstr.2023.1241901 KEYWORDS primary biliary cholangitis, treatment, ursodeoxycholic acid, obeticholic acid, ﬁbrates peroxisome proliferator-activated receptors primary biliary cholangitis, treatment, ursodeoxycholic acid, obeticholic acid, ﬁbrates, peroxisome proliferator-activated receptors OPEN ACCESS OPEN ACCESS EDITED BY Atoosa Rabiee, United States Department of Veterans Affairs, United States REVIEWED BY Valentina Cossiga, University of Naples Federico II, Italy Ciro Celsa, University of Palermo, Italy CORRESPONDENCE Sandra Naffouj snaffo2@uic.edu RECEIVED 17 June 2023 ACCEPTED 17 November 2023 PUBLISHED 04 December 2023 CITATION Naffouj S and Wang J (2023) Current and future opportunities for the management of primary biliary cholangitis. Front. Gastroenterol. 2:1241901. doi: 10.3389/fgstr.2023.1241901 EDITED BY Atoosa Rabiee, United States Department of Veterans Affairs, United States Sandra Naffouj and Jennifer Wang Division of Gastroenterology and Hepatology, Department of Medicine, University of Illinois, Chicago, IL, United States Division of Gastroenterology and Hepatology, Department of Medicine, University of Illinois, Chicago, IL, United States Primary biliary cholangitis (PBC) is a rare immune-mediated chronic cholestatic liver disease that can progress to liver ﬁbrosis and, ultimately, cirrhosis if left untreated. Since the pathogenesis of PBC is not well understood, curative therapies have yet to be established. Ursodeoxycholic acid (UDCA), the standard of care treatment for PBC, has been proven to reduce disease progression and improve transplant-free survival. However, one third of patients have no response or partial biochemical response to UDCA and are at increased risk for disease progression. In such cases, second-line therapy with obeticholic acid (OCA) or peroxisomes proliferator-activated receptors (PPARs) should be considered in conjunction with UDCA. In this review article, we aim to provide an overview of the most recent data on PBC treatment in patients with inadequate response to UDCA, as well as novel therapies in the early stages of development. CITATION Naffouj S and Wang J (2023) Current and future opportunities for the management of primary biliary cholangitis. Front. Gastroenterol. 2:1241901. doi: 10.3389/fgstr.2023.1241901 Frontiers in Gastroenterology frontiersin.org COPYRIGHT © 2023 Naffouj and Wang. 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. Introduction Primary biliary cholangitis (PBC) is an uncommon chronic cholestatic liver disease, characterized by immune-mediated inﬂammatory destruction of the small and medium intra-hepatic bile ducts (1). It predominantly affects women, and its peak incidence occurs in the fourth and ﬁfth decades of life. If left untreated, it can progress to liver ﬁbrosis, and ultimately, cirrhosis with its related complications (2). Since the pathogenesis of PBC is not very well understood, curative therapies have yet to be established, and the focus has been on preventing disease progression and ameliorating symptoms. Ursodeoxycholic acid (UDCA) is the standard of care and recommended for all patients with PBC by the American Association for the Study of Liver Disease (AASLD) and the European Association for the Study of the Liver (EASL) (3, 4). It has been shown to remarkably improve the progression of PBC as well as clinical outcomes (5, 6). The recommended dose is 13-15 mg/kg of body weight per day. It is a hydrophilic bile acid with an excellent safety proﬁle, even in pregnancy (7). Additionally, a recent retrospective study showed that UDCA use as a preventative therapy post-liver transplant in PBC patients was associated Frontiers in Gastroenterology Frontiers in Gastroenterology 01 frontiersin.org 10.3389/fgstr.2023.1241901 Naffouj and Wang receive 10 mg of OCA, 5 mg of OCA with up-titration to 10 mg, or placebo in addition to UDCA. A small group of patients received OCA as monotherapy due to intolerance to UDCA. The primary endpoint of achieving ALP reduction and normal T. bili occurred more frequently in both OCA groups (46 – 47%) compared to the placebo group (10%). There was no difference in ﬁbrosis staging between the groups at the end of the study, and dose-dependent pruritis was the most common side effect related to OCA. The recommended starting dose is 5 mg, which can be increased to 10 mg if reduction in ALP and/or bilirubin does not occur within 3 months (17). OCA can also be started earlier in the course for patients who are intolerant to UDCA, more commonly due to GI symptoms such as unexplained diarrhea (18). In 2021, the FDA updated the label on OCA, restricting its use in patients with advanced cirrhosis deﬁned as current or prior episodes of decompensation events or with compensated cirrhosis who have evidence of portal hypertension. Prognostic tools for PBC It is imperative to risk stratify PBC patients and identify high- risk groups who may progress to cirrhosis with its associated complications. Poor prognostic factors prior to starting treatment are young age at disease onset (<45 years), male gender, advanced ﬁbrosis, and abnormal total bilirubin (T. bili) and albumin (4). Additionally, stratifying patients based on their biochemical response to UDCA after one year of therapy offers a readily available measure to identify high-risk groups that may beneﬁt from additional treatment. The two most used prognostic models are GLOBE and UK-PBC scores (11, 12). Biochemical markers including alkaline phosphatase (ALP) and T. bili have been shown to independently predict outcomes in PBC patients, including need for liver transplantation (LT) and death (13). Therefore, they have been widely used as surrogate markers for disease outcomes in clinical trials given rarity of the disease and its slow progressive nature (14). Fibrosis assessment in PBC patients also provides an important prognostic factor irrespective of the biochemical response, suggesting that ﬁbrosis staging should be incorporated into risk stratiﬁcation models (15). Considering all these ﬁndings, patients who do not achieve targeted biochemical response after one year of UDCA and those with advanced ﬁbrosis are at high risk for disease progression and should be evaluated for second-line treatments. A summary of the most recent clinical trials on novel therapies in PBC is demonstrated in Table 1. Real-world data continue to emerge, demonstrating the safety and efﬁcacy of OCA on clinical outcomes such as persistent improvement in ALP, stabilization of bilirubin, ﬁbrosis, and transplant-free survival (21–23). For example, the 5-year open- label extension study of the POISE trial showed that long-term OCA treatment was associated with improved histological features, including ﬁbrosis and ductular injury (22). Interim analyses from the same study conﬁrmed the efﬁcacy and safety of OCA at three years with continued improvement in ALP levels and stabilization of T. bili with no serious adverse events reported. Pruritis and fatigue remained the two most common adverse events (23). Additionally, patients in the POISE group had signiﬁcantly better transplant-free survival when compared to the Global PBC control group at the 6-year mark with hazard ratios (HRs) of 0.29 (95% CI, 0.10-0.83) (24). A phase 4 clinical trial is ongoing to evaluate the effect of OCA on long-term clinical outcomes (COBALT NCT02308111). Obeticholic acid Obeticholic acid (OCA) is a synthetic bile acid that acts as a potent farnesoid X receptor (FXR) agonist, impairing bile acid synthesis and promoting bile acid secretion. It is the only approved second-line therapy in adult PBC patients in the US, recommended to be used in conjunction with UDCA in partial responders or as monotherapy in those who are intolerant to UDCA (3, 4). The medication was approved by the Food and Drug Administration (FDA) and European Medicines Agency (EMA) based on results of the POISE trial in 2016, showing its efﬁcacy in improving biochemical markers in high-risk subgroups (16). The trial was a 12-month, double-blinded, placebo-controlled phase III trial that included 217 patients who were partial responders or intolerant to UDCA. Subjects were randomized to Introduction The AASLD then published an updated practical guidance statement reporting the contraindicated use of OCA in advanced cirrhosis and recommending cautious use in select patients with compensated cirrhosis and without features of portal hypertension with close monitoring (19). Similarly, the EMA updated the OCA label in 2022 prohibiting its use in decompensated cirrhosis including Child-Pugh Class B and C (20). with decreased risk of PBC recurrence, graft loss, and all-cause mortality (8). However, it has been estimated that 30-40% of patients with PBC have no response or partial biochemical response to UDCA and are at high risk for disease progression, highlighting the necessity for novel therapies in the management of PBC (9). In such cases, a second-line treatment is recommended in conjunction with UDCA (10). In this review, we aim to summarize the most recent evidence on PBC therapies in patients with inadequate response to UDCA as well as developing therapies on the horizon. Prognostic tools for PBC In addition to OCA, newer FXR agonists are under investigation in PBC treatment, such as cilofexor (NCT02943447) and ASC 42 (NCT05190523). Frontiers in Gastroenterology Peroxisomes proliferator- activated receptors Fibrates are a class of hypolipidemic agents that act on the peroxisomes-proliferator-activated receptors (PPARs), a transcription factor involved in fat catabolism and inﬂammatory response, resulting in decreased bile acid synthesis in the liver, detoxiﬁcation of bile acids, and increased bile acid output as well as suppressing inﬂammation (25). Fenoﬁbrate is the only ﬁbrate that is FDA- approved in the United States (US) for dyslipidemia and primary 02 frontiersin.org 10.3389/fgstr.2023.1241901 Naffouj and Wang TABLE 1 Summary of the most recent clinical trials on novel therapies in PBC. TABLE 1 Summary of the most recent clinical trials on novel therapies in PBC. Medication Trial/ duration Subjects’ Groups Outcomes/primary end point Considerations Obeticholic acid (OCA) POISE RCT(16) 12 months . OCA 10 mg group + UDCA . OCA 5-10 mg group + UDCA . Placebo + UDCA . ALP <1.67 ULN with 15% reduction from baseline and normal T.bili . Pruritis is the most common side effect . No change in ﬁbrosis . OCA is contraindicated in advanced or decompensated cirrhosis and biliary obstruction Bezaﬁbrate (pan- PPAR agonist) BEZURSO RCT(26) 24 months . Bezaﬁbrate 400 mg daily + UDCA . Placebo + UDCA Biochemical response (normal Tbili, ALP, aminotransferase, albumin, prothrombin index) . Improved pruritis, fatigue, and liver ﬁbrosis . Limited use in advanced cirrhosis . Concerns regarding kidney function, liver enzyme elevation, and myalgias Bezaﬁbrate (pan- PPAR agonist) FITCH RCT(27) 21 days . Bezaﬁbrate 400 mg daily . Placebo Patients with moderate-severe pruritis from cholestatic disease (VAS>5 of 10) . >50% reduction of pruritis . Short term study with unclear long-term safety . Increase in serum creatinine in 3% of treated patients Elaﬁbranor (dual-PPAR agonist) Phase II RCT(29) 12 weeks . Elaﬁbranor 80 mg . Elaﬁbranor 120 mg . Placebo . Primary: % change in ALP . Composite end point: ALP <1.67 ULN, ALP reduction >15%, Tbili < 1 ULN . Improved pruritis . Improved gamma-glutamyl transferase (GGT), high-sensitivity C-reactive protein . Side effects: nausea, diarrhea, fatigue, headache . Phase III RCT ELATIVE is underway; it will include markers of ﬁbrosis Saroglitazar (dual-PPAR agonist) Phase II RCT(31) 16 weeks . Saroglitazar 4 mg . Saroglitazar 2 mg . Placebo . Endpoint: ALP reduction . 4 patients in the 4 mg group developed elevated liver enzymes (DILI), withdrew from study . Peroxisomes proliferator- activated receptors Further studies at 1 mg and 2 mg are underway Seladelpar (PPAR-delta agonist) Phase II open label study(32) 52 weeks .Seladelpar 5 mg (n=53) .Seladelpar 10 mg (n=55) .Seladelpar 2 mg (N=11) 12-week dose-ranging period 40-week extension period . Primary endpoint: ALP change at week 8 . Secondary endpoints: biochemical response (ALT, AST, GGT, LDL, TG, C4, FGF-19) . Pruritis (VAS score) . The criteria for dose increase were not standardized . Improved pruritis . Improved lipid proﬁle . Phase III trial (RESPONSE) seladelpar 10 mg vs. 5 mg vs. placebo 12 months Budesonide Phase III RCT(36) 36 months . Budesonide 9 mg daily + UDCA (12-16 mg/kg/day) . Placebo + UDCA (12-16 mg/ kg/day) . Histological improvement and preventing ﬁbrosis progression (not achieved) . POISE end point of biochemical response were reached in treatment group . Budesonide may improve biochemical markers in non-cirrhotic PBC patients . Consider steroid related side effects NOX Inhibitor Phase II RCT(48) 24 weeks . Setanaxib 400 mg daily + UDCA . Setanaxib 400 mg BID + UDCA . Placebo . Primary endpoint: change from baseline in GGT . Secondary endpoint: change from baseline in ALP, liver stiffness via elastography, and fatigue. . Preliminary results suggest possible anti- cholestatic and anti-ﬁbrotic effects trial enrolled 100 PBC patients with inadequate response to UDCA deﬁned by the Paris-2 criteria (i.e., a serum level of ALP or AST >1.5 times the ULN or an abnormal T.bili after 6 months or more of treatment, excluding patient with T. bili level >3 mg/dl) (26). Patients were randomized to receive 400 mg of bezaﬁbrate or placebo in addition to UDCA for 24 months. The primary endpoint deﬁned as a complete biochemical response (normal levels of T.bili, ALP, aminotransferase, albumin, and prothrombin index) was met in 31% of patients in the treatment group compared to none in the placebo group. Normal ALP level was achieved in hypercholesterolemia, with ongoing phase II clinical trials to assess its efﬁcacy in the management of PBC with and without UDCA (NCT01141296 and NCT00575042). Frontiers in Gastroenterology Seladelpar Seladelpar, a PPAR-delta agonist, was assessed in PBC patients who were non-responders or intolerant to UDCA, in a 52-week phase II, dose-ranging, open-label study randomizing patients in 1:1 ratio to seladelpar 5 mg/day (n=53) or 10 mg/day (n=55) or 2 mg/day (n=11) for 12 weeks (32). Doses could be titrated to 10 mg/ day at or after 12 weeks of initiation. 21% of patients had compensated cirrhosis, and 71% had pruritis. The mean ALP levels were signiﬁcantly reduced from baseline, and the reduction was dose-dependent; 23% in the 2 mg cohort, 35% in the 5 mg cohort, and 43% in the 10 mg cohort (p<0.005). Biochemical responses and further improvements were maintained until week 52. ALP normalization occurred in 31% of patients in the 10 mg cohort at week 12 and was durable at 33% through week 52. On the other hand, ALP normalization occurred in 0% and 11% of patients in the 2 mg and 5 mg groups at week 12, respectively, and in 9% and 13% at week 52, respectively. Furthermore, mean pruritis VAS scores improved from baseline in all three cohorts at week 12. Symptoms remained stable or continued to improve until the end of the study in a dose-dependent fashion. A pivotal phase III, randomized, placebo-controlled trial is ongoing to further assess the treatment effect and safety of Seladelpar (NCT04620733). Although the data are limited on the long-term effect of ﬁbrates on transplant-free survival in PBC patients with inadequate response to UDCA, Tanaka et al. showed in a large retrospective Japanese nationwide cohort study that bezaﬁbrate-UDCA combination treatment was associated with reduction in all-cause mortality and need for LT compared to UDCA alone (HR 0.33, 95% CI 0.19-0.54, P<0.001) (28). Elaﬁbranor Elaﬁbranor is another promising ﬁbrate that is a dual-PPAR (alpha-delta) agonist. In a recent phase II double-blind RCT, 45 non-cirrhotic PBC patients with inadequate biochemical response to UDCA were randomized to elaﬁbranor 80 mg, elaﬁbranor 120 mg, or placebo (29). Patients who received either dose of elaﬁbranor had a signiﬁcant reduction in ALP level compared to the placebo group, achieving the composite endpoint of ALP <1.67-fold the ULN, a decrease of ALP >15%, and normalization of T.bili at 12 weeks. A favorable trend of improved pruritis was noted in patients who received elaﬁbranor. Patients who received the 120 mg dose had a signiﬁcant increase in sCr attributed to increased Cr release from muscle rather than a decrease in GFR since cystatin C level, a more accurate marker for GFR, remained unchanged. This study prepared the foundation for a larger prospective phase III trial, ELATIVE, that will determine the efﬁcacy and safety of elaﬁbranor as a second-line therapy for PBC (NCT04526665). Despite convincing data on the efﬁcacy of ﬁbrates in the treatment of PBC, more data are needed on their safety in patients with advanced liver disease. Saroglitazar 67% of the patients in the bezaﬁbrate group compared to 2% in the placebo group. Additionally, parallel improvement in pruritis, fatigue, and liver stiffness was observed in the bezaﬁbrate group. Features of portal hypertension and ALP level >2.53 the upper limit of normal (ULN), were independent predictors of treatment failure; thus, advanced cirrhosis and cholestasis are limiting factors to the use of bezaﬁbrate. Although the rate of adverse events was not signiﬁcant between the two groups, myalgia happened more frequently in the bezaﬁbrate group, serum creatinine increased in 5% of patients in the treatment group compared to 3% in the placebo group, and ﬁnally ALT increased >5 times the ULN within the ﬁrst 6 months of therapy initiation leading to treatment discontinuation, suggesting the importance of monitoring liver enzymes and kidney function after starting ﬁbrates. Saroglitazar is a novel dual-PPAR (alpha-gamma) agonist. It is currently approved outside the US for treatment of diabetic dyslipidemia and hypertriglyceridemia with type 2 diabetes mellitus that is not controlled with statin therapy as well as non- alcoholic steatohepatitis (NASH) (30). Due to similarities in the mechanistic pathogenesis, the efﬁcacy of saroglitazar is being evaluated in the management of PBC as a second-line therapy. Saroglitazar was effective in reducing ALP levels at doses of 2 mg and 4 mg compared to placebo in a recent phase II double bind RCT that included 37 PBC patients who are UDCA resistant or intolerant after 16 weeks of treatment. Of note, the study was terminated in 4 patients in the 4 mg group due to suspected drug- induced liver injury (DILI). Currently, investigations on doses of 1 mg and 2 mg are underway (31). Bezaﬁbrate also has beneﬁcial effect on pruritis as demonstrated in the FITCH trial which included 79 patients with moderate to severe pruritis (visual analog scale >5) due to cholestatic liver disease including PBC, primary and secondary sclerosing cholangitis who were randomized to receive bezaﬁbrate vs. placebo (27). 55% of patients in the PBC group achieved >50% reduction in pruritis compared to 13% in the placebo group (p=0.04) after 21 days. Furthermore, there was a signiﬁcant reduction in ALP level associated with improved pruritis in the treatment group. No signiﬁcant difference in creatinine change was noted between the two groups, concluding that bezaﬁbrate is an effective short-term treatment of moderate to severe pruritis due to cholestatic diseases. Bezaﬁbrate Bezaﬁbrate, a pan-PPAR agonist, is the third approved drug outside the US after UDCA and OCA based on the result of BEZURSO study. This double-blind, placebo-controlled phase III 03 frontiersin.org 10.3389/fgstr.2023.1241901 Naffouj and Wang 10.3389/fgstr.2023.1241901 Budesonide In the real- world setting, budesonide is rarely used as a second-line therapy for PBC as fewer biopsies are being performed to make the diagnosis, its contraindication in cirrhotic patients, and the high likelihood of achieving adequate response to the standard PBC therapies without immunosuppression even if overlap inﬂammatory features are present. at 5 years in 49 patients (39). However, larger randomized controlled trials are needed to conﬁrm the efﬁcacy of this combination. There are reports recommending the use of triple therapy (UDCA, OCA, and ﬁbrates) in patients who fail to achieve biochemical response with UDCA and OCA or ﬁbrates (40). Furthermore, CNP-104 is a novel proprietary system that combines disease-speciﬁc pathogenic antigens with pharmaceutical nanoparticles that mimics the routine removal of dying cells from the body. It is being studied in phase II double-blind placebo- controlled trial (NCT05104853). Several other promising therapies are in the early stages of development with a focus on both immune and non-immune pathways. features are usually corticosteroid-responsive, similar to classic autoimmune hepatitis (9). Additionally, in-vitro data showed that budesonide and UDCA have a synergistic effect on increasing biliary secretion of bicarbonate with a protective effect on the biliary epithelium from toxic bile acids (34, 35). Based on these facts, Hirschﬁeld et al. conducted a placebo-controlled randomized phase III trial on non-cirrhotic PBC patients who had histologically conﬁrmed inﬂammatory activity based on the Ishak score and inadequate response to UDCA to evaluate the role of budesonide as an additional therapy to improve liver histology including inﬂammation and ﬁbrosis (36, 37). Budesonide is not recommended in patients with cirrhosis due to the risk of portal vein thrombosis and uncontrolled systemic shunting of the medication (38). Patients were randomized to receive budesonide 9 mg daily in addition to UDCA 12-16 mg/kg/day vs. placebo and the same dose of UDCA. The study was underpowered due to recruitment challenges for the primary efﬁcacy analysis. In the intention-to-treat analysis, there was no difference in the primary endpoint between the treatment and placebo groups at 36 months. However, the proportion of patients in the budesonide group who met the POISE endpoint of biochemical response (ALP <1.67 the ULN with a 15% reduction from baseline and normal T.bili) was higher compared to the placebo group. The two groups did not differ in the frequency of side effects or serious adverse events. NOX inhibitors The nicotinamide adenine dinucleotide phosphate oxidases (NADPH oxidases, NOX) is the primary enzymatic source that regulates the production of reactive oxygen species (ROS), which have been implicated in a variety of disease processes including PBC (45). NOX1, NOX2, and NOX4 are expressed by the hepatocytes and play a signiﬁcant role in mediating liver ﬁbrosis and hepatocyte apoptosis (46). NOX4 additionally promotes the signaling of transforming growth factor beta (TGFb-1), which contributes to ﬁbrinogenesis in PBC (47). This suggests that selective NDAPH oxidase inhibitors, such as NOX4 inhibitors, might be a promising therapeutic agent. A recent phase II randomized trial evaluating the efﬁcacy and safety of setanaxib, a NOX1/NOX4 inhibitor, in UDCA non-responsive patients suggested promising anti-cholestatic and anti-ﬁbrotic effects for this agent as well as a role in improving fatigue warranting further evaluation in PBC patients with advanced ﬁbrosis (48). An ongoing placebo-controlled double- blind clinical trial assessing biochemical response at 52 weeks in patients who are intolerant or have an inadequate response to UDCA with elevated liver stiffness (≥8.8 kPa) started in 2021 with an estimated completion date in 2024 (NCT05014672). Budesonide Side effects such as hypertension, osteopenia, cataract, and weight gain were more common in the budesonide group and likely contributed to a higher number of discontinuations. Although this study did not show that budesonide improved histological outcomes in the UDCA-treated PBC patients, it showed that budesonide was associated with improved biochemical surrogate markers for disease progression, suggesting that it may be effective in PBC patients with marked histologic inﬂammatory activity. In the real- world setting, budesonide is rarely used as a second-line therapy for PBC as fewer biopsies are being performed to make the diagnosis, its contraindication in cirrhotic patients, and the high likelihood of achieving adequate response to the standard PBC therapies without immunosuppression even if overlap inﬂammatory features are present. Nor-urodeoxycholic acid Nor-ursodeoxycholic acid (nor-UDCA) is a side-chain- shortened derivative of UDCA with special pharmacological features, making it a promising therapy for cholestatic liver disease (41). It has shown potent anti-cholestatic, anti- inﬂammatory and anti-ﬁbrotic properties (42, 43). It is also distinguished by its intrahepatic enrichment, leading to a possible role in non-cholestatic metabolic and inﬂammatory liver disease (41). A recent phase II clinical trial showed that nor-UDCA was effective in reducing ALP levels within 12 weeks in a dose- dependent manner in primary sclerosing cholangitis (PSC) patients with an excellent safety proﬁle (44). Phase III clinical trial is underway (NCT03872921). NorUDCA is currently undergoing phase II trial for PBC in Europe (EudraCT number: 2021-001431-56). Frontiers in Gastroenterology Budesonide Biliary cirrhosis in PBC occurs due to progressive cholestatic hepatitis caused by destructive cholangitis and interface hepatitis (33). The presence of autoimmune hepatitis features is often associated with increased serum aminotransferases and positivity of serum auto-antibodies, such as antinuclear antibodies (ANA) and anti-smooth muscle antibodies (ASMA). These autoimmune Frontiers in Gastroenterology 04 frontiersin.org 10.3389/fgstr.2023.1241901 10.3389/fgstr.2023.1241901 Naffouj and Wang features are usually corticosteroid-responsive, similar to classic autoimmune hepatitis (9). Additionally, in-vitro data showed that budesonide and UDCA have a synergistic effect on increasing biliary secretion of bicarbonate with a protective effect on the biliary epithelium from toxic bile acids (34, 35). Based on these facts, Hirschﬁeld et al. conducted a placebo-controlled randomized phase III trial on non-cirrhotic PBC patients who had histologically conﬁrmed inﬂammatory activity based on the Ishak score and inadequate response to UDCA to evaluate the role of budesonide as an additional therapy to improve liver histology including inﬂammation and ﬁbrosis (36, 37). Budesonide is not recommended in patients with cirrhosis due to the risk of portal vein thrombosis and uncontrolled systemic shunting of the medication (38). Patients were randomized to receive budesonide 9 mg daily in addition to UDCA 12-16 mg/kg/day vs. placebo and the same dose of UDCA. The study was underpowered due to recruitment challenges for the primary efﬁcacy analysis. In the intention-to-treat analysis, there was no difference in the primary endpoint between the treatment and placebo groups at 36 months. However, the proportion of patients in the budesonide group who met the POISE endpoint of biochemical response (ALP <1.67 the ULN with a 15% reduction from baseline and normal T.bili) was higher compared to the placebo group. The two groups did not differ in the frequency of side effects or serious adverse events. Side effects such as hypertension, osteopenia, cataract, and weight gain were more common in the budesonide group and likely contributed to a higher number of discontinuations. Although this study did not show that budesonide improved histological outcomes in the UDCA-treated PBC patients, it showed that budesonide was associated with improved biochemical surrogate markers for disease progression, suggesting that it may be effective in PBC patients with marked histologic inﬂammatory activity. The pipeline for future PBC therapies There is a strong interest in developing newer therapies for PBC, and early initiation of combination therapy in high-risk patients is also under investigation. Most recently, a ﬁxed dose of OCA and bezaﬁbrate has received orphan drug designation by the FDA; this novel combination therapy may provide greater efﬁcacy in bile acid synthesis and metabolism as it targets two different pathways in the pathogenesis of PBC. Two ongoing phase II trials (NCT04594694 and NCT05239468) are investigating a range of therapeutic doses of this combination treatment. An additional promising combination therapy is bezaﬁbrate and UDCA in UDCA non-responders which showed signiﬁcant improvement in biochemical markers, ﬁbrosis, and inﬂammatory histological score Frontiers in Gastroenterology 05 frontiersin.org 10.3389/fgstr.2023.1241901 10.3389/fgstr.2023.1241901 Naffouj and Wang Naffouj and Wang Nudt1 a promising therapeutic target for PBC (57–59). Some of the proposed mechanisms through which the gut-liver axis contributes to PBC pathogenesis include the effect of dysbiosis on the intestinal mucosal immune system balance increasing gut permeability leading to increased bacterial translocation and abnormal immune system activation, which can cause tissue damage through molecular mimicry mechanisms and the lipopolysaccharide (LPS)-Toll-like receptor 4 (TLR 4) signaling pathway (60, 61). Another mechanism is the impaired bile- microbiota interactions, leading to poor bile acid metabolism and cholestasis (60). Based on these ﬁndings, probiotics and fecal microbiota transplantation (FMT) may provide new avenues for future PBC therapies by restoring healthy microbiota and repairing intestinal barrier function (62, 63). Current evidence is limited, and further studies are warranted to better understand and harness their therapeutic potential (64). T-cell dysfunction is another contributing factor to the pathogenesis of PBC (49). Cholangiocytes sustain cell-mediated autoimmune injury via clusters of autoreactive CD4+ CD8+ pyruvate dehydrogenase complex (PDC-E2) speciﬁc T cells in the liver (50). A recent study found that Nudt1 mediates the inappropriate expansion, hyperactivation, and long-term survival of CD 103+ cells, the primary autoreactive T cells demonstrating cytotoxicity against cholangiocytes in patients with PBC. Additionally, blocking Nudt1 resulted in ameliorating cholangitis in mice models (51). These investigations propose a new paradigm of developing highly selective immunomodulating therapies for treating PBC. Ustekinumab The interleukin (IL)-12 pathway has been implicated in PBC pathogenesis. An open-label clinical trial evaluating the efﬁcacy of ustekinumab did not result in a signiﬁcant reduction in ALP and failed to establish the proof-of-concept for this therapy (56). Future directions Fibroblast Growth Factor 19 (FGF)-19 is an ileum-derived post- prandial enterokine that reduces bile acid synthesis in the hepatocytes via direct feedback, hence its potential role in PBC treatment (52). FGF-19 agonists have been shown to have anti- steatotic, anti-inﬂammatory, and anti-ﬁbrotic properties (53). Aldafermin (NGM 282), an engineered FGF-19 analog, has achieved signiﬁcant ALP reduction at 28 days in UDCA non- responders compared to placebo with an acceptable safety proﬁle in a randomized, double-blind placebo-controlled phase II clinical trial (54). Further studies are needed to prove the durability of biochemical response and its effect on preventing ﬁbrosis and liver decompensation and improving transplant-free survival. Despite the evidence of PBC being an autoimmune disease, the exact etiology remains unclear, making the establishment of surrogate endpoints difﬁcult. Using histological response as a primary endpoint, like in the budesonide trial, remains challenging due to difﬁculties in recruitment. Gamma glutamyl transferase (GGT) is rising as an additional biochemical marker being assessed as a primary endpoint in clinical trials to predict clinical outcomes of liver transplantation and liver related death even in patients with low serum level of ALP (65). Furthermore, transient elastography-derived liver stiffness measurement has emerged as a surrogate marker for liver ﬁbrosis in PBC, providing a non-invasive modality in monitoring disease progression and response to therapy over time and is being incorporated in recent trials. A large international retrospective multicenter study has shown that liver stiffness measurement by vibration-controlled transient elastography is an independent predictor for PBC clinical outcomes including liver transplantation and death with a measurement of 15 kilopascal (kPa) as a cut off for high-risk group (66). Frontiers in Gastroenterology Janus kinase inhibitors Barcitinib, a JAK 1 and JAK 2 inhibitor, is hypothesized to downregulate multiple cytokines involved in PBC pathogenesis. It was investigated in a clinical trial (NCT03742973) and showed efﬁcacy in reducing ALP at 12 weeks in UDCA non-responders (55). However, the study was terminated due to a lack of enrollment. This mechanistic pathway remains a potential target for novel therapies in the future and further studies are warranted. Other issues that contribute to the challenges in the execution of clinical trials in PBC and setting hard clinical outcomes include the rare nature and slow progression of the disorder, hesitation of patients to stay on placebo as part of long-term outcome trials, which limit the achievement of adequate sample sizes and the ability to determine the efﬁcacy of medications on transplant-free survival and mortality. Additionally, the signiﬁcant heterogeneity in clinical trials limits the generalizability and interpretation of metanalysis results. Therefore, using a standard design in larger clinical trials and relying on proper endpoints is crucial in advancing clinical research in PBC. frontiersin.org Author contributions 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. Both authors contributed to the study conception and design. SN drafted the initial portions of the manuscript. JW provided a critical revision of the manuscript. All authors contributed to the article and approved the submitted version. Conclusion Therapies for PBC continue to progress with possible targets of normalizing liver enzymes, minimizing symptoms, and avoiding Many studies have reported the link between gut dysbiosis and the pathophysiology of PBC, suggesting that gut microbiota may be 06 frontiersin.org Naffouj and Wang 10.3389/fgstr.2023.1241901 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. Funding liver transplantation. A new paradigm is creating a personalized treatment approach depending on the individual risk factors, symptom burden, liver biochemistries, degree of ﬁbrosis and potentially starting combination therapy with a step-down approach to prevent disease progression. To date, OCA is the only approved second-line therapy for PBC patients in the US. Fibrates have been shown to be effective in improving the biochemical surrogates for disease progression, pruritis, and potentially clinical outcomes compared to UDCA alone in high- risk patients, and its approval for PBC treatment is eagerly awaited. Multiple other promising ﬁbrates are under investigation in phase II and III clinical trials as demonstrated in Table 1. The pipeline for additional second-line therapies is rapidly growing, with potential treatments targeting underlying mechanistic pathways in PBC pathogenesis, such as NOX inhibitors. This review demonstrates that recent advances have provided a better understanding of PBC pathogenesis, inspiring new treatment approaches beyond UDCA. However, unmet needs remain, especially for patients with high- risk features and symptomatic disease, emphasizing the importance of ongoing clinical trials to ﬁnd more effective therapies. 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